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September 17, 2014

Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital

Highlights in Recent Literature | Clinical Immunology Highlights | Basic Immunology & Novel Therapies | ImmunphenotypingPDF VersionPrevious Issues

Highlights from Recent Literature

Increased MicroRNA-7 Levels and Decreased PTEN Signaling May Drive B Cell Activation in Lupus

A review of Xiang-ni Wu., et al. Defective PTEN regulation contributes to B cell hyperresponsiveness in systemic lupus erythematosus. Sci Transl Med 6, 246ra99 (2014). PMID: 25101889

Systemic erythematosus (SLE) is an autoimmune disease characterized by B cell production of autoantibodies that deposit in the tissues and give rise to tissue inflammation. Phosphatase and tensin homolog (PTEN) is a molecule known to regulate B signaling through the B cell receptor. In these studies, the authors report that PTEN activity in the B cells of patients with SLE is abnormally low and that this may contribute to lupus pathogenesis.

  • PTEN plays a critical role in regulating B cells by suppressing the activity of phosphatidylinositol 3-kinase (PI3K), one of the pathways involved in signal transduction initiated by signaling though the B cell receptor.

  • The authors studied PTEN expression by flow cytometry and found that PTEN levels were decreased in all B cells from SLE patients, except for memory B cells, but were normal in healthy controls.

  • The levels of PTEN expression inversely correlated with disease activity, with periods of active disease coinciding with the lowest levels of PTEN expression.

  • The authors then studied PTEN regulation in SLE patients and found it was abnormal. In healthy controls, IL-21 induced PTEN expression after B cell receptor signaling but IL-21 did not increase PTEN protein levels in B cells from SLE patients.

  • IL-21 signaling did increased mRNA for PTEN in SLE B cells, but there was no corresponding increase in PTEN protein levels, suggesting an interfering RNA may be responsible.

  • The authors then studied microRNAs that could regulate PTEN and found increased levels of miR-7, miR-21 and miR-22 in SLE B cells.

  • Treatment of SLE B cells with an anti-microRNA (antagomir) targeted against miR-7 corrected the PTEN abnormalities in B cells from SLE patients.

PTEN is a key negative regulatory of B cell activation in healthy individuals. The authors report that PTEN levels are decreased in B cells from patients with SLE and suggest that decreased PTEN levels may contribute to B cell activation and autoantibody production in SLE. The authors found inappropriately high expression of the microRNA miR-7 in SLE B cells, leading to decreased protein levels of PTEN. This work is significant because it identifies decreased PTEN signaling may contribute to chronic B cell activation in SLE and suggests that agents that decrease miR-7 levels could be effective new therapeutics for SLE.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


Immune Cells Annexin A1 Production Drives Keratinocyte Death in Toxic Epidermal Necrolysis

A review of Saito N., et al. An annexin A1–FPR1 interaction contributes to necroptosis of keratinocytes in severe cutaneous adverse drug reactions. Sci Transl Med 6, 245ra95 (2014).
PMID: 25031270

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, potentially fatal drug reactions that involve widespread keratinocyte death and sloughing of the skin. Because of widespread loss of skin, TEN patients must be managed in burn units and this drug reaction is uniformly fatal when advanced facilities are not available. In this study, the authors find that immune cell production of annexin A1 induces keratinocyte death in these patients and that inhibition of annexin A1-keratinocyte signaling may be an effective therapy for TEN and SJS.

  • The authors identified patients who had survived SJS/TEN, treated PBMC from these patients with the causative drug in vitro, and collected the supernatants. They also collected supernatants from treated PBMC of patients who previously had non-threatening, non-SJS/TEN drug eruptions.

  • The authors incubated these supernatants with keratinocytes from the same patients and found that SJS/TEN supernatants induced keratinocyte death but supernatants from non-SJS/TEN patients did not.

  • Mass spectrometry studies identified annexin A1 as the key mediator of keratinocyte death in SJS/TEN supernatants. Neutralizing antibodies against annexin A1 blocked keratinocyte death in vitro.

  • Keratinocyte cell death receptors for annexin A1 were expressed on keratinocytes. Keratinocyte death was found to be caused by necroptosis, signaling induced cell death, and was mediated by the RIP1/RIP3 complex.

  • Inhibition of necroptosis completely blocked SJS/TEN-like responses in a mouse model of SJE/TEN.

SJS/TEN are severe, life threatening drug reactions with no specific therapy. The authors found that keratinocyte cell death in these patients occurs via necroptosis- a type of signaling induced cell death that under normal conditions is thought to be a defense against viral infection. The authors identified that annexin A1 was produced by drug-reactive T cells after exposure to the causative drug and it was this factor that mediated keratinocyte death. These studies are important because they identify annexin-A1 signaling as a causative agent in the life threatening loss of skin viability and integrity in SJS/TEN patients. These results suggest that annexin-A1 neutralizing or inhibitors of necroptosis may be effective new therapies for patients with SJS and TEN.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


Epigenetics Underlying CD4 T Cell Differentiation and Asthma Pathogenesis

A review of Seumois G., et al. Epigenomic analysis of primary human T cells reveals enhancers associated with Th2 memory cell differentiation and asthma susceptibility. Nature Immunology. (2014) PMID:24997565

Over the past few decades we have seen global increases in asthma in both adults and children. Asthma is a chronic inflammatory illness with significant morbidity and mortality, which is associated for many patients with atopy. However, though it is the most common chronic condition in childhood and has been the subject of deep investigation, the fundamental mechanisms remain unclear. CD4 T cells that secrete type 2 cytokines (Th2 cells) have been implicated as a key cell subset in the pathogenesis of asthma. In this study Seumois et al compare the genome wide histone modification profiles of a single epigenomic mark (H3K4me2) using ChIP-seq in naïve, T helper 1 (Th1) and T helper 2 (Th2) patients with asthma versus healthy controls. This single mark was chosen as an assay that could be used to identify enhancers that are either active or poised to become active in rare cell subsets from human samples with inherently limited volumes of blood available for research. The investigators isolated peripheral blood CD4+ T cells from 12 healthy controls and 12 asthmatic patients and then isolated naïve and memory cells, and divided the memory cells into CCR4 expressing (and denoted these as Th2, given that they were enriched for Th2 cells) and CCR4 negative (and denoted these as Th1, given that they were depleted of Th2 cells and enriched for Th1 cells). Within these three cell subsets in each patient group, ChIP-seq was used to determine the DNA regions associated with H3K4me2. The assay was extensively optimized and microscaled (to be reproducible down to 10,000 cells). The major findings were:

  • Enhancers associated with memory differentiation as well as enhancers associated with asthma were identified.

  • They next investigated differentially enriched regions (DERs) between the cell types they were studying.

  • 90% were associated with the transition from naïve to memory T cells, only 10% with the difference between Th1 and Th2 cells.
  • Many of the lineage associated enhancers were conserved between human and mouse.

  • DERs were then associated with genes, including 1400 protein coding genes, 70 microRNAs and 78 long non-coding RNAs using enrichment of H3K4me2 at their promoters. These genes were assigned to six groups based on gain or loss of enrichment with differentiation into Th1, Th2 (or shared). These included genes that are known to be associated with those subgroups, which were assigned to the correct groups by this process (i.e. IL-4 and Th2) as well as genes that had not previously been associated with this differentiation step.

  • Much more frequently, DERs were outside promoters (referred to as putative enhancers), and these DERs were connected to genes based on connecting them with genes sharing insulator bounded regions of the genome and performing computational analysis of those relationships.

  • Genome wide association studies have identified disease associated SNPs. Asthma associated SNPs were more enriched in Th2, rather than Th1, cells. Interestingly, SNPs associated with systemic lupus erythematosus (SLE) were more enriched in Th1, rather than Th2, cells.

  • Finally, the asthmatic patients were compared to the healthy controls to find CD4+ T cell enhancers that varied between the two groups, and 200 enhancers were identified, of which 163 were Th2 specific. Interestingly, 42% had at least one transcription factor binding site, with significant overrepresentation of T cell differentiation associated TF.

  • Candidate genes associated with asthma status included the Toll-like receptor and chemokine receptor pathways.

This epigenome-wide study of 24 patients, including 12 healthy controls, 6 mild asthmatics (never treated with corticosteroids) and 6 moderate asthmatics (treated with inhaled corticosteroids) presents evidence that this microscaled assay of a single epigenomic mark could be used to identify novel pathways (and potentially therapies) in human disease. As with any initial study demonstrating a novel technique, there are caveats. For example, the use of CCR4 as the differentiating factor for Th2 vs. Th1 cells provides enrichment, rather than pure populations of Th2 and Th1 cells. In addition, asthma is a very inclusive term and we are only beginning to understand the sub-phenotypes within that diagnosis (which have different ages of onset, key cell type and response to therapy) and this study included mild and moderate patients. It is possible that severe adult patients (or moderate pediatric patients) would have different results. However, this is a very powerful technique for deepening our understanding of the genomics underlying human disease, and it will likely be applied broadly in the next few years.

Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia


Hematopoetic Stem Cell Transplantation in Severe Combined Immunodeficiency

 A review of Pai, S.Y., et al. Transplantation outcomes in severe combined immunodeficiency, 2000-2009. NEJM. (2014) 371: 434-436.
PMID: 25075835

In severe combined immunodeficiency, infants have reduced T cell counts and impaired T and B cell immune responses, and the many etiologies of the condition are fatal in infancy if left untreated. Allogeneic hematopoetic stem cell transplantation can be curative, and there are many possible types of transplants (from bone marrow, T cell depleted bone marrow, peripheral blood derived stem cells and umbilical cord blood) and donors (siblings, parents and unrelated donors). With so many variables, including age at transplant and whether or not to condition (and what type of conditioning), a study surveying the current landscape of practice and outcomes was undertaken to identify important clinical factors.

The records from 240 infants with SCID who were transplanted at 25 centers who participate in the Primary Immune Deficiency Treatment Consortium (PIDTC) 1from 2000-2009 were collated to study the factors associated with better outcomes.

Outcomes investigated included survival at 5 years, CD3+ T cell recovery, lack of need of intravenous immunoglobulin, IgA recovery and phytohemagglutinin-induced T cell proliferation and factors collected included age at transplantation, whether the infant had ever had a significant infection (or was now infection free after having been treated for a significant infection), as well as the relationship to the transplant donor and the use of conditioning prior to transplant. The major findings were:

  • Overall, 72% had engraftment of donor T cells (graft failure was lowest with matched sibling donors)Of note, donor type, genotype and use of conditioning (and regimen if used) were not significant in their association with graft failure necessitating retransplant
  • Infants with SCID who were transplanted at less than 3.5 months of age had an impressive 94% survival rate at 5 years, which decreased with age (90% in older, never-infected infants) or the history of a treated infection (down to 82 % in infants who had been treated for an infection). Infants with active infections at the time of transplant benefited most with haploidentical T cell depleted transplants without conditioning.

  • Of note, given that this sample was focused prior to the initiation of NBS, the infants identified earlier than 3.5 months were generally in families with a prior SCID history

  • Patients with active infections at the time of transplant had worse outcomes with use of conditioning with many types of donors, most clearly with mismatched unrelated donors.

  • Patients receiving matched sibling donors had a 97% survival rate, followed by patients receiving mismatched related donors without conditioning (79%).

  • Immune reconstitution, as measured by CD3+ T cells > 1000/microliter was correlated with matched sibling donors and with myeloablative or reduced intensity regimens (versus no conditioniongconditioning or immune suppression) in other scenarios. Interestingly, B+ SCID was correlated with a greater likelihood of T cell reconstitution and NK+ SCID with poor T cell reconstitution.

  • Acute GVHD (grades 2-4) at 100 days was seen in 20% of patients, while chronic GVHD at 2 years was seen in 15% and its rate did not vary based on donor type.

The improved outcomes of infants less than 3.5 months of age (and those without prior infection) emphasize the importance of SCID newborn screening, improving our ability to identify and transplant infants prior to significant infections. These programs continue to spread and this data further supports the importance of these programs. This study permits an assessment of many key questions in SCID transplantation and provides a rich dataset for consideration

Reviewed by Sarah Henrickson, MD, PhD, Children’s Hospital of Philadelphia 


Hyperactivation of Phosphoinositide 3-Kinase Causes Primary Human Immunodeficiency

 A review of Deau MC., et al. A human immunodeficiency caused by mutations in the PIK3R1 gene. J Clin Invest. 2014 Sep 2;124(9):3923-8. PMID:25133428

Phosphoinositide 3-kinase (PI3K) is a heterodimeric enzyme composed of a catalytic p110 subunit (P110α, P110β, or P110δ) bound to a regulatory subunit (P85α/P55α, P85β, or P55γ). PI3K coordinates signals emanating from a wide variety of receptors and modulates an array of cellular functions including proliferation, differentiation, survival, and metabolism in multiple cell types. While the P110α and β subunits are ubiquitously expressed, the P110δ subunit is restricted to lymphocytes. Deletion of P110δ in mice results in impaired B cell development and loss of T and B cell activation and function. It was thus notable when two recent studies identified activating mutations in the P110δ subunit of PI3K in several unrelated patients with primary immunodeficiency (PID). The syndrome was named activated P110δ syndrome (APDS). New work by Deau et al. suggests that hyperactivation of the PI3K signaling axis may be a more common theme in PID syndromes. There are several key findings in this study:

  • Whole exome sequencing of 4 patients with PID from 3 unrelated families reveals two distinct PIK3R1 heterozygous splice site mutations. Both resulted in aberrant splicing of exon 10 leading to truncation of the p85α subunit of PI3K and loss of its PI3K p110-binding domain.

  • Western blot analysis of p85α confirmed a smaller protein with decreased expression, suggesting that the mutant protein was also less stable.

  • Expression of P110δ in T cells was similar to controls. However, consistent with loss of the p85α inhibitory domain, phosphorylation of the PI3K target AKT was elevated in both the basal state and after stimulation through the TCR. Likewise, phosphorylation of the downstream target ribosomal protein S6 was also elevated in unstimulated cells.

  • Further substantiating the splice site mutation as a disease-causing allele, ectopic expression of mutant but not wildtype p85α resulted in augmented in Pi3K/AKT/mTORC1 signaling.

  • Phenotypically, all four patients had recurrent respiratory bacterial tract infections, hypogammaglobulinemia, and decreased naïve T and memory B cell counts. T cell activation-induced cell death was increased in vitro and could be corrected by addition of a PI3Kδ B cell proliferation was also reduced, consistent with a functional B cell defect.

These findings phenocopy those in APDS syndrome leading the authors to suggest naming PID syndromes due to p85α splice mutations activated PI3Kδ syndrome 2 (APDS2) since uncontrolled activation of the p110 subunit is responsible for many of the observed phenotypes. These data are especially intriguing given that mutations that decrease PI3K activity can also cause a PID phenotype. Together, these data suggest that careful titration of PI3K activity is critical for normal immune cell function and have important implications for drugs targeting this pathway. The data also suggest that this pathway should be interrogated in patients with abnormal immune cell function or recurrent infections. One could envision treating APDS1 and 2 patients with PI3K pathway inhibitors. However, given that aberrant PI3K activity has been implicated in lymphomagenesis, further studies modeling the phenotypic consequences of these mutations in murine models would be of value.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco


Treating Alopecia Areata with Janus Kinase Inhibitors

A review of  Xing L., et al. Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition. Nat. Med. 2014 Sep;20(9):1043-1049. PMID: 2529481

Alopecia areata (AA) is a T-cell-mediated autoimmune disease characterized by hair loss. While not life threatening, the psychological impact for those with severe disease can be significant. Current therapy for AA most commonly involves broad-acting steroids and is met frequently with limited success. The study by Xing, et al. provides new hope for these patients. Building upon a previous genome-wide association study showing that AA is associated with polymorphisms of the NKG2D receptor and additional work demonstrating infiltration of CD8+NKG2D+ T cells in the peribulbar region of human AA hair follicles, the authors explored the role of these cells in disease pathogenesis. They exploit the C3H/HeJ mouse model that develops spontaneous alopecia and recapitulates many histopathologic features of human AA. There are several interesting findings from this work.

  • CD8+NKG2D+ T cells were found to infiltrate the epithelial layers of the hair follicle. These cells had an effector memory phenotype (CD8hi, αβ+, CD44hi, CD62Llow, CD103+) and were also found in draining lymph nodes. Gene expression analysis of sorted CD8+NKG2D+ T cells demonstrated a transcriptional profile characteristic of cytotoxic T lymphocytes and revealed several NK-specific transcripts. Functional studies demonstrated high levels of IFNγ and cytotoxicity to syngeneic dermal sheath target cells.

  • Adoptive transfer of purified CD8+NKG2D+ T cells or total LN cells from diseased mice could transfer disease to healthy recipients. However, transfer of LN cells depleted of NKG2D+ cells failed to cause disease, demonstrating that CD8+NKG2D+ T cells were both necessary and sufficient for AA.

  • Gene expression profiling of skin lesions from mice or humans with AA or healthy control skin revealed signatures indicative of IFNγ response genes, CTL-specific transcripts, and γc specific cytokines (IL-2 and IL-15). Immunofluorescence studies demonstrated high levels of IL-15 expression in follicular epithelial cells of disease, but not normal, hair follicles and high levels of IL-15 receptor expression on infiltrating T cells.

  • Systemic administration of blocking antibodies to IL-2, IL-15R, and IFNγ (but not IL-21) blocked disease development but did reverse established AA in mice.

  • Systemic treatment of AA mice using the JAK1/2 inhibitor ruxolitinib or the JAK1/3 inhibitor tofacitinib prevented AA and reversed established AA in mice. Interestingly, topical treatment with either JAK inhibitor also enabled hair regrowth, an effect that persisted for 2-3 months after treatment cessation.

  • Finally, the authors treated 3 human AA patients with oral ruxolitinib (FDA approved for the treatment of myelofibrosis). All three patients had almost complete hair regrowth after 3-5 months of therapy. Comparison of skin biopsies at baseline and 12 weeks after therapy showed reduced T cell infiltration and reversal of the IFNγ signature.

Based on these findings, the authors propose a feed-forward model for AA where CD8+NKG2D+ T cells produce IFNγ that then induced production of IL-15 by follicular epithelial cells. This IL-15 further promotes survival and expansion of the infiltrating CD8+NKG2D+ T cells and loss of immune privilege in the hair follicle. The authors postulate that JAK inhibitors break this cycle by interfering with cytokine-mediated signals. Further clinical trials that address frequency of response and long-term safety issues in AA patients are clearly warranted.

Reviewed by Michelle Hermiston, MD, PhD, University of California San Francisco


Immune Checkpoint Therapy Boosts the Efficacy of BRAF Inhibition

A review of Cooper, Z.A., et al. Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade. Cancer Immunol Res, 2014; 2(7): 643-654. PMID: 24903021

Therapies targeting the oncogenic BRAF V600E mutation have yielded impressive but short-lived responses, with half of cases progressing after only 6 months. The authors have shown previously that treatment with BRAF inhibitors (BRAFi) leads to an upregulation in known melanoma antigens, resulting in T cell infiltration. The tumors seem primed for an immune response, however high levels of the inhibitory ligand PD-L1 in the microenvironment, along with other immunosupressive signals stall this response. Combining the two approaches of BRAF inhibition and immune checkpoint blockade is a promising strategy and, indeed clinical trials are ongoing. In this study, Cooper et al. show that this combination has strong potential for clinical success using a novel mouse model.

  • First, the authors evaluated intratumoral CD8+ T effector (CD8): FoxP3+ T regulatory (Treg) cell ratios in a melanoma patient treated with BRAFi followed by treatment with anti-CTLA4 antibody. They show that treatment with BRAFi alone resulted only in a temporary increase in the CD8:Treg ratio; however, subsequent treatment with anti-CTLA-4 rescued the effect and resulted a durable and greater increase in the intratumoral Teff:Treg ratio.

  • Next, the authors developed a syngeneic immunocompetent implantable murine melanoma model to study the combination of BRAFi with checkpoint inhibition. The model they established recapitulates the clinical observations of increased T cell infiltration and cytokine production, tumor antigen upregulation, and PD-L1 expression following BRAFi treatment.

  • Using their model, they demonstrate that treatment with BRAFi1 results in a dose-dependent increase in tumor-free survival that is abrogated when CD8+ T cells are depleted.

  • Combining PD1 blockade with BRAFi led to better survival and reduced tumor growth when compared with either monotherapy.

  • Finally, they show that the synergy between these two treatments is the result of increased tumor infiltration and function of CD8 T cells.

In this study, the authors demonstrate that BRAFi treatment leads to enhanced CD8+ Teff cell infiltration into tumors, but immune regulatory signals, such as the PD1-PD-L1 axis, oppose the infiltrating T cells and suppress the antitumor response. The possibility of combining the two powerful therapies to achieve a durable tumor response has inspired several clinical trials for which data is not yet available . Cooper et al. provide further evidence that these approaches are on the right track, while also creating a model system for elucidating mechanisms of synergy and resistance, and also determining optimal dosing schedules.

Reviewed by Alexander Hopkins and Eric Lutz, PhD, Johns Hopkins University


Peptibodies as a New Approach for Inhibiting Myeloid-Derived Suppressor Cells

A review of Hong Qin, et al., Generation of a New Therapeutic Peptide That Depletes Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice. Nature Medicine. 2014; 20(6): 676-81. PMID:24859530

Induction of a potent antitumor response requires circumventing the immunosuppressive environment of the tumor, including myeloid-derived suppressor cells (MDSCs). However, there are few defined surface markers for MDSCs, which limits studies of MDSCs within the tumor microenvironment. Using a peptide phage display platform, Qin et al. identify two peptides that specifically bind murine MDSCs and, when fused with the Fc region of mouse IgG2b to form a peptibody, can deplete MDSCs and delay tumor growth in mice. Immunoprecipitation experiments indicate that the proteins S100A9 and S100A8 may be the targets of these two peptibodies.

  • A commercially available peptide phage library was panned against Gr-1 and CD11b labeled splenocytes for three rounds, resulting in two predominant peptide clones, G3 and H6. These clones were selected for peptibody development for further characterization.

  • Peptibodies specifically stained CD11b+Ly6G-Ly6Chigh (monocytic) and CD11b+Ly6G-Ly6Cint/low (granulocytic) MDSCs by flow cytometry, but did not stain Ly6G-CD11c+ dendritic cells (DCs) or B, T, or NK lymphocytes (a small portion of T and NK lymphocytes were stained with the G3 peptibody).

  • Intravenous injections of the peptibodies depleted intratumoral, blood, and splenic monocytic and granulocytic MDSCs in mouse tumor models (EG.7 and EL4 thymomas), but did not affect DC or lymphocyte (B, T, or NK) populations. Treatment with control peptibody (irrelevant peptide-Fc fusion) had no effect on cell populations, and a Gr-1-specific monoclonal antibody (mAb) resulted in depletion of only granulocytic MDSCs. Cellular depletion was transient with systemic MDSC populations recovering 3 days after a single treatment.

  • Tumor development, as measured by tumor size and mass, was delayed when tumor challenged mice were treated with either G3- or H6-peptibody. Treatment with Gr-1-specific mAb also resulted in tumor growth inhibition, but was less consistent than inhibition by peptibodies.

  • Immunoprecipitation of MDSC surface proteins using G3- or H6-peptibodies followed by proteomic analysis of eluted proteins suggested that S100A9 and S100A8 were the binding partners of both peptibodies. As S100A9 and S100A8 form heterodimers, it is possible that the peptibodies recognize the dimer, or that there is cross-reactivity with the two proteins. Peptibody treatment of S100A9-deficient mice (which still express S100A8) resulted in partial depletion of MDSCs, consistent with the cross-reactivity hypothesis.

MDSCs are potent inhibitors of T cell function with few identified surface markers. The peptides identified by the competitive phage display platform identify two potential molecular markers of both monocytic and granulocytic MDSCs, S100A9 and S100A8, with limited or no cross-reactivity with proinflammatory DCs and lymphocytic cell populations. Furthermore, treatment with MDSC-specific peptibodies resulted in the depletion of both granulocytic and monocytic MDSCs; and delayed tumor growth with no visible off-target effects when compared to control tumor-bearing mice. These data suggest that this platform may be useful for identifying therapeutic targets and cell surface markers for specific cell subsets, including human MDSCs.

Reviewed by Heather Kinkead and Eric Lutz, PhD, John Hopkins University

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Highlights From Clinical Immunology, the Official Journal of FOCIS

TLR Ligands Induce IL-17RA Expression in Neuroglia in EAE

A review of Liu, et al. Toll-like receptor signaling directly increases functional IL-17RA expression in neuroglial cells. Clinical Immunology. 154:127–140. 2014. PMID:25076485 

IL-17 and Th17 responses are now considered central to the immunopathology of several autoimmune diseases including multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis. Previous work has shown that IL-17 receptor (IL-17R) is widely expressed on neuroglial cells and neuroglia respond to IL-7 by expressing various pro-inflammatory cytokines and chemokines. Previous work has also shown that IL-17R is up-regulated in EAE. The major pro-inflammatory receptor for IL-17a and f is a heterodimer composed of IL-17RA and IL-17RC subunits. The authors of this paper explored the regulation of IL-17R expression by neuroglial cells in response to innate signals, using a MOG peptide/CFA immunization protocol to induce EAE. The major findings are:

  • IL-17RA mRNA expression is up-regulated in mice brains and spinal cord during EAE induction, but IL-17RC expression remains at a constant level.

  • qRT-PCR was done on neurons, astrocytes, microglia, and oligodendrocytes isolated from the brains of normal and EAE mice, and increased IL-17RC message was elevated in all three neuroglial cell types, and was not detected in neurons.

  • IL-17R expression in the CNS was induced in mice treated with CFA without MOG peptide, even though these mice did not develop EAE.

  • IL-17R expression was up-regulated, shown by immunohistochemistry and qRT-PCR, in CFA immunized Rag1−/− mice, indicating that T cells were not necessary for induced expression.

  • In vitro studies showed that LPS and IL-17A synergistically stimulated secretion of CCL2, CXCL8 and IP-10 chemokines by astrocytes, as did IL-17 made by CD4+ T cells.

  • IL-17 was shown to down-regulate the induced levels of IL-17RA in astrocytes.

This paper helps clarify the progression of inflammatory events that occur during the development of EAE. An early event is innate signaling in microglial cells, via TLRs, to induce IL-17RA expression, allowing these cells to be receptive to IL-17 secreted by antigen-specific Th17 cells. The microglial cells then respond to the IL-17 by producing a variety of chemokines that promote further recruitment of inflammatory cells. IL-17 may also promote down-regulation of IL-17RA in astrocytes as a negative feedback mechanism.

 Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital


IFNα in Lupus Nephritis

A review of Dai C., et al. Interferon alpha on NZM2328.Lc1R27:Enhancing autoimmunity and immune complex-mediated glomerulonephritis without end stage renal failure. Clinical Immunology. 154, 66–71. 2014. PMID: 24981059

A significant role for type I interferons, especially interferon α, in the pathogenesis of SLE has been inferred from gene expression profiles of blood leukocytes of SLE patients. Furthermore, some patients treated with IFNα show ANA, anti-dsDNA antibody responses and some develop SLE. However, longitudinal studies of IFNα levels do not appear to reflect disease activity in SLE patients, and trials of anti-IFNα antibody treatment have not been successful. The authors of this paper previously described the NZM2328 and NZM2328.Lc4 mouse models of lupus nephritis (LN), which they used to show that LN and end stage renal disease (ESRD) are under separate genetic control from autoantibody production. Those previous studies indicated that ANA and anti-dsDNA antibodies in their mice are not necessary for LN/ESRD. The authors also previously described a congenic derivative of NZM2328 line, called R27, which spontaneously develop immune complex-mediated glomerulonephritis (GN) but, unlike the parent strain, do not progress to chronic GN/ESR. In the current paper, the authors use the NZM2328 and R27 mouse lines, together with IFNα treatment via an adenovirus vector, to explore the genetic influence on IFNα induced SLE and LN. The major findings are:

  • IFNα treated young NZM2328 mice developed accelerated GN including severe proteinuria, chronic GN, and early mortality, which mimics the disease that occurs spontaneously in older NZM2328 mice.

  • In contrast, the R27 congenic mice treated with IFNα did not develop chronic GN, yet they did develop autoantibodies and immune complex deposition in glomeruli similar to IFNα-treated NZM2328 mice.

  • IFNα treated NZM2328 and R27 mice both developed anti-dsDNA antibodies.

The implications of this study are that IFNα can induce autoimmunity in mice, as previously described, but end organ damage is not directly mediated by IFNα and is regulated by distinct genetic factors. The authors suggest that the IFN signature in SLE patients and the lack of correlation of IFNα levels with clinical activity may reflect the importance of IFNα in initiating autoimmunity, but secondarily induced cytokine/inflammatory responses and susceptibility to end organ damage depend on independent genetic factors, as in their mice studies.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

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Human Immunophenotyping Update

Cell Ontologies: Concepts and Issues

Holden T. Maecker, PhD, Stanford University

There exists a standard ontology, or definition and description, of human genes (1), which is administered by the Gene Ontology Consortium (http://geneontology.org). This is an extremely useful tool, because genes are discreet, and mostly shared within individuals of a species (aside from allelic variation, mutations, duplications, or deletions). Thus, their definition by a standard nomenclature and association into functional groups provides a framework for standardized genomic analyses.

Building on the concept of the gene ontology, efforts have been made to construct a cell ontology (https://code.google.com/p/cell-ontology), which has been updated for hematopoietic cell types (2). This is a most noble effort, because the current state of cellular immunology lacks standard definitions for the different types of cells that it studies. Ideally, a cell ontology would provide minimal essential definitions of immune cell types based on their expressed markers and/or functions. Imagine the help that this would provide the field, if every time we referred to, for example, a regulatory T cell, one could be sure that it was a cell defined by a discreet set of commonly agreed-upon markers and/or functions.

Unfortunately, cells are much more diverse than genes. With high-dimensional techniques such as mass cytometry, hundreds of different phenotypes of CD8+ T cells can be defined by combinatorial expression of cytokines, for example (3). Similar diversity has been demonstrated in the NK cell compartment using combinations of stimulatory and inhibitory receptors (4). One could postulate that the number of discreet cell types is limited only by the number of markers being analyzed (and of course, by the total number of cells in the organism).

The fact that one cell type can give rise to another further complicates the definitions. Are differences that result from short-term activation sufficient to define a cell as a new type? What degree of stability is required to define a discreet cell type? And how do we handle markers that have a continuous distribution of expression, like CD38 in activated T cells? These are just some of the questions that complicate the ability to create a true cell ontology.

This is not to say that cells can’t be grouped into categories based on shared markers and/or functions. For example, most of us would agree that it’s useful to define CD4+ T cells as distinct from CD8+ T cells. And the definition of these groups is relatively easy, requiring only a few markers (CD3, CD4, and CD8). But even this simple case illustrates some difficulties of definitions. For example, CD4+ and CD8+ T cells can have overlapping functions: subsets of both cell types can be cytolytic, and they can produce common cytokines like IFNγ, TNFα, and IL-2. So, an ontology built strictly on functions would look very different from one based only on phenotypic markers.

As we progress to more esoteric cell types, the definitions get harder and harder. Precisely because of this, we have previously used this column to try to create some standardization in the way that important and controversial cell types are defined (e.g., Tregs, dendritic cells, Th17 cells, and B cells; see http://www.focisnet.org). Upcoming issues will continue this effort. Furthermore, the FOCIS Human ImmunoPhenotyping Consortium (HIP-C) has created standard 8-color panels for definition of basic immune cell types (5). Our experience with using these panels in lyophilized format across multiple labs is currently being prepared for publication. Although these panels were chosen by consensus of a large group of experts from academics, industry, and NIH, they don’t necessarily reflect agreement across the entire field of cellular immunology. And differences in how specific cell types are gated (and even how many different cell types to gate) can still be debated.

Another issue of course is the incorporation of new knowledge. At what point do we determine that a particular new marker is “essential” for defining a certain cell type? Is one publication demonstrating its utility enough? In some ways, what the cell ontology attempts to do is to synthesize the whole of the immunology literature, as it relates to cell types, in a single document/database. Such an enormous effort is sure to cause contention, and will require constant curation.

Given all these caveats, some suggestions for the advancement of the cell ontology are perhaps in order. First, it makes sense to concentrate first on the largest and most easily defined cell types. Dissecting the flavors of CD8+ T cells into ever-smaller and more narrowly defined subtypes, for example, is probably not that useful. But standard definitions for naïve, central memory, effector memory, and effector CD8+ T cells could be very helpful.

Second, there needs to be room for multiple options and equivalency in definitions. Building on the above example, the classical definition of a central memory T cell might be given as CD45RA-CCR7+, but under the right conditions, CD62L might be substituted for CCR7 (5). I say “under the right conditions”, because CD62L is labile and can be greatly reduced with cryopreservation or other perturbations (6). Other markers, like CD27 or CD127, might be options as well, but with their own caveats; they are not 100% equivalent to CCR7, but might be suitable for some purposes.

This brings up a final point, namely, the concept of “fit for purpose”. The HIP-C panel, by design, uses only cell-surface markers, for ease of use and standardization. This precludes, for example, the use of FoxP3 for definition of Tregs. Are surface markers truly sufficient to define Tregs? That is debatable, though I would say the loss of resolution is minor. The surface panel is, in effect, fit for the purpose of using a quick and easy staining panel. Another example: Are CD19 and CD20 both required to define mature B cells? Arguably it’s better to have both markers if possible, particularly if the labels used for either of them are dim, and might not provide sufficient resolution on their own. But if one is constrained to designing, say, a 4-color panel, one would probably chose just one of these markers for defining B cells.

All of this is to say that it’s not easy to synthesize the many nuances of immunophenotyping into a single document. Nevertheless, the discussions that take place in attempting this are probably themselves reason enough to pursue the effort. Furthermore, tools that help to apply the knowledge compiled in the cell ontology could also be helpful to the field. In that vein, the Brinkman lab has recently developed a tool, called FlowCL, that can automatically label populations with suggested cell types from the cell ontology (Courtot et al., manuscript submitted).

To summarize, we need to proceed with caution when attempting to summarize the immense diversity and plasticity of immune cells. Just as we don’t want to discount a study that fails to use one specific marker for definition of Tregs, we also don’t want to assume that a minimal definition of such cells is suitable for all applications.

References

  1. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, M. A. Harris, D. P. Hill, L. Issel-Tarver, A. Kasarskis, S. Lewis, J. C. Matese, J. E. Richardson, M. Ringwald, G. M. Rubin, and G. Sherlock. 2000. Gene Ontology: tool for the unification of biology. Nature genetics 25: 25–29.

  2. Diehl, A. D., A. D. Augustine, J. A. Blake, L. G. Cowell, E. S. Gold, T. A. Gondré-Lewis, A. M. Masci, T. F. Meehan, P. A. Morel, A. Nijnik, B. Peters, B. Pulendran, R. H. Scheuermann, Q. A. Yao, M. S. Zand, and C. J. Mungall. 2011. Hematopoietic cell types: prototype for a revised cell ontology. Journal of Biomedical Informatics 44: 75–79.

  3. Newell, E. W., N. Sigal, S. C. Bendall, G. P. Nolan, and M. M. Davis. 2012. Cytometry by Time-of-Flight Shows Combinatorial Cytokine Expression and Virus-Specific Cell Niches within a Continuum of CD8+ T Cell Phenotypes. Immunity 36: 142–152.

  4. Horowitz, A., D. M. Strauss-Albee, M. Leipold, J. Kubo, N. Nemat-Gorgani, O. C. Dogan, C. L. Dekker, S. Mackey, H. Maecker, G. E. Swan, M. M. Davis, P. J. Norman, L. A. Guethlein, M. Desai, P. Parham, and C. A. Blish. 2013. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry. Science Translational Medicine 5: 208ra145.

  5. Maecker, H. T., J. P. McCoy, and R. Nussenblatt. 2012. Standardizing immunophenotyping for the Human Immunology Project. Nat. Rev. Immunol. 1–10.

  6. Weinberg, A., L. Y. Song, C. Wilkening, A. Sevin, B. Blais, R. Louzao, D. Stein, P. Defechereux, D. Durand, E. Riedel, N. Raftery, R. Jesser, B. Brown, M. F. Keller, R. Dickover, E. McFarland, T. Fenton, for the Pediatric ACTG Cryopreservation Working Group. 2009. Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization. Clinical and Vaccine Immunology 16: 1176–1186.

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December 19, 2014

Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital

Highlights in Recent Literature | Clinical Immunology Highlights | Basic Immunology & Novel Therapies | ImmunphenotypingPDF VersionPrevious Issues

Highlights from Recent Literature

Metformin a Surprising New Ally in the War Against Drug-resistant Tuberculosis

A review of Singhal A., et al. Metformin as adjunct antituberculosis therapy. Sci. Transl. Med. 6, 263ra159 (2014).Sci Transl Med 6, 246ra99 (2014). PMID: 25411472

Tuberculosis (TB) remains one of the biggest killers worldwide and the emergence of drug-resistant strains has complicated therapy. Novel agents that control TB by enhancing host immune responses are urgently needed. In this study, authors report that the anti-diabetic medication metformin enhanced anti-TB immune responses via multiple pathways and may be a powerful new weapon in the war against drug-resistant tuberculosis.

  • Metformin is known to have multiple effects including activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK), an energy-sensing molecule that regulates cell growth and survival, alteration of host derived mitochondrial reactive oxygen species (ROS) production and modulation of autophagy mediated by mTOR signaling.

  • The authors studied the effect of various medications to inhibit intracellular growth of the TB vaccine strain BCG and pathogenic strains of TB.

  • Metformin was found to inhibit the growth of vaccine strains of TB, pathogenic strains of TB, and even multi-drug-resistant TB strains in vitro. This antibacterial function depended on AMPK activity and metformin also increased mitochondrial ROS production and enhanced autophagy.

  • Metformin was also effective in a mouse model of acute and chronic TB. Gene expression analyses determined that metformin down regulated genes associated with inflammation, including interferons, and acted to balance the immune response.

  • The authors then studied patients with both type II diabetes and TB and found that metformin use was associated with reduced severity of pulmonary lesions and with increased survival.

The authors found that metformin modulates several aspects of host immune responses to TB, acidifying the phagosome to enhance microbial killing, inducing autophagy and reducing overt inflammation which helps to ameliorate tissue damage. This comprehensive study, including in vitro analyses, animal models and retrospective analysis of patient responses, sets the stage for adoption of metformin as a new and powerful adjuvant therapy for TB.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


MicroRNA and Skewing of the T Cell Polarization in Asthmatic Lung

A review of Simpson LJ., et al. Epigenomic analysis of primary human T cells reveals enhancers associated with Th2 memory cell differentiation and asthma susceptibility. Nature Immunology. (2014) 15: 777. doi: 10.1038/ni.3026. PMID: 25362490

Asthma is a chronic inflammatory disease characterized by episodes of airflow limitation and bronchial hyperreactivity and is the most common chronic disease of childhood. While there is an increasing appreciation that there are multiple phenotypes within asthma, T helper 2 cells play a clear role in a subset of patients. In this study, Simpson et al profile miRNA expression in human airway T cells in asthma patients versus healthy controls.

SUMMARY

  • They isolated CD4+ T cells from bronchoalveolar lavage (BAL) fluid from 8 healthy controls and 12 steroid naive asthmatic patients and 21 ‘steroid using’ asthmatic patients and profiled 190 miRNAs, of which nearly 90 were present in at least 60% of subjects.

    • miRNA-19a (part of the miR-17~92 cluster) was the most correlated with asthma (both steroid naïve and steroid using)

    • The miR-17~92 cluster positively regulated secretion of type 2 cytokines IL-13, IL-4 and IL-5. This effect was found to be cell-intrinsic.

  • Rather than a brief requirement for miR-19a during Th2 differentiation, miR-19a was found to be needed throughout Th2 polarization. 

    • Using inhibitors and mimics of miR-19a in murine and human T cells, they show that altering miR-19a alone is sufficient to affect IL-13

  • In order to determine the relevant targets of miR-19a in Th2 polarization, they used siRNAs to inhibit the 38 previously known target genes of miR-19a and measured the effect on IL-4 and IL-13. They also used miR 17~92 deficient cells and showed that rescue of the phenotype was possible with inhibition of PTEN, SOCS1 and TNFAIP3.

    • All three are negative regulators of signaling, but for three separate signaling pathways (PI3K, Jak/STAT and NFkB respectively).

  • Finally, they demonstrated in mice that the reduction in Th2 cytokine production found in miR 17~92 T cells leads to a decrease in Th2 inflammation in vivo using an OVA/OT-II TCR transgenic model of airway hyperreactivity. They found that with antigen specific cells that lacked miR17~92 there was a decrease in eosinophilia in BAL fluid.

This group was able to use profiling of miRNA in airway infiltrating T cells to identify a miRNA, miR-19a, that plays a mechanistic role in encouraging Th2 polarization of T cells, which may contribute to pathogenesis in a subset of asthmatics. While this is fascinating, it will require additional confirmation and mechanistic studies to further elucidate the details of this process (including possible roles in using this mediator to predict prognosis, define phenotype and even possibly provide a therapeutic target for asthmatics with Th2 predominant disease).

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


Using Systems Analysis to Investigate Pathogen Susceptibility in Primary Immunodeficiency

A review of Alsina L., et al. A narrow repertoire of transcriptional modules responsive to pyogenic bacteria is impaired in patients carrying loss-of-function mutations in MYD88 or IRAK4. Nature Immunology. (2014) 15: 1134-42. PMID: 25344726

Many components of innate and adaptive immunity have been implicated in primary immunodeficiency syndromes, including selective pathogen associated molecular pattern recognition abrogation due to Toll-like receptor (TLR) pathway dysfunction due to MyD88 and IRAK4 loss of function mutations. Of the 10 TLRs in humans, all but one signals via MyD88, and IRAK4 is recruited by MyD88 to signal downstream of those receptors. Interestingly, patients with these defects do not have the broad increased susceptibility to pathogens that might be expected, and instead have a relatively narrow range of pathogens that they are susceptible to (i.e. pyogenic gram positive and gram negative bacteria) and they show clinical improvement in adolescence. Rather than use a traditional panel approach, this team compares responses of patients to purified TLR ligands and whole pathogens using transcriptional readouts and cutting edge systems analysis.

SUMMARY

  • They began by characterizing the effect of purified TLR agonists and PMA-ionomycin on whole blood transcriptome analysis in 14 healthy controls. Transcripts with consistent changes were retained. Pathway analysis was undertaken and shared programs (i.e. inflammatory response) were examined.

  • They next assessed the responses of (n=4) IRAK4 and (n=4) MyD88 deficient patients to purified TLR agonists.

    • While responses to PMA/ionomycin were preserved and LPS responses were only partially reduced, transcriptional response to LPS, PAM3, PAM2, flagelln IL-1b and IL-18 and R-848 were dramatically reduced

    • No differences seen between IRAK4 and MyD88 deficient patients

  • Interestingly, when patient samples were stimulated instead with heat killed whole bacteria (S. pneumonia or S. aureus) they retained the ability to have > 50% of the transcriptional activity of healthy control cells responding to these infections.

    • In addition, MyD88 deficient patients had consistently lower responses versus IRAK4 deficient patients

  • To better understand the exact effect of the IRAK4 or MyD88 deficiency, the group clustered their genome-wide data and were able to use clustering to define modules of transcriptional change in the setting of infection. The 1088 transcripts yielded 66 modules, which were then arranged into 7 larger clusters. These included a cluster (5) which contained genes that were associated with bacterial infection but not part of the TLR pathway (consistent with compensatory processes being available to protect the host from these pathogens), including the inflammasome.

    • They also demonstrated strain specific susceptibility to bacteria in some patients

    • In addition, responses to fungi and viruses ranged from normal (Candida) to impaired (HSV) in these patients

This group is able to use whole transcriptome analysis of responses of whole blood from IRAK4 or MyD88 deficient patients to demonstrate that while responses to purified TLR ligands is blocked, as expected, they retain the ability to respond to selective whole pathogens (even to the pathogens that they are clinically susceptible to), likely secondary to compensatory inflammatory responses (e.g. inflammasome). This application of genome wide transcriptional analysis to individualized pathogen susceptibility in primary immunodeficiency is interesting and could be broadened to the more detailed assessment of responses to immune stimuli in other patient groups (i.e. autoimmunity or malignancy) or other infectious agents. If feasible for broader application, it might yield a more nuanced view of the mechanisms underlying susceptibility in a given patient and potentially more individualized prophylaxis or treatment regimens in those patients.

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


Naughty or Nice? Potential Role of Tumor-associated Neutrophils in Early Stage Human Lung Cancer

 A review of Erusalnov EB., et al. Tumor-associated neutrophils stimulate T cell responses in early stage human lung cancer. J. Clin. Invest. 2014 Dec. 124(12)5466-5480. PMID:25384214

While several recent studies have highlighted the role of adaptive immunity and the value of enhancing cytotoxic T cell function with checkpoint inhibitors such as those targeting PD1 or CTLA4 in oncogenesis, the role of the innate immune system in modulating human malignancy is less well characterized. A recent study by Eruslanov, Signhal, and colleagues sheds new light on a potential role for tumor-associated neutrophils (TANs) in the cancer microenvironment. Most prior studies employing immunohistochemistry have suggested that TAN infiltrates are associated with a poor prognosis in human cancers. However, studies in murine models have suggested that TANs could have both pro-tumor as well as anti-tumor effects. Moreover, in human gastric cancer, neutrophils were associated with a good prognosis. To address these conflicting results, the authors employed both immunohistochemical as well as functional studies of TANs in early stage human lung cancer. Contrary to the investigators initial hypothesis, TANs surprisingly appear to have an anti-tumor effect by stimulating T cells in early stage lung cancer. There are several intriguing findings from this work.

  • Using both immunohistochemistry as well as flow cytometry to analyze single cell populations, TANs constituted 5-25% of the cells isolated from 25 squamous cell carcinoma (SCC) and 45 adenocarcinoma (AC) stage 1 or 2 non-small cell lung cancer (NSCLC) samples. TANs were more prevalent in NSCLCs with a SCC phenotype. In both histologic subtypes, TANs were often co-localized with T cells. Interestingly, there was no correlation between the frequency of TANs and whether or not the patient was a smoker.

  • Compared with blood neutrophils or neutrophils obtained from non-malignant lung tissue from the same patient, TANs had a more activated phenotype (CD62LloCD54hi) with a distinct chemokine receptor repertoire (CCR5, CCR7, CXCR3, and CXCR4 high). TANs also failed to express inhibitory receptors or ligands such as PD-L1. These correlations were not impacted by histologic subtype or smoking history.

  • Relative to blood neutrophils or neutrophils from non-malignant sites, TANs produced increased quantities of proinflammatory cytokines upon stimulation, had high phagocytic activity in vitro, and produced high levels of reactive oxygen species (ROS). While the isolation process is an important caveat, these data suggest TANs are functionally active.

  • TANs stimulated antigen-nonspecific T cell proliferation and activation. Intriguingly, TANs from smaller tumors had a more robust impact than those isolated from larger tumors.

  • While the proportion of TANs in both SCC and AC did not correlate with tumor size, TANs obtained from larger tumors were associated with a lower capacity to augment T cell proliferation.

Naughty or nice? These findings suggest that TANs may potentially play a nice role in early stages of lung cancer by stimulating T cell responses to the tumor. While quite intriguing, additional work is clearly needed. Although the impact of TANs on T cells suggests an anti-tumor effect, it is conceivable that other TAN functions could simultaneously serve a pro-tumor function. For example, do TANs have a direct impact on tumor cell proliferation, metastasis, or other aspects of the tumor microenvironment such as angiogenesis or infiltration of other immune cell populations? It will also be important to test whether the functional properties of TANs change during tumor progression or at relapse to a more immunosuppressive phenotype and whether these findings are generalizable to other tumor subtypes. If this is the case, TANs could become another target in the immuno-oncology arsenal.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco


Naughty or Nice? Role of Tumor-infiltrating Myeloid Cells in Tumor Cell Senescence

A review of Di Mitri, et al. Tumor- infiltrating Gr-1+ myeloid cells antagonize senescence in cancer. Nature. 2014 (Nov 6) 515:134-137.  Nat. Med. 2014 Sep;20(9):1043-1049. Epub 2014 Aug 17. PMID:25156255

Cellular senescence is the induction of permanent loss of the ability of a cell to divide. It can be induced by a variety of mechanisms including aberrant activation of oncogenes or loss of tumor suppressor genes such as Pten or exposure to chemotherapy. In this regard, stable cell growth arrest due to cellular senescence is an important anti-cancer mechanism for the host. Recent work by Di Mitri, Tosso, Alimonti, and colleagues addresses the question of whether tumor-infiltrating immune cells can modulate whether tumor cells become senescent. The authors had previously demonstrated that loss of Pten in the mouse prostate epithelium induces the formation of benign tumors characterized by a strong senescence response that opposed tumor progression. Over time, these tumors overcame this senescence and adopted an aggressive and invasive phenotype. In the current work, the authors address the hypothesis that immune cells might enable Pten deficient tumor cells to evade senescence in a non-cell autonomous manner. Using both mouse models and human prostate cancer samples, they find a new role for the innate immune system in regulating cellular senescence and chemotherapy resistance. There are several intriguing findings from this work.

  • Characterization of Pten deficient prostate (PtenPC) tumors revealed a large infiltration of CD45+CD11b+Gr-1+ myeloid cells. Intriguingly, these cells tended to co-localize with Ki-67 positive proliferating cells that appeared to have overcome the senescence induced by loss of Pten.

  • Adoptive transfer for GFP-labeled bone marrow precursors into PtenPC mice resulted in infiltration of GR-1+ cells into prostate tumors, again with a preference for close proximity to proliferating tumor cells, raising the possibility that these cells prevented senescence in a paracrine manner.

  • Consistent with this notion, co-culture of Pten deficient mouse embryonic fibroblasts (MEFs) with conditioned media from GR-1+ cells prevented senescence in these cells. Using a variety of strategies, the authors identified interleukin-1 receptor antagonist (IL-1RA) as a potential GR-1+ cell secreted factor mediating this effect.

  • Validating a role for IL-1RA, Pten-loss induced senescence was enhanced in vivo when IL1ra deficient cells were adoptively transferred. Likewise, preventing migration of CD11b+ Gr1+ myeloid cells using a CXCR2 antagonist resulted in increased chemotherapy-induced senescence.

  • Finally, the authors evaluated a panel of human prostate cancer specimens. They found that patients with high levels of intratumoral IL-1RA levels had poor responses to chemotherapy and decreased progression-free survival. Importantly, high levels of IL-1RA correlated with increased numbers of tumor-infiltrating myeloid cells and aggressive phenotype. Conversely, low IL-1RA levels correlated with fewer infiltrating myeloid cells and increased expression of senescence markers in the tumor cells.

Are innate immune cells naughty or nice? In this context, they are naughty. Taken together, these data are consistent with a model whereby infiltrating innate immune cells are detrimental, promoting tumor progression by opposing senescence, perhaps via production of IL-1RA. It will be important to determine whether these findings can be expanded to other tumor types. If validated, these data suggest that blocking recruitment of Gr-1+ myeloid cells or inhibiting IL-1RA may be of therapeutic benefit.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco


New Bioinformatic Approaches for Neoepitope Identification

 A review of Duan, F., et al. Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity. J Exp Med. 2014; 211(11):2231-48. PMID:25245761

Cancer cells carry tens to hundreds of unique mutations. Each mutation has the potential to generate a neoepitope capable of being recognized by T cells and contributing to anti-tumor immunity; but in reality, only a small fraction of the total pool of mutations are immunoprotective. Despite the relative ease of identifying tumor-specific mutations using next-generation sequencing, predicting which mutations generate immunogenic neoepitopes that can drive protective antitumor responses has proven to be challenging. While NetMHC and other algorithms capable of predicting MHC:peptide binding affinities provide promising screening tools, they only tell part of the story. Screens using predicted MHC binding affinity as a sole metric omit contributions of the TCR, as well as tolerance mechanisms which may be preventing anti-tumor immunity. Duan et al. examine this problem by testing the efficacy of vaccines targeting putative neoepitopes selected based either on NetMHC scores alone (which predicts MHC:peptide binding affinities), or based on the authors’ newly proposed differential agretopicity index (DAI, which compares the MHC affinity of the mutant peptide to the wild-type peptide).

  • The authors first identify single nucleotide variants predicted to generate strong MHC binding peptides (identified using the NetMHC algorithm) from two chemically induced implantable murine tumor cell lines, CMS5 and Meth A. They identified 112 and 823 strong binding single nucleotide variants, respectively.

  • Next, the authors found that 56% of the top NetMHC predicted peptide neoepitopes had high MHC affinity (IC50 > 500nM) when measured experimentally. Despite this, when the authors vaccinated mice with these peptides, only 17% of the mutant peptides induced a specific T cell response (IFNγ secretion measured by FACS or ELIspot relative to the wild-type peptide). Furthermore, none of the highest NetMHC scoring peptides was able to protect mice from tumor challenge.

  • From these initial results, the authors conclude that the NetMHC score alone is insufficient to predict the immunogenicity or anti-tumor efficacy of a peptide. To identify peptides more likely to illicit a response, they created a new score called the differential agretopic index (DAI) which is calculated as the difference between the NetMHC scores for the wild-type and mutant peptides.

  • Neoepitope peptides identified based on DAI scores were much more successful in protecting mice from tumor challenge in a CD8-dependent manner, however the hit rates were still quite low (30% of peptides from CMS5 and 14% Meth A). The authors note that nearly all peptides identified by DAI bear anchor residue mutations.

  • Next, the authors performed structural analysis of the mutant and wild-type peptide:MHC complexes to determine if any physical properties correlated with immunogenicity. While no trend was observed in the overall magnitude of the structural differences induced by the mutation, peptides with greater structural stability (measured by root mean square fluctuation of the peptide's α carbons) were found to be more likely to induce an immune response.

Duan et al. suggests that MHC:peptide affinities, predicted or calculated, are insufficient for predicting the immunogenicity of putative mutant neoepitopes. They identify two new principles which will likely guide future work in this field: first, the affinity of the mutant peptide must be considered relative to the wild type peptide; and second, increases in the structural rigidity of the peptide resulting from mutations may increase immunogenicity. These insights provide a foothold for the development of more reliable neoepitope prediction methods, which could have far reaching implications for the clinical development of mutant neoepitope-targeted immunotherapy.

Reviewed by Alexander Hopkins and Eric Lutz, PhD, Johns Hopkins University


Anti-CTLA-4 Therapy Broadens the Melanoma-Reactive CD8+ T Cell Response

A review of Kvistborg, P. et al., Anti-CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response, Science Translational Medicine 6, 254ra128–254ra128 (2014). PMID:25232180

Treatment of melanoma patients with the T cell checkpoint inhibitor ipilimumab (an anti-CTLA-4 antibody) has led to improved patient survival; however, it is unclear whether this benefit is derived from an increased magnitude of pre-existing T cell responses, or the induction of novel tumor-specific T cell responses, resulting in a broader tumor-specific CD8+ T cell repertoire. Kvistborg et al. show that patients with advanced-stage melanoma who received ipilimumab had an increase in the number of different T cell responses while the overall magnitude of pre-existing T cell responses was on average unchanged.

  • Peripheral blood mononuclear cells (PBMCs) from HLA-A*0201 positive, advanced-stage melanoma patients who received ipilimumab were probed for CD8+ T cell reactivity to peptide-major histocompatibility (pMHC) complexes for 145 defined melanoma epitopes as well as 3 viral epitopes (influenza A, CMV, and EBV). T cell reactivity was assessed from pre-treatment and several post-treatment samples, and revealed induction of novel melanoma epitope reactivity as soon as 3 days post-infusion of ipilimumab, while pre-existing responses generally did not change in magnitude.

  • CD8+ T cell responses were more commonly reactive against epitopes derived from cancer/germ-line and melanocyte differentiation antigens than epitopes from overexpressed antigens.

  • Comparisons of the kinetics of T cell response from time of diagnosis to treatment initiation and from the first 12 weeks of treatment demonstrated that T cell responses increased from 0.029 T cell responses per month prior to treatment to 0.321 T cell responses per month after the induction of ipilimumab therapy.

  • Melanoma epitope-specific T cell receptors that appeared after the induction of ipilimumab therapy were cloned and shown to recognize several melanoma tumor cell lines expressing the corresponding antigens, demonstrating the potential of the new T cell responses induced by anti-CTLA-4 to control tumor growth.

The T cell checkpoint inhibitor ipilimumab has shown a survival benefit for patients with advanced melanoma, but the mechanism by which targeting CTLA-4 confers a survival benefit remains to be determined. Kvistborg et al. demonstrate that while the magnitude of pre-existing melanoma-specific CD8+ T cell responses remains constant, new melanoma antigen-specific T cell responses arise shortly after the induction of ipilimumab therapy. Thus, these data suggest that anti-CTLA-4 works by priming new T cell responses and broadening the repertoire of the tumor-specific T cell response, rather than by amplifying pre-existing T cell responses. Likewise, other strategies capable of broadening the tumor-specific T cell response may induce similar antitumor effects as anti-CTLA-4.

Reviewed by Heather Kinkead and Eric Lutz, PhD, John Hopkins University

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Human Immunophenotyping Update

Phenotyping Assessment of Human Blood T Follicular Helper Cells by Flow Cytometry

Salah-Eddine Bentebibel and Hideki Ueno, MD, PHD, Baylor Institute for Immunology Research

T follicular helper (TFH) cells represent the subset of CD4+ T cells that helps B cells in germinal centers (GCs) and supports their differentiation into memory B cells or long-lived plasma cells. GC TFH cells express the chemokine receptor 5 (CXCR5), which guides their migration into B cell follicles. They express inducible co-stimulator (ICOS), a molecule essential for their development and function. GC TFH cells also express high levels of immune-regulatory molecule PD-1, signaling adaptor SLAM-associated protein (SAP), and the transcription repressor B cell lymphoma 6 (Bcl-6).

CXCR5+ CD4+ T cells are also found in human blood, and share functional properties with GC TFH cells in secondary lymphoid organs. Recent multiple lines of evidence shows that blood CXCR5+ CD4+ T cells are likely a memory compartment of TFH cells. Accordingly these cells have been termed as blood (or circulating) TFH cells (1). Blood TFH cells are defined as CD4+CD45RA-CXCR5+ and constitute 15-20% of memory CD4+ T cells in humans. The majority of these cells express CD62L, CCR7, and CD45RO and thus belong to central memory cells. In contrast to GC TFH cells, a vast majority of blood TFH cells do not express activation markers such as CD69 and ICOS, and express PD1 only at low levels. Blood memory TFH cells also lack the expression of Bcl-6. Thus, while TFH cells in secondary lymphoid organs are in active state, blood TFH cells are generally in resting state. Recent studies show that human blood memory TFH are composed of heterogeneous subsets with different phenotypes and functions. Currently, there are two main approaches to identify human blood TFH cell subsets.

The first approach consists of using the chemokine receptors CXCR3 and CCR6 (2). This approach permits the dissection of blood memory TFH cells into three major subsets: CXCR3+CCR6- cells, CXCR3-CCR6- cells, and CXCR3-CCR6+ cells. The CXCR3+CCR6- cells resembles to TH1 cells (hereafter called blood TFH1 cells). The CXCR3-CCR6- subset resembles to TH2 cells (hereafter called blood TFH2 cells). The CXCR3-CCR6+ cells resembles to TH17 cells (hereafter called blood TFH17 cells). An example of the analysis of peripheral blood mononuclear cells (PBMCs) from a healthy individual is shown in Figure 1. While TFH2 and TFH17 cells produce interleukin-21 (IL-21) upon interactions with naïve B cells, and are capable of inducing B cell differentiation into immunoglobulin-producing cells, TFH1 cells lack this capacity (2).

The second approach consists of using the immune-regulatory molecule PD1, the inducible co-stimulatory molecule ICOS, and the chemokine receptor CCR7. This approach permits the dissection of another three subsets, one activated subset (ICOS+PD-1++CCR7lo), and two quiescent subsets (ICOS-PD-1+CCR7int, and ICOS-PD-1-CCR7hi) (3, 4). ICOS expression defines blood memory TFH cells with activated phenotype that highly express PD-1 and the cell cycle marker Ki-67. ICOS- cells are further divided into PD-1+CCR7int cells and PD-1-CCR7hi cells, both of which lack the expression of Ki-67 and therefore are in a quiescent state.

We propose that human blood memory TFH cells are composed of nine distinct subsets (1), which are defined by combining the two approaches. It is feasible to construct a flow cytometry panel containing CD3, CD4, CD8, CXCR5, CD45RA, CXCR3, CCR6, ICOS, PD1, and CCR7. A damp channel to exclude any contamination of other cells such as CD8+ T cells, dendritic cells, and natural killer cells and a viability dye such as Live/Dead also can be added to the panel. In order to assess whether CXCR3 and CCR6 staining is properly working, we recommend to compare the expression of these two chemokine receptors between naïve CD4+ T cells and blood memory TFH cells, as naïve CD4+ T cells do not contain cells that highly express CXCR3 or CCR6. When a 10-color flow cytometer is not available, the panel can be simplified by removing CCR7. CCR6 can also removed from the panel as the usage of CXCR3 alone can separate TFH1 (non-efficient helpers) and non-TFH1 cells (efficient helpers).

An increasing number of studies have already demonstrated that analyses of human blood TFH cell subsets provide insights regarding the type and the magnitude of ongoing Tfh and antibody response in patients with autoimmune or infectious diseases, and in subjects after vaccinations. For instance, patients with juvenile dermatomyositis, a pediatric autoimmune disease, are found to display an alteration in the balance of blood memory TFH1, TFH2, and TFH17 subsets (less TFH1 and more TFH2 and/or TFH17), which correlates with disease activity and circulating plasmablast (2). Similar finding was reported in studies of adult SLE, Sjogren’s syndrome, and multiple sclerosis (1). In a study with a large cohort of HIV infected subjects, subjects with broadly neutralizing antibodies against HIV are found to display relatively higher frequency of ICOS-PD-1+ TFH2 and TFH17 cells among blood TFH cells (4). Influenza vaccine was found to induce the emergence of the ICOS+CXCR3+ TFH1 cells at 7 days post-vaccination, which positively correlates with the generation of protective antibody responses (5).
Currently the strategy to define subsets in human blood TFH cells is often different among laboratories. We believe that a standardized approach for the analyses and definition of blood Tfh cell subsets will have a great advantage to the field, because it will permit an integration of results from multiple studies performed on different diseases at different sites. Such effort might facilitate the process in the identification of new biomarkers associated with antibody responses in vaccinations, and of similarities and differences in the type of alterations in the TFH response in patient with autoimmune diseases.

Picture1

 

References

  1. Schmitt N, Bentebibel SE, Ueno H. Phenotype and functions of memory Tfh cells in human blood. Trends Immunol. 2014;35(9):436-42.

  2. Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, et al. Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity. 2011;34(1):108-21.

  3. He J, Tsai LM, Leong YA, Hu X, Ma CS, Chevalier N, et al. Circulating Precursor CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T Cells Indicate Tfh Cell Activity and Promote Antibody Responses upon Antigen Reexposure. Immunity. 2013;39(4):770-81.

  4. Locci M, Havenar-Daughton C, Landais E, Wu J, Kroenke MA, Arlehamn CL, et al. Human Circulating PD-1(+)CXCR3(-)CXCR5(+) Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses. Immunity. 2013;39(4):758-69.

  5. Bentebibel SE, Lopez S, Obermoser G, Schmitt N, Mueller C, Harrod C, et al. Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination. Science Translational Medicine. 2013;5(176):176ra32.a

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Developments in Basic Immunology and Novel Therapies

Regulatory T Cells and Tolerance of the Fetus

Abul K. Abbas, MD, University of California San Francisco

The development of placentation in eutherian mammals enabled the fetus to mature and remain in a protected environment for much longer than was possible in less evolved species. But the free vascular connections between the fetus and the pregnant mother enabled the maternal immune system to readily access fetal tissues, including paternal antigens that were expressed in the fetus. These antigens are obviously foreign for the mother but have to be tolerated to prevent rejection of the fetus. The problem of tolerance of the fetus has fascinated immunologists for over fifty years, and has been addressed by a variety of experimental and clinical studies. Many explanations have been proposed, with varying degrees of experimental and clinical support. Recent discoveries implicating a central role of regulatory T cells promise to give meaningful answers that may not only solve a biological riddle but may also suggest approaches for elucidating the basis of spontaneous fetal loss in humans, and ultimately to provide therapies for this condition.

Mechanisms of fetal tolerance: the early findings
As the mechanisms of allograft rejection and acceptance were defined over the last twenty or so years, these same concepts were tested for their relevance to fetal tolerance. The principal mechanisms that have been proposed to explain fetal tolerance have been reviewed recently (1). These include: low expression of MHC molecules in the fetus and trophoblast; immaturity of antigen-presenting dendritic cells in the fetus; defects in accumulation of maternal dendritic cells in the decidua; imbalance of T cell activation, resulting in excess of anti-inflammatory Th2 over pro-inflammatory Th1 cells; production of the enzyme indoleamine 2, 3-dioxygenase (IDO) in the placenta, which generates tryptophan metabolites that are toxic to proliferating lymphocytes. Although all of these are plausible explanations, it has proved difficult to translate some of the experimental results to humans or, in some cases, to show that aberrations in these inhibitory pathways consistently result in fetal loss. Thus, the contribution of these mechanisms to fetal tolerance remains uncertain.

A role for Foxp3+ regulatory T cells: the evolutionary perspective
Regulatory T cells (Tregs) that express the Foxp3 transcription factor have emerged as a dominant mechanism of tolerance. It is now established that Tregs consist of multiple subsets. Some, called thymic Tregs (often referred to as “natural” Tregs) arise in the thymus upon recognition of self antigens expressed in this organ, and others, called peripheral Tregs (also called “adaptive” or “induced” Tregs) arise from mature CD4+ T cells upon recognition of newly encountered antigens in peripheral tissues, such as commensal microbes and tumors. One of the most intriguing clues about the role of Tregs in fetal tolerance came from a landmark study by Samstein et al (2). These authors had previously shown that the Foxp3 gene contained a regulatory region called the consensus noncoding sequence 2 (CNS2) that was required for the stable expression of Foxp3 in peripheral Tregs and, hence, for the development and maintenance of this population. They went on to demonstrate that during evolution, the CNS2 region appeared in eutherian mammals at precisely the same time as placentation, and it was not present in mammals such as wallabies and opossums that do not have a placenta or in non-mammalian species. The concordant appearance of two apparently unconnected features – the CNS2 genetic region in the canonical Treg gene Foxp3 and placentation – strongly implies a causal relationship between these. The authors went on to use a mouse model to show that T cells from CNS2-expressing mice developed into Tregs in a pregnant female and the fetus survived normally, but if the T cells lacked CNS2, T cells were activated and a significant fraction of the fetuses were resorbed. These biological experiments nicely complemented the evolutionary data and strongly suggested that peripheral Tregs induced by recognition of “foreign” antigens in the fetus were responsible for protecting the fetus from attack by the maternal immune system.

A role for Foxp3+ regulatory T cells: experimental studies
Many other experimental studies have explored the involvement of Tregs in fetal tolerance. In one elegant study (3), the investigators used transgenic technology to express a model antigen in male mice and, thus, in the fetus after breeding with normal females. Tregs specific for this antigen were detected in the pregnant females. When these Tregs were eliminated and replaced with Tregs from normal mice (not containing any cells specific for the paternal antigen), there was a high rate of fetal resorption. (The approach of replacing fetal antigen-specific Tregs with Tregs lacking this specificity was an important step because it prevented the development of systemic autoimmunity that invariably results from depletion of Tregs.) Thus, fetal antigens induce specific peripheral Tregs in the mother, and elimination of these cells results in fetal loss.

Other studies have indicated that even Tregs that develop in the thymus can protect the fetus from rejection (4). Since most (or all) thymic Tregs are specific for self antigens expressed in the thymus, these results imply that the fetus must contain some of the same self antigens that are present in the pregnant female – obviously a not unreasonable idea. However, these self antigens are unlikely to be targets of rejection by the mother’s immune system. Perhaps self recognition activates Tregs in the fetus that suppress responses not only against the self antigens but also against paternal antigens that are foreign to the mother, a form of “bystander suppression”.

A role for Foxp3+ regulatory T cells: observations in humans
A large number of studies have examined the frequencies of Tregs in the blood and trophoblast of individuals with normal pregnancies, pre-eclampsia, and pre-term fetal loss. These have been well reviewed recently (5). Despite considerable variations in the results, the bulk of the available evidence is consistent with the generation or expansion of Tregs in healthy pregnancies and quantitative or qualitative defects in this population in conditions associated with premature fetal loss. One of the major challenges in this field is to define the antigen specificity of the Tregs that may mediate fetal tolerance. This challenge reflects the technical difficulty of establishing the antigen specificity of all lymphocytes in humans.

Perspectives and future prospects
Recent data briefly reviewed above are leading to an increasing acceptance of Foxp3+ Tregs as a key mechanism responsible for tolerance of the fetus. These results have led to the hypothesis that thymic Tregs evolved to prevent harmful immune reactions against self antigens (many of which are expressed in the thymus) and peripheral Tregs evolved to prevent responses against commensal microbes and fetal antigens. Such a hypothesis is reasonable, but a role for thymic Tregs in fetal tolerance cannot be excluded.

Among the many questions that remain to be answered are how Tregs suppress immune responses against the fetus. Many mechanisms of Treg-mediated suppression of immune responses have been proposed. Of these, the ones that are supported by the most compelling evidence are blockade or elimination of B7 costimulators (CD80, CD86) on antigen-presenting cells by CTLA-4 expressed on Tregs; production of immunosuppressive cytokines, such as IL-10 and TGF-ß, by Tregs; and absorption of the T cell growth factor IL-2 by the high levels of IL-2 receptors expressed on Tregs. These mechanisms are, of course, not mutually exclusive, and at present there is little evidence to implicate or exclude any of them in Treg-mediated fetal tolerance.

Another important mechanistic question is why the trophoblast may favor the development of Tregs. It has been known for decades that while paternal antigens in the fetus are tolerated, the same antigens expressed in a tissue graft at a distant site are targets of rejection. Thus, the fetal environment is especially prone to activate mechanisms that suppress immune responses. Elucidating these mechanisms may provide new ideas about ways of inducing and harnessing Tregs to control harmful immune reactions at any location. A corollary to the postulated special role of the trophoblast is that Treg-mediated fetal tolerance is not associated with generalized immunosuppression.

Finally, the contribution of defective Treg generation or maintenance to recurrent abortions is a question of obvious clinical importance. As methods for inducing and expanding Tregs are entering clinical testing, this question becomes one of increasing significance. Preventing fetal loss by promoting Treg-mediated suppression without compromising protective immunity against infections remains a distant goal, but the recent basic research raises hopes that one day, the goal may be achieved.

References

  1. Erlebacher A. Mechanisms of T cell tolerance towards the allogeneic fetus. Nat Rev Immunol 13:23, 2013.

  2. Samstein RM, Josefowicz SZ, Arvey A, Treuting PM, Rudensky AY. Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell 150:29, 2012.

  3. Rowe JH, Ertelt JM, Xin L, Way SS. Pregnancy imprints regulatory memory that sustains anergy to fetal antigen. Nature 490:102, 2012.

  4. Chen T, Darrasse-Jeze G, Bergot AS, Courau T, Churlaud G, Valdivia K, Strominger JL, Ruocco MG, Chaouat G, Klatzmann D. Self-specific regulatory T cells protect embryos at implantation in mice. J Immunol 191:2273, 2013.

  5. Jiang TT, Chaturvedi, E,Ertelt JM, Kinder JM, Valent AM, Xin L, Way SS. Regulatory T cells: new keys for further unlocking the enigma of fetal tolerance and pregnancy complications. J Immunol 192:4949, 2014.

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March 23, 2015

Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital

Highlights in Recent Literature | Clinical Immunology Highlights | Basic Immunology & Novel Therapies | ImmunphenotypingPDF VersionPrevious Issues

Highlights from Recent Literature

Modulation of CD4+ T Cell Metabolism Improves Lupus

A review of Julia Cuende et al. Monoclonal antibodies against GARP/TGF-b1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo. Science Translational Medicine (2015) 7, 274ra18. PMID: 25904740

Regulatory T cells (Treg) play critical roles in initiating and maintaining self tolerance but they can also interfere with tumor immunity and permit the persistence of chronic infections. One of the mechanisms by which Treg suppress other T cells is by presenting inactive TGFb1 on the cell surface that becomes activated to immunosuppressive active TGFb1 by unknown mechanisms. The authors demonstrate that the protein GARP plays a key role in TGFb1 activation and that a monoclonal antibody that blocks this conversion inhibits Treg function.

  • GARP is a transmembrane protein expressed by Treg but not by other T cell subsets and it binds to latent TGFb1 on the Treg cell surface.
  • The authors generated 31 new mouse and llama-anti human GARP antibodies and cloned these onto the human IgG1 backbone, creating humanized anti-GARP antibodies.
  • Two of these antibodies inhibited the production of active TGFb1 by Treg, suggesting that GARP is directly involved in production of active TGFb1 by human Treg.
  • These two inhibitory antibodies both mapped to a similar domain of the molecule, hGARP101-141, a part that is involved with TGFb1 association on the cell surface.
  • The two inhibitory antibodies blocked Treg mediated suppression in in vitro assays and in a model of human anti-mouse xenogenic graft vs. host disease (GvHD).

By creating and studying antibodies against GARP, a protein that associates with latent TGFb1 on the surface of Treg, the authors demonstrated that GARP is involved in the activation of TGFb1 by Treg and that inhibition of this activity abrogates Treg mediated suppression in vitro and in an in vivo human anti-mouse GvHD model. Neutralizing anti-GARP antibodies are therefore a novel new class of Treg inhibitory therapeutics that may be valuable in the treatment of cancers and chronic infections.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


Inhibition of the mTOR Pathway Improves Immune Function in Older Individuals

A review of Mannick J., et al. mTOR inhibition improves immune function in the elderly. Science Translational Medicine. 6, 268ra179 (2014). PMID:25540326

Inhibition of the mammalian target of rapamycin (mTOR) pathway extends the life spans of multiple animal species but its effect on human aging is poorly characterized. In this report, the authors took the first steps towards characterizing mTOR inhibition in human aging by studying its effect on immune function in elderly individuals.

  • Adults 65 years and older experience 90% of influenza related deaths in the United States and also have lower antibody responses following influenza vaccination than younger individuals
  • Decreased immune functioning with age is associated with a number of immune deficits, including a decreased production of naïve T cells and increased numbers of PD-1-expressing exhausted T cells
  • The authors evaluated the effect of the MTOR inhibitor RAD001 versus placebo in elderly volunteers by administering the drug for six weeks before influenza vaccination.
  • RAD001 enhanced anti-influenza vaccine responses by approximately 20% and reduced the percentage of both CD4+ and CD8+ T cells that expressed PD-1

The authors found that a relatively short course of mTOR inhibitor therapy significantly increased the responses to influenza vaccination and reduced the number of PD-1 expressing T cells. Moreover, the authors noted efficacy at lower doses than those used in organ transplantation, thereby minimizing side effects. Further studies to characterize the effect of mTOR inhibition in younger individuals following vaccination and on other aspects of the immune system in elderly individuals are warranted.

Reviewed by Rachel A. Clark, MD, PhD, Brigham and Women's Hospital


Nature or Nuture? T Cell Differentiation?

A review of Becattini, et al. Functional heterogeneity of human memory CD4+ T cell primed by pathogens or vaccines. Nature Immunology. 2015; 347: 400-405. PMID:25477212

The nature and breadth of heterogeneity in human T cell responses has been the focus of many recent exciting studies. In this study, Becattini et al examine whether individual pathogens (including a fungus, bacteria and a vaccine) instigate one predominant subtype of human CD4+ helper cells and whether a single antigen specific clone can differentiate to multiple subtypes.

SUMMARY

  • They isolated CD4+ T cells from PBMCs from healthy donors and defined four T cell subsets (Th1, Th1*, Th2, Th17) based on chemokine receptors (CXCR3+CCR4- CCR6-; CXCR3+CCR6+CCR4-; CCR4+CXCR3-CCR6-; CCR4+CCR6+CXCR3-).
  • They began by stimulating CFSE labeled PBMCs with Candida albicans and then characterized the memory (CFSElo) cells.
    • Th17 and Th1* predominated (though Th1 and Th2 also were found). In addition, when the TCRβ repertoire was examined in 5 donors, the overall clonotype diversity was comparable among the four CD4+ T cell subsets.
    • They next examined whether clonotypes were shared among T cell subsets within a given donor (i.e. does a given clonotype have T cell subset specificity?). They found that there were shared TCRβ clonotypes among each possible pair of CD4+ T cell subsets (and some clones were even found in all four CD4+ T cell subsets).
  • Interestingly, when Mycobacterium tuberculosis specific T cells were examined, they found Th1* and Th17 predominance, with few clonotypes shared between Th1* and Th17 (unlike Candida albicans).
  • Finally, they turned to an antigen with less epitopes, tetanus toxoid vaccine. PBMCs were sorted into the four T cell subsets and stimulated with tetanus toxoid and autologous monocytes. They saw differentiation of all four subsets and remarkable diversity of TCRβ clonotypes which was shared extensively among subsets, contrary to expectations (given that this antigen was a purified protein rather than an intact microbe).
  • Next, individual T cell clones from the CFSElo population responding to Candida albicans were further characterized. Of note, 80 of 242 clones were ‘sister clones’ (most often in both Th1* and Th17 or Th2 and Th17).
  • In order to understand the origin of diversity of cells responding to a pathogen (among and within clones) they primed a small number of isolated naïve CD4+ T cells with Candida albicans and recovered cells with various patterns of cytokine secretion (IL-17+, IFNγ+ or IL-4+), cloned them via limiting dilution and expanded in IL-2 cultures. Of note, while most progeny produced the same cytokine profile as the progenitor, progeny with divergent cytokine expression were also produced. The three CD4+ T cell subsets also shared approximately 40% of their clonotypes among each possible pair (though some clones were found only in one T cell subset).

This group was able to delve deeper than previous studies into the nature and extent of flexibility of CD4+ T cells responses to pathogens and vaccinations. They demonstrated that diverse repertoires can be stimulated to expand in response to a given pathogen, and that individual clonotypes can be driven by a given antigenic experience to differentiate among each of the four examined CD4+ T cell subtypes. Different pathogens have an effect on the nature of the skewing of the CD4+ T cell subsets, but the mechanism remains unclear. This study emphasizes diversity and complexity at all levels in the immune response and raises many questions, from the mechanism and timing and signals that tip two given naïve T cells with the same clonotype down two different differentiation pathways, to the varied signals from a given pathogen that yield multiple T cell subsets. Becattini et al have further clarified in this elegant study the extent to which the human immune system is an amalgam of dynamic, interconverting, unique cells, wherein each variable (amplitude and type of antigenic stimulation, APC type, APC number, cytokine environment, etc.) has fundamental impact on both the population level as well as on each individual cell.

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


Divergence: Twin Study into Immune Variation

 A review of Brodin, et al. Variation in the human immune system is largely driven by non-heritable influences. Cell. 2015; 160: 37-47. PMID:2594173

Investigation of twins is a fundamental tool in the study of the genetic component of human disease. By comparing monozygotic (MZ) and dizygotic (DZ) twins, it is possible to isolate the role of inherited factors on immune function. While these types of studies have previously been undertaken, this study allows for systems level analysis because many factors were studied in parallel. While the nature of the adaptive immune system, with its inherent somatic diversification of antigen receptors, makes possible an important role for somatic and environmental factors, the degree to which heritable factors are important in human immunology is a fascinating question.

SUMMARY

  • 78 monozygotic and 27 dizygotic twins (ages ranging 8-82 years) were studied in the twin cohort of SRI international, including differences in 51 cytokines, chemokines and growth factors; the responses of multiple cell types to cytokine stimulation; the relative abundances of 72 defined cellular subsets as well as anti-influenza antibody responses. Of note, they attempted to minimize time delay between sample acquisition within a twin pair. In addition, they ran assays at the Human Immune Monitoring Center
    • 77% of the many factors examined are ‘dominated’ and 58% ‘almost completely determined’ by non-heritable factors (and some become more variable with age)
  • The estimate of heritability of each factor was calculated by comparing the measured covariance matrices for the MZ and DZ groups to the expected values from their model. They corrected for age and gender and calculated 95% CI.
  • Interestingly, this study serves as a resource for cell subset labeling for flow cytometry.
  • The cell population frequencies and protein levels were only partially affected by heritable factors. Naïve, central memory and CD27+ CD4+ T cells have the greatest effect of heritable factors. IL12p40 was most affected by heritable factors. However, for both cell subsets and serum proteins, the majority are not affected greatly by heritable factors.
  • The effect of cytokine stimulation on a subset of cell types, using STAT1, 3 and 5 phosphorylation, was also assessed. Baseline and stimulation levels were assessed. It was noted that IL-2 and IL-7 mediated STAT5 was highly heritable. However, 69% of signaling responses were not heritable.
  • They next constructed a network, which can be viewed online (http://www.brodinlab.com/twins.htmlwww.brodinlab.com/twins.html).
  • Recognizing that older individuals are always assumed to have more effect of their (cumulative) environment, they compared the oldest to the youngest twins. A number of interesting factors became more divergent with age, especially Tregs, CXCL10.
  • Interestingly, CMV plays a significant role the immune response. For example, CMV discordant MZ twins have reduced correlations with Teff and γδ-T cells
  • Finally, seasonal flu vaccine responses did not have a heritable component. 

This group was able to show that the immense variation in the human immune response is primarily non-heritable, merging the most traditional genetic technique (twin study) with cutting edge evaluation of immune system components, function and production and systems level analysis of that data. This study is fascinating both from the analysis of the data and the definitions it provides for cell populations and analysis technique.

Reviewed by Sarah Henrickson, MD, PHD, Children's Hospital of Philadelphia


How Do Serum IgE Levels Correlate with Eosinophil Function in Asthma?

A review of Liang L., et al. An epigenome-wide association study of total serum immunoglobin E concentration. Nature Immunology. 2015. PMID:2570780

Asthma and allergy are IgE-related diseases that affect millions of patients worldwide. The role of genetics in IgE regulation in human allergy and asthma is poorly understood. Genome-wide association studies (GWAS) have revealed polymorphisms in STAT6. FCER1A, IL4/RAD50 and the MHC locus that only account for 1-2% of the variation in IgE levels. By using 95 nuclear family pedigrees and methylation arrays, Liang and colleagues examine the genome-wide epigenetic associations between serum IgE levels and methylation at loci with CpG islands. The authors used Illumina HumanMethylation27 arrays to examine individual CpG loci within the proximal promoter regions of 14,475 genes. Models were fitted with log-normalized IgE as a dependent variable and methylation status of each Illumina probe as a predictor. Their epigenomic analysis implicates eosinophils and their associated proteins in governing IgE concentration. There are several intriguing observations from this work:

  • Genes that encode eosinophil proteins have lower levels of methylation in subjects with asthma and high serum IgE. The authors identified 36 significant loci. Supporting their approach, the authors validated their statistical methods by confirming IL-4, a known regulator of IgE, has epigenetic associations with high IgE asthma. Of the 36 loci identified, several loci were annotated to genes that encode proteins important for eosinophil function. The authors identified IL5RA, CCR3, IL1RL1, PGR2 and GATA1 and confirmed that asthma patients with high serum IgE had lower levels of methylation and these loci. Asthmatic patients with low serum IgE had intermediate methylation compared to healthy controls.
  • Eosinophils, but not B and T cells, govern IgE production. The authors isolated DNA from peripheral blood leukocytes (a mixture of cells) and examine correlations between cell counts and serum IgE levels. The authors’ models indicate that their top IgE associations were not correlated with lymphocyte counts but instead were confined to eosinophils.
  • Differential methylation of genes associated with eosinophils identified by the authors account for 13.5% of IgE variation in their patient cohort. Eosinophil counts explain an additional 8.8% of IgE variation. Therefore, both the methylation status of eosinophil-associated proteins and the absolute number of eosinophils are correlated with serum IgE levels.
  • The most significant locus in the authors’ analysis was cg01998785, which is adjacent to LPCAT2. LPCAT2 encodes lyso-platelet-activating factor (PAF) acetyltransferase, which induces formation of PAF, a potent pro-inflammatory mediator. Compounds targeting the PAF pathway could provide novel therapeutics for the treatment of asthma.

These data suggest that eosinophil counts and methylation at genes encoding eosinophil proteins contribute to serum IgE levels. The top three loci in the authors’ analysis account for 13% of IgE variation in their cohort and represent novel druggable targets for the treatment of allergy and asthma. This work also supports epigenome approaches as a useful way to understand disease pathogenesis and to identify new therapeutic approaches.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco


Plasmablasts Induce Tfh Differentiation via IL-6 Production

 A review of Chavele K.M., et al. Circulating plasmablasts induce the differentiation of human T follicular helper cells via IL-6 production. Journal of Immunology. 2015; 194(6):2482-5. PMID:25681343

Previous work has shown that CD4+ T follicular helper (Tfh) B cell interactions in the follicles of lymphoid organs are required for B cell survival, high-affinity B cell responses to antigen, and facilitating differentiation into plasma cell and memory B cells. Production of IL-21 by Tfh cells is critical for optimal B cell responses to antigen. In turn, murine studies indicate B cells production of IL-6 can influence Tfh differentiation. However, the role of B cells in human Tfh cell differentiation is poorly understood. In this study, Chavele and colleagues demonstrate the role of human B cell production of IL-6 on Tfh differentiation in healthy controls and elucidate a novel mechanism of action for the anti-IL-6 biologic agent Tocilizumab in Rheumatoid Arthritis (RA) patients. There are several important findings from this work.

  • Human B cells are required for Tfh development. Depletion of B cells from peripheral blood mononuclear cells (PBMCs) resulted in a significant reduction in Tfh cell development after stimulation with anti-CD3/CD28. To determine if B cells directly supported Tfh differentiation, naïve T cells were stimulated with IL-4 or anti-CD3 in the presence or absence of B cells. The presence of B cells correlated with increased Tfh cell differentiation. Interestingly, the degree of Tfh differentiation correlated with the frequency of plasmablasts in the culture.
  • To determine if plasmablasts are sufficient for Tfh differentiation, the authors isolated plasmablasts and naïve B cells from health human PBMCs. Plasmablasts potently expanded Tfh cells compared to monocytes, naïve B cells, and memory B cells. Tfh cells expanded via plasmablasts expressed high levels of Bcl-6, IL-21, and were functional as they induced naïve and memory B cells to produce increased levels of IgM and IgG.
  • To determine how plasmablasts induce Tfh differentiation, the authors evaluated plasmablast cytokine production and found they produced high levels of IL-6 compared to either naïve or memory B cells. However, plasmablasts did not produce IL-12, a cytokine that has been implicated in other studies in Tfh differentiation. Culture of purified naïve T cells with IL-21, IL-6 or a combination of both and showed each cytokine could induce Tfh cells alone and that the effect was greatest in combination. Further supporting a role for these cytokines, addition of anti-IL-21R and anti-IL-6 blocking antibodies to the plasmablast-naïve T cell cocultures significantly reduced Tfh differentiation and IL-21 production.
  • To determine if the anti-IL-6R therapeutic agent tocilizumab induces reductions in Tfh and plasmablasts through this pathway, the composition of RA patient’s blood pre and post treatment with tocilizumab was examined. Tocilizumab was associated with a significant decrease in Tfh cells and plasmablasts.

Taken together, these data support a model for a positive feedback loop between Tfh cell and plasmablast differentiation in which plasmablasts induce Tfh cell differentiation by IL-6 production and Tfh cells augment plasmablast differentiation by IL-21 production. While such a feedback loop could be beneficial in infection, it could be detrimental in autoimmune disease due to the rapid proliferation and differentiation of antibody-secreting cells in RA. An important next step is to determine how this feedback loop is initially triggered. This work also supports a novel mechanism of action for the anti-IL6R antibody Tocilizumab in RA and potentially other autoimmune diseases. Conversely, augmenting this pathway could be beneficial in boosting adaptive immune responses to vaccines.

Reviewed by Melissa, Ruck, MD, PhD, and Michelle Hermiston, MD, PhD, University of California, San Francisco


IL-9 in Peanut Allergy

A review of Brough, H.A., et al. IL-9 is a key compontent of TH cell peanut-specific responses from children with peanut allergy. J Allergy Clinical Immunology 134: 1329-1338.
PMID:25112699

Peanut allergy affects ~1.4% children in the United States, and its prevalence has been reported to be on rise. Definition of criteria that help distinguish between peanut-allergic (PA) and peanut-sensitized (PS) individuals safely is an unmet need in the clinical diagnosis of peanut allergy. In this study, the authors carried out an exploratory microarray investigation of gene expression in peanut-activated memory Th (CD4+ CD69+ CD45RO+) subsets from children, who are PA, or PS, or atopic without peanut allergy (NA) to identify diagnostic biomarkers. The following results were reported:

  • Among 12,257 differentially expressed genes, IL-9 emerged as the most differentially expressed marker, followed by IL-5 and IL-13.
  • Microarray findings were confirmed by real-time qPCR, while the flow cytometry data showed that IL-9 and IL-5 were expressed by two distinct Th (Th9 and Th2) subsets.
  • Skin- and gut-homing Th cells from PA children expressed similar Th2- and Th9-associated genes.
  • Interestingly, a machine-learning approach employed in this study could most accurately distinguish between PA and NA children based on IL-9 expression.

The findings from this study thus have important implications with regards to diagnosis and clinical desensitization of peanut-allergic children.

Reviewed by Kari Nadeau, MD, PhD, Stanford School of Medicine


Biopolymer Implants Enhance the Efficacy of Adoptive T Cell Therapy

 A review of Stephan S.B., et al. Biopolymer implants enhance the efficacy of adoptive T cell therapy. Nature Biotechnology 33, 94-98. 2015. PMID:25503382

Adoptive T cell therapy has shown promise for the treatment of some cancers; but unfortunately for most solid tumors is limited by the inefficient trafficking of transferred lymphocytes to the tumor site and poor expansion of the infused cells within the immunosuppressive tumor microenvironment. In this study, Stephan and colleagues describe a bioactive polymer implant capable of improving the delivery and expansion of tumor reactive T cells in the tumor microenvironment, thereby inducing more potent antitumor responses when compared to more traditional methods of adoptive T cell therapy.

  • The authors initially described the development of a porous biopolymer scaffold with stimulatory and migratory signals to facilitate T cell activation and migration. They used polymerized alginate (a naturally occurring polysaccharide approved by the US Food and Drug Administration) infused with a synthetic collagen-mimetic peptide (CMP, to enhance T cell migration via the α2β1 collagen receptor) and porous silica microparticles encapsulating a superagonist IL-15/IL-15Rα fusion protein and coated with lipid bilayers bound with anti-CD3, anti-CD28, and anti-CD137 antibodies to support T cell activation, proliferation and migration out of the scaffold.
  • Using a 4T1 mouse breast cancer model (a model for incomplete resection), they evaluated the antitumor efficacy of implanting scaffolds containing tumor-reactive T cells into the resection bed compared to conventional delivery strategies. Tumor recurrence was completely prevented only when scaffolds were used to deliver tumor-specific T cells. Other methods of T cell administration failed to prevent tumor recurrence: intravenous administration of tumor-specific T cells provided no protection; intracavitary administration resulted in a 4 day survival advantage; and intracavitary administration of pre-activated T cells prolonged survival by only 9 days. The antitumor effect was also abrogated when scaffolds infused with non-specific T cells were implanted.
  • The authors showed that T cells delivered via biopolymer implants maintain low expression of the exhaustion markers Tim-3 and 2B4, migrate to tumor draining lymph nodes, and develop a central memory phenotype (CD44+ and CD62L+); intravenously administered T cells do not adequately traffic into tumors, but instead accumulate in the spleen and liver, while intracavitary administered T cells do not persist at the tumor site and acquire an exhausted phenotype (Tim-3high and 2B4high).
  • They also showed that the scaffold-delivery of NKG2D-CAR T cells was capable of eliminating ID8-VEGF ovarian carcinomas (a model for advanced unresectable tumors) in 60% of mice, whereas conventional methods of T cell delivery failed to induce tumor regression.

This study shows that the antitumor efficacy of adoptive lymphocyte transfer may be improved by delivering them in biopolymer scaffolds engineered to promote lymphocyte expansion and migration in the tumor microenvironment; and suggests that scaffold-based T cell delivery may provide an effective therapy for incompletely resected and/or inoperable tumors. In addition, the use of scaffolds does not require patient irradiation, systemic cytokine administration, or chemotherapeutic lymphodepletion; and therefore also has the potential to minimize toxicity.

Reviewed by Heather Kinkead and Eric Lutz, PhD, Johns Hopkins University


Whole Cell Variation Primes Pancreatic Cancer for PD-1 Targeted Checkpoint Immunotherapy

A review of Soares K.C., et al. PD-1/PD-L1 blockade together with vaccine therapy facilitates effector t cell infiltration into pancreatic tumors. Journal of Immunotherapy 38, 1-11. 2015. PMID:25415283

Pancreatic cancer is predominantly infiltrated with immunosuppressive cells and signals and has historically been considered as a non-immunogenic tumor In concordance with this notion, pancreatic cancer has not responded to treatment with single-agent checkpoint inhibitors targeting PD-1, PD-L1 or CTLA-4 that have shown promise for other cancers, including melanoma, renal cell carcinoma, and lung cancer. Treatment with a GM-CSF-secreting allogeneic whole pancreatic cancer cell vaccine (GVAX) can activate tumor specific T cells and induce the formation of intratumoral tertiary lymphoid aggregates, and thereby convert the immunologically quiescent pancreatic cancer microenvironment into an active one. However, like single-agent checkpoint inhibitors, treatment with GVAX alone produces limited survival benefit to patients with pancreatic cancer. In this study, Soares et al show that treatment with GVAX induces both the infiltration of activated effector T cells, and the upregulation of PD-L1 expression in the pancreatic cancer tumor microenvironment; and that the combination of GVAX with PD-1-targeted checkpoint blockade is more effective than either single therapy alone.

  • The authors examined PD-L1 expression in tumors resected from patients treated with or without GVAX. Tumors resected from vaccinated patients two weeks following GVAX treatment were twice as likely to express PD-L1compared to tumors resected from unvaccinated patients, and the intensity of PD-L1 staining was significantly higher in tumors from vaccinated patients. Intratumoral lymphoid aggregates (germinal center-like structures) were observed only in tumors resected from vaccinated patients, and uniformly contained PD-L1+ cells.
  • The authors next evaluated PD-L1 expression in a preclinical mouse model of metastatic pancreatic cancer. Liver metastases in untreated mice did not express PD-L1, whereas liver metasteses in GVAX-treated mice expressed high levels of membranous PD-L1, indicating that GVAX also induces PD-L1 expression in murine pancreatic cancers.
  • Using the preclinical metastatic model, the authors examined whether PD-1 blockade could augment the antitumor activity of GVAX. They showed that the combination of PD-1 inhibition with GVAX (with cyclophosphamide to deplete Tregs) yielded a significant survival advantage and increase in the number of mice cured when compared to either monotherapy. The same trends were observed whether anti-PD-1 or anti-PD-L1 was used.
  • Treatment with GVAX increased the number of tumor infiltrating lymphocytes (TIL) observed, and the addition of anti-PD-1 shifted the population, increasing the percentage of TIL that were CD8+, and the percentage of tumor-infiltrating CD8+ T cells secreting IFNγ.
  • TIL from mice treated with the combination therapy contained fewer Tregs, and fewer CTLA-4+ cells, suggesting that the combination of GVAX with PD-1-targeted checkpoint therapy may reduce immunosuppression broadly through multiple mechanisms.

This study demonstrates that vaccination can convert a non-immunogenic tumor that does not respond to PD-1-targeted checkpoint therapy into a tumor that does respond to PD-1-targeted checkpoint therapy by inducing the infiltration of T cells and the upregulation of PD-L1 expression. This study may explain why vaccines and immune checkpoint inhibitors as single agents have failed against pancreatic cancer, and possibly other non-immunogenic cancers (vaccines alone fail because the T cells they induce are inhibited by immunosuppressive mechanisms, such as the PD-1 pathway; and immune checkpoint inhibitors alone fail because there are too few effector T cells for them to act on). Most importantly, this study supports a new approach for evaluating checkpoint inhibitors in “non-immunogenic” cancers, like pancreatic cancer.

Reviewed by Alexander Hopkins and Eric Lutz, PhD, Johns Hopkins University

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Highlights From Clinical Immunology, the Official Journal of FOCIS

Double Negative Epigenetics

A review of Renauera A.,et al. The DNA methylation signature of human TCRαβ+CD4-CD8- double negative T cells reveals CG demthylation and a unique epigenetic architecture permissive to a broad stimulatory immune response. Clinical Immunology 156, 19-27, 2015. PMID:25451162

Peripheral TCR αβCD4-CD8- “double negative” T cells (DN T) are a poorly understood subset of T cells which appear to have TCR-dependent regulatory functions against single positive effector T cells in healthy individuals but are expanded and have a pro-inflammatory phenotype in patients with various auto-immune diseases. In this paper, the authors characterized DNA methylation (the “methylome”) of DN T cells and used bioinformatics tools to look for patterns of of immune activity and regulation. They FACs isolated TCR αβCD4-CD8-, TCR αβCD4+CD8- and TCR αβCD4-CD8+ cells from healthy women, ages 25-60 years, purified DNA, and performed a chip based assessment of methylation of both CGs and non CG sites, across the genome. The major findings included the following:

  • DNA methylation was significantly different in double negative T cells compared single positive effector T cells
  • Overall, there was increased hypomethylation in the DN T cells. Compared to CD4+ T cells, 84.59% of the 2912 RefGene database genes assayed were hypomethylated and 15.41% were hypermethylated. Compared to CD8+ T cells, 91.1% of the genes were hypomethylated and 8.9% were hypermethylated.
  • Most of the differentially methylated sites detected in DN T cells vs. the single positive cells were at CG sites, and most of these were hypomethylated, but most of the differentially methylated non-CG sites were hypermethylated in DN T cells.
  • Consistent with the overall reduction in DNA methylation, there was lower expression of DNA methyl transferases (DNMT1, DNMT3A, and DNMT3B) in double negative T cells, by qRT-PCR analysis, in the DN T cells compared to the single positive T cells.
  • Methylation status of several CG sites in the CD4, CD8A and CD8B genes were assessed, and fond to be hypermethylated in the DN T cells, consistent with the suppression of CD4 and CD8 expression in these cells.
  • Functional patterns of the differentially methylated genes was performed by gene annotation enrichment. Hypomethylated genes in the DN T cells were enriched for genes related to cell communication , cell adhesion-mediated, signal transduction, cell junction, and cadherin.
  • 17 cytokine-related genes were differentially methylated in DN T cells compared to CD4+ and CD8+ T cells; most of these were hypomethylated in DN T cells, including IFNG, IL17F, IL12B, and IL18, RANKL and TNFSF13B. IL19 and TGFB2 were hypermethylated.

DNA methylation has been shown to be an important epigenetic mechanism for regulation of T cell subset lineages, such as TH1, TH2, and TH17 cells. Epigenetic suppression of CD8 expression in DN T cells has been previously shown as well. This study broadens our knowledge of the DNA methylation status of DN T cells, and establishes that there is a global hypomethylation status of many immune genes, consistent with a pro-inflammatory function of these T cells. Furthermore, the study suggests that methylation of CD4 may be related to DN T cell differentiation. It would be interesting to know of the methylaltion status of genes in DN T cells from patients with SLE, or other autoimmune diseases, in which these cells are expanded, compared to the healthy women studied here.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital


No Tears for a Sick Mouse?

A review of Young NA., et al. A chimeric human-mouse model of Sjögren’s Syndrome. Clinical Immunology 156, 1-8, 2015. PMID:25451161

Sjögren's Syndrome (SjS) is a systemic autoimmune disease that mainly affects women. Manifestations typically include dry eyes and mouth, but also systemic manifestations, such as arthritis, neuropathy and lung fibrosis. The underlying mechanisms of Sjögren's Syndrome are not known, and although there are mouse models of the disease, there translatability to testing therapies for the human disease is questionable. To this end, Young et al. have developed a humanized mouse SjS model, by adoptively transferring peripheral blood mononuclear cells (PBMCs) from SjS patients into immunodeficient NOD-scid γc-null mice (NSG). NSG mice are known to stably engraft human lymphcoytes, and have been used to study T1D, but not other human autoimmune diseases. The authors compared several parameters in NDG mice with SjS PBMC transfers vs. NSD mice with healthy control PMBM transfers:

  • The only detectable human blood cells in SjC or control chimeras were CD4+ and CD8+ T cells with no difference in numbers or distribution between the two groups.
  • Serval human cytokine were elevated in 28d after PBMC transfer in the SjC group compared to controls, including IFN-γ by 2.4-fold, IL-10 by 2.7-fold, IL-17 by 6-fold, IL-2 by 3-fold, IL-6 by 74-fold, and TNF-α by 21-fold.
  • Mice with SjS PBMC had enhanced lacrimal and salivary gland inflammation, with primarily CD4+ infiltrates and few CD8+ T cells and B cells. There was sialoadenitis in mice with healthy PBMCs, as reported in previous studies of human PBMC transfer into NSG mice, but the degree of infiltrate was greater in the SjS group. There was minimal inflammation in sections of other tissues including skin, intestine, kidney, and liver, with no difference between SjS and control groups.
  • At 4 weeks post SjS PBMC transfer, saliva production in response to pilocarpine was significantly 36% less then control mice.

This new humanized mouse model of SjC syndrome recapitulates some important features of the human disease, especially salivary and lacrimal gland inflammation by CD4+ T cells. The model has the potential to enable studies of immunopathogenesis and therapy. It is not clear for this report how the human T cells become specifically recruited to or activated in the mouse lachrymal and salivary glands. If the CD4+ T cells in the tissues are typical TCR αβ T cells, it will be of interest to know if they being activated mouse tissue antigens homologous to human antigens, and if so, what the antigen presenting cells are.

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Human Immunophenotyping Update

Publishing Flow Cytometry Data

J. Phillip McCoy, Jr. PhD, National Institute of Health

All too often I have read papers in highly respected journals where data from flow cytometric experiments are published in a manner that makes me cringe. Generally, the methods section reads ‘we performed FACS on these cells’ with no other experimental detail. The results generally show a figure with one or more dot plots having no scales or labels on the axes. Why do these make me cringe? I cringe because there is little chance of anyone being able to duplicate the authors’ results given this paucity of information, and the lack of replication would lead to skepticism over the data and conclusions.

Let’s begin with the methods. The statement “We performed FACS” gives little information and what information is given might be misleading. The acronym FACS stands for Fluorescent-Activated Cell Sorting. If you did not physically sort the cells, you did not perform FACS, rather you performed immunophenotyping or flow cytometry. Semantics aside, to reproduce these data much more information is needed – even if it is provided as supplemental data. To start, the reagents and fluorochromes (or dyes) used should be specified. Ideally, for antibodies, this would include the clone or catalog number for the reagent. Why? Not all clones, even for the same CD are identical. For example, as we discovered in our preparation for the FOCIS HIPC Lyoplate study, not all CD38 clones give equivalent results. Furthermore, fluorochromes have different quantum yields, or “brightness”. Staining relatively dim markers, such as intracellular cytokines, with a relatively dim fluorochrome such as FITC will yield substantially less cytokine detection than staining with a brighter fluorochrome such as phycoerythrin (PE). Similarly, information about the cytometer would be highly useful. For example, what laser(s) were used to excite the fluorochromes? This is important because particular laser wavelengths may better excite fluorochromes than other wavelength, thus affecting the brightness of fluorochromes. An example of this would be the excitation of PE. PE will excite off of a 488nm laser, but is brighter when excited with a 532nm laser, and brighter still when excited at 561nm. Referring again to the cytokine example, the laser excitation will potentially influence the number of cells determined to be positive for staining. PMT voltages and compensation values will vary on every instrument and would not need to be included in the methods, but the filter configuration in the cytometer would be useful. To summarize, including complete information is vital to the credibility of the publication. Including the information as a table when publishing flow cytometry experiments, even as a supplement can be quite powerful as seen in Tables 1 and 2. In addition, the clarity gained from using this format to publish the information rather than publishing no information at all or listing the information as an endless paragraph of CDs, clones and fluorochromes is obvious.


                                                  Table 1. Reagents Used                           Table 2. Cytometer Configuration

rsz 3tables

Moving to the results and presentation of the flow cytometry data, what sense does it make to publish dot plots with no scales or labels? Can you think of any other data, chart, or graph that you would publish without a scale or labels? Probably not. Flow cytometers can collect, and present data, on either logarithmic or linear scales, and log scales may cover differing numbers of decades. Axis scales can even bi-exponential or log-linear. Thus the scale on a dot plot is very informative, particularly if you want others to be able to replicate your work. Labels such as “FL1” or “FL2” have little meaning in comparison to labels such as “FITC CD3” and “PE CD4” and therefore it is recommended that the labels include the antibody and fluorochrome (or dye) (Figure 1). It is also quite useful to know precisely how a gate was determined, or how ‘positive’ events were calculated. Was it through the use of an “N-1” control, an isotype control, or perhaps by cluster analysis? It is common practice to publish only the final, most pertinent dot plot showing the data of interest. This is understandable considering the page and figure constraints imposed by many journals. Nonetheless, it would be most useful to include dot plots of the entire gating scheme leading to the final dot plot as supplemental figures.

Figure 1. Examples of uninformative (left) and informative (right) dot plots of the same data

Picture1

Reporting of flow cytometry data should also be very specific on what data are being presented. For example, stating that there are “x % memory T cells” does not tell the reader if the denominator is all T cells or all lymphocytes, or even all leukocytes. A clearer manner of stating this would be to say “of the CD3+ T cells, x% displayed a memory phenotype.” If data are presented concerning the mean fluorescence intensity (MFI), a statement should be included to explain how the MFI was obtained and whether or not these values were normalized based on a negative population.

The suggestions given here for publishing flow cytometry data permit accurate reporting of these data, enabling other investigators to more readily reproduce your findings. The current suggestions largely reiterate those made in a previous publication (1). Even more rigorous publication guidelines for flow cytometry data can be found in MiFlowCyt (minimum information about a flow cytometry experiment), recommended by the International Society for the Advancement of Cytometry (2).

References

  1. Alvarez DF, Helm K, DeGregori J, Roederer M, and Majka S: Publishing flow cytometry data. Am J Physiol Lung Cell Mol Physiol 298:L127-L130, 2010.

  2. Lee JA, Spindlen J, Boyce K, Cai J, et al; MIFlowCyt: The minimum information about a flow cytometry experiment. Cytometry Part A, 73A (10): 926-930, 2008.

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Developments in Basic Immunology and Novel Therapies

B Cell Depletion Therapy for Autoimmune and Inflammatory Diseases

Shiv Pillai, MD, PhD, Ragon Institute of MGH, MIT and Harvard; Harvard Medical School

B cell depletion using a monoclonal antibody to CD20, a B cell surface protein whose function remains poorly defined, was first conceived of as a therapeutic strategy to treat B cell lymphomas and chronic lymphocytic leukemia. Although this approach is still utilized for the treatment of B cell malignancies, B cell depletion has since emerged as a powerful way to treat a number of autoimmune and inflammatory conditions, including certain disorders that are generally believed to be caused by T cells. The initial anti-CD20 antibody used for therapy was a chimeric antibody called Rituximab. Rituximab remains the major B cell depleting reagent in clinical use today. The success of Rituximab therapy has spawned the generation of a number of newer therapeutics that also target B cells.

During B cell development, CD20 is first expressed on pre-B cells and can be more readily detected on immature, transitional, mature and memory B cells but is lost when activated B cells differentiate into plasmablasts. It is not normally found on plasma cells though malignant cells in a small subset of multiple myelomas express CD20. Depletion of normal or malignant B cells is influenced by polymorphisms of the FcγRIIIA gene, suggesting that antibody dependent cellular cytotoxicity mediated by NK cells or macrophage mediated antibody dependent phagocytosis contributes to B cell removal. One study has shown that most of the clearance of B cells actually occurs in the liver and is mediated by Kupffer cells.

Rituxan mediated B cell depletion has proved to be an efficacious therapy for a number of autoimmune disorders. An incomplete list would include autoimmune thrombocytopenia, rheumatoid arthritis, relapsing remitting multiple sclerosis, Wegener's granulomatosis, IgG4 related disease and myasthenia gravis. This approach has proved less useful overall in systemic lupus erythematosus, although some subjects with lupus do respond to B cell depletion. It is relatively easy to understand why Rituxan might be useful in diseases like myasthenia gravis, autoimmune thrombocytopenia and pemphigus for instance, all disorders in which autoantibodies play a causal role. Depleting B cells may be considered to be one way in which the titers of disease causing antibodies could be induced to drop.

B cell depletion using anti-CD20 targets B cells but not plasmablasts or plasma cells. If an auto-antibody is largely made in the context of an ongoing immune response and is largely secreted by plasmablasts and short-lived plasma cells then anti-CD20 mediated B cell depletion would likely be clinically effective. Auto-antibodies made by long-lived plasma cells would not be depleted by such a therapy. We can therefore assume that in auto-antibody mediated diseases in which clinical improvement is seen with B cell depletion, the majority of the relevant autoantibodies are in fact secreted by plasmablasts and short-lived plasma cells. The reason why lupus may often not be responsive to anti-CD20 mediated B cell depletion may reflect the central role of nucleic acids and chromatin as the auto-antigens in this disease. The ability of these antigens to also activate endosomal Toll like receptors in B cells may facilitate the differentiation of activated lymphocytes emerging from the germinal center reaction into long-lived plasma cells. It is also possible that in some diseases, B cell depletion, apart from depleting cells poised to differentiate into plasmablasts, also depletes regulatory B cells that may normally function to constrain autoimmunity. In such a context, B cell depletion may indeed cause disease exacerbation rather than remission.

Why does anti-CD20 mediated B cell depletion lead to remission in conditions like multiple sclerosis and other T cell mediated autoimmune diseases? It is likely that some diseases that many immunologists assume are T cell mediated are actually primarily linked to T cell help for B cells that secrete pathogenic autoantibodies. A case in point may be rheumatoid arthritis, a disease in which antibodies against citrullinated proteins and immune complexes incorporating these antibodies may be critical for disease onset and progression. Follicular helper T cells may well be relevant for HLA class II dependent help to disease causing B cells, and Th17 cells may contribute to inflammation in a secondary context. In other diseases like multiple sclerosis in which T cells likely play a predominant role, B cells may provide “help” to T cells. It is well established that effector/memory CD4+ T cells are sustained by B cells. How exactly this occurs is unclear. It is assumed that high affinity somatically mutated memory B cells may be the most effective antigen presenting cells for the activation of rogue effector and effector/memory CD4+ T cells that drive autoimmunity and chronic inflammation.

Although Rituxan is still widely used there are a number of different anti-CD20 agents that have been developed. These include humanized and fully human versions of anti-CD20. Some examples include Ocrelizumab, a humanized anti-CD20 antibody, Obinutuzumab, another humanized anti-CD20 which has been glyco-engineered to facilitate Fc receptor engagement and B cell clearance and Ofatumumab, a fully human version of anti-CD20. Many other B cell therapies have also been developed but a therapeutic that can effectively clear long-lived plasma cells has so far not yet emerged.

References:

  1. Reddy V, Jayne D, Close, D and Isenberg D. B cell depletion in SLE: Clinical and trial experience with Rituximab and Ocrelizumab and implications for study design. Arthritis Research and Therapy 2013, 15 (Suppl 1) S2.

  2. Edwards JCW and Cambridge G. B cell gtargeting in rheumatoid arthritis and other autoimmune diseases. Nature Reviews Immunology 2006 6, 396-403.
Selected Recent Clinical Trial Results

Rixuximab Versus Azathioprine for Maintenance in ANCA-associated Vasculitis

Clinical Trial:
Guillevan, L., et al. Rituximab versus Azathioprine for Maintence in ANCA-Associated Vasculitis. N Engl J Med. 2014; 371(19): 1771-80.
PMID:PMC3888603

Disease: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

Drug: Rituximab is a human monoclonal antibody directed against CD 20 (B-cells.) Azathioprine is a purine analogue that inhibits DNA synthesis and affects proliferating B cells and T cells.

Study design:

  • 115 patients with ANCA-associated vasculitis, ages 18-75.
  • Newly diagnosed or in complete remission after cyclophosphamide-glucocorticoid regimen treatment for 4-6 months
  • Randomly assigned (not blinded) to either daily tapering doses of azathioprine (N=58) until month 22 or 500 mg rituximab infusions (N=57) on days 0 and 14 and at months 6, 12, and 18
  • Endpoint at month 28: rate of major relapse, defined as the reappearance of disease activity or worsening, with a Birmingham Vasculitis Activity Score > 0, and involvement of one or more major organs, disease-related life-threatening events, or both.

Results:

  • At month 28, major relapse occurred in 17 patients (29%) in azathioprine group and in 3 patients (5%) in rituximab group (P=0.002)
  • No significant differences in rates of minor relapse or adverse events, including cancer, between groups

Why the Trial is of Interest to the Broader FOCIS Community:
Remission in ANCA-associated vasculitis usually can usually be achieved with cyclophosphamide and glucocorticoids, but maintenance of remission remains a therapeutic challenge. Currently azathioprine is used for maintenance therapy, due to its low cost and acceptable side effect profile. Previous trials of maintenance strategies evaluated azathioprine vs. cyclophosphamide (1), and azathioprine vs. methotrexate (2), but neither cyclophosphamide nor methotrexate demonstrated greater efficacy or safety compared to azathioprine for maintenance. The reviewed study is the first RCT to evaluate rituximab therapy compared to azathioprine for maintenance of remission. The study suggests that rituximab is superior to azathioprine in maintenance of remission at 28 months.

Recently, the Chapel Hill Consensus Conference proposed its 2012 classification system which divides ANCA-associated vasculitis (AAV) into multiple subgroups defined by clinical symptoms and pathophysiology. These include granulomatosis with polyangiitis (GPA; formerly termed Wegener’s), microscopic polyangitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA; formerly termed Churg Strauss Syndrome). EGPA is considered separately from GPA and MPA as it has a different clinical presentation and prognosis. GPA is most frequently associated with proteinase 3 (PR3) specific ANCA, and myeloperodiase (MPO) ANCA is associated with MPA, but with significant overlap. Clinically, those with renal-limited disease can be considered another subgroup. In North America and Europe, GPA (Wegener’s) has a higher prevalence than MPA.
In this study, 87/115 subjects had GPA disease [granulomatosis with polyangiitis (Wegener’s)]. While other classifications of ANCA disease were included (23/115 with MPA, 5/115 with renal-limited disease), there were too few subjects to evaluate whether subgroups had different outcomes. This highlights a common conundrum in the design of clinical trials – lumping heterogeneous populations together can facilitate enrollment and prevents the exclusion of a population that could potentially benefit from therapy; however, enrolling more homogenous populations may result in more pronounced effects – particularly if there is a mechanistic associated rationale. In this case, additional studies will be needed to understand the response to therapy by subgroups.

References:

  1. Jayne D, Rasmussen N, Andrassy K, et al. A randomized trial of maintenance therapy for vasculitis associated with anti-neutrophil cytoplasmic autoantibodies, N Engl J Med 2003;349:36-44

  2. Pagnoux C, Mahr A, Hamidou MA, et al. Azathioprine or methotrexate maintenance for ANCA-associated vasculitis, N Engl J Med 2008;359:2790-803

Reviewed by Sandra Lord, MD, Benaroya Research Center


Brodalumab, An Anti-IL17RA Monoclonal Antibody in Psoriatic Arthritis

Clinical Trials:
Mease, P., et al. An anti-IL19RA monoclonal antibody, in psoriatic arthritis. N Engl J Med, 2008; 370:2295-306. PMID:24918373

Disease: Psoriatic Arthritis

Drug: Brodalumab, a human monoclonal antibody directed at the IL17 receptor. IL17 is a mediator of several pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α/β.

Study design:

  • Phase 2 randomized, double-blind, placebo-controlled multicenter study
  • 168 patients aged 18-75 years with active psoriatic arthritis: 57 in brodalumab 140 mg group, 56 in brodalumab 280 mg group, 55 in placebo group
  • Patients received sc injection of placebo or brodalumab on day 1 and at weeks 1, 2, 4, 6, 8, 10. At week 12, subjects offered option of open-label administration of brodalumab 280 mg every 2 weeks for an additional 40 weeks.
  • Primary endpoint was a 20% improvement in American College of Rheumatology response criteria (ACR20) at week 12

Results:

  • At week 12, brodalumab 140 mg and brodalumab 280 mg groups had higher rates of ACR 20 than the placebo group: 37% (P=0.03) and 39% (P=0.02) vs 18%. Treatment groups also had higher rates of ACR 50 (50% improvement): 14% (P=0.05) and 14% (P=0.05) vs 4%. Rates of ACR 70 were similar: 3% in each group.
  • At week 24, ACR 20 rates were 51% and 64% in the brodalumab 140 mg and 280 mg groups respectively, compared to 44% of participants who switched from the placebo group to open label 280 mg brodalumab after 12 weeks. 
  • Outcomes continued to improve in the 40 week open label portion except in the 280 mg group.
  • Similar results were seen in subjects who had received previous biologic therapies compared to those who had not.
  • Adverse event rates were similar in all groups. Most common adverse event reported in all groups was upper respiratory infection

Why The Trial is of Interest to the Broader FOCIS Community:
IL-17 and IL-23 are thought to be central to the pathogenesis of both isolated skin psoriasis and Psoriatic arthritis (PsA). PsA is a systemic inflammatory condition that affects 20-30% of people with psoriasis, and it is generally more treatment-resistant as compared to isolated skin psoriasis. The IL-17A monoclonal antibodies (secukinumab and ixekizumab), the IL-17R antibody brodalumab (the agent used in the reviewed paper) and ustekinumab (antibody directed against the p40 subunit common to both IL-12 and IL-23) have shown similar efficacy in isolated skin psoriasis as measured by improvement in the Psoriasis Area and Severity Index (PASI), which is a clinical measurement of disease activity. PSAI 75 indicates a 75% improvement in disease activity and is the primary endpoint in most psoriasis trials. Multiple phase II and III trials show that PASI 75 scores in the range of 65-85% can be achieved with all of these agents. (1-4)

In contrast, IL-17/IL-23 inhibition has not been as effective for PsA as measured by the ACR (American College of Rheumatology) score, a clinical measure of arthritis activity. ACR 20 indicates a 20% improvement in disease activity and is the endpoint in most inflammatory arthritis trials. Phase III trials of secukinumab (5,6) and ustekinumab (7,8) showed similar treatment response rates: ~50% in treated groups met the ACR 20 at 24 weeks vs ~20% in placebo groups. In the reviewed study, 64% of subjects in the highest-dose brodalumab group met the ACR 20 at 24 weeks. However, it is difficult to compare the results of this study, in which the primary endpoint was at 12 weeks, followed by open label extension, to the secukinumab and ustekinumab studies, in which the primary endpoint was at 24 weeks. Without this comparison, it is unclear whether IL-17R blockade with brodalumab provides a greater clinical response in PsA than do either IL-17A inhibition with secukinumab, or IL-12/IL-23 inhibition with ustekinumab. The continued improvement seen in all treated groups between week 12 and 24 suggests that a full clinical response in psoriatic arthritis might require longer than 12 weeks of treatment.

References:

  1. Langley RG, et al. Secukinumab in plaque psoriasis—results of two phase 3 trials, N Engl J Med 2014;371:326-338

  2. Papp KA, et al. Efficacy and safety of ustekinumab, a human interleukin 12/23 monoclonal antibody, in patients with psoriasis: 52-week results from a randomized, double-blind, placebo-controlled trial (PHOENIX 2) Lancet 2008;371:1675-1684

  3. Leonardi C, et al. Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis, N Engl J Med 2012;366:1190-9

  4. Papp KA, et al. Brodalumab, an inti-interleukin-17-receptor antibody for psoriasis, N Engl J Med 2012;366:1181-9

  5. Mease P, et al. Secukinumab, a human anti-interleukin-17A monoclonal antibody, improves active psoriatic arthritis and inhibits radiographic progression: efficacy and safety data from a phase 3 randomized, multicenter, double-blind, placebo-controlled study. Arthritis Rheum 2014; 66(Suppl):S423. Abstract 953

  6. van der Heijde D et al. Secukinumab, a monoclonal antibody to interleukin-17A, provides significant and sustained inhibition of joint-structural damage in active psoriatic arthritis regardless of prior TNF inhibitors or concomitant methotrexate: a phase 3 randomized, double-blind, placebo-controlled study. Arthritis Rheum 2014; 66(Suppl):S424. Abstract 954

  7. McInnes IB, et al. Efficacy and safety of ustekinumab in patients with active psoriatic arthritis: 1 year results of the phase 3, multicenter, double-blind, placebo-controlled PSUMMIT 1 trial. Lancet 2013;382:780-789

  8. Ritchlin C et al. Efficacy and safety of the anti-IL12/23 p40 monoclonal antibody, ustekinumab, in patients with active psoriatic arthritis despite conventional nonbiological and biological antitumour necrosis factor therapy: 6-month and 1-year results of the phase 3, multicenter, double-blind, placebo-controlled, randomized PSUMMIT 2 trial. Ann Rheum Dis 2014;73:990-999

Reviewed by Sandra Lord, MD, Benaroya Research Institute


Anti-thymocyte Globulin/G-Csf Treatment Preserves B Cell Function in Patients with Established Type 1 Diabetes

Clinical Trials:
Haller, M., et al. Anti-thymocytes globulin/G-CSF treatment preserves β cell function in patients with established type 1 diabetes. Journal of Clinical Immunology. 15 December 2014. PMID:25500887

Disease: Type 1 diabetes

Drug:

  • Anti-thymocyte Globulin (ATG,) or Thymoglobulin, a rabbit-derived antibody against human T-cells, resulting in a substantial reduction in the number of circulating T-lymphocytes, but with a relative preservation of Tregs
  • Granulocyte Colony Stimulating Factor (G-CSF), a glycoprotein that stimulates the bone marrow and stem cells to produce granulocytes or neutrophils and in particular favors repletion of Tregs.

Study design:

  • Phase IIa randomized, single-blinded, placebo-controlled trial
  • 25 subjects (17 in ATG/GCSF group, 8 placebo) with mean age 24.5 years
  • Participants with “established” diabetes, diagnosed within 4 months to 2 years of enrollment
  • Participants received an infusion of ATG (2.5 mg/kg) or placebo followed by GCSF or placebo injections (6 mg sc every 2 weeks for 6 doses)

Results:

  • Primary endpoint measurement was the difference in area under the curve (AUC) C peptide as measured by 2 hour mixed meal tolerance test (MMTT) between treated and placebo groups at 1 year.
  • The difference in AUC C peptide between treated and placebo groups was 0.28nmol/L (P=0.05)
  • Cytokine release syndrome (CRS) and serum sickness (SS) were seen in the majority of treated subjects (82% CRS and 76% SS.) There were no differences in infections rates between treated and placebo groups.

Why the Trial is of Interest to the Broader FOCIS Community:
The reviewed article describes a combination therapy approach to established TID, those with disease duration of 4 months to 2 years. This was a pilot study with relatively small enrollment numbers (n=25); hence it was difficult for the investigators to determine whether disease duration could have affected response to therapy. Although baseline C-peptide AUC was identical in both groups, subgroup analysis of those with disease duration < 1 year vs those > 1 year revealed that among subjects with disease duration > 1 year, baseline C-peptide was higher in those randomized to receive ATG/GCSF. In contrast, among subject with disease duration < 1 year, baseline C-peptide was lower in those randomized to receive ATG/GCSF. Most, but not all Phase 2 trials to preserve beta cell function in T1D enroll subjects within 3 months of diagnosis; the investigators chose otherwise for this pilot study so as not to compete with ongoing studies. Nonetheless, the results are compelling enough that a fully powered 3 arm trial (ATG+GSCF; ATG alone; placebo) enrolling subjects within 3 months of diagnosis has recently been launched by Diabetes TrialNet in T1D. With increasing information about persistence of insulin secretion in some individuals further from diagnosis, more studies may be designed in the future to formally test the impact of time from diagnosis as differentiated from baseline level of insulin secretion on outcomes.

Combination therapy may have several advantages, such as reduced risk of adverse effects if the combination permits lower doses or shorter duration of treatment and allowing for more specific targeting of different pathogenic pathways. Thus, a key point of interest from this study is that these positive results are in contrast to results from other studies using the same agents individually. In higher doses than employed in this trial, ATG alone did not preserve insulin secretion in a previously reported fully powered, phase 2, placebo controlled trial of individuals within a few months of diagnosis (1). A smaller study with GCSF alone was also negative (2). By trialing the arms individually in the upcoming TrialNet study, investigators may be able to sort out whether there is therapeutic synergy with the combination, or whether administration of a lower dose of ATG could limit untoward effects on Tregs suggested in the previous negative ATG study.

References:

  1. Gitelman SE et al. Antithymocyte globulin treatment for patients with recent-onset type 1 diabetes: 12-month results of a randomized, placebo-controlled, phase 2 trial Lancet Diabetes Endocrinol. 2013;1(4):306-316

  2. Haller M et al. Granulocyte colony stimulating factor (GCSF) fails to preserve β cell function in patients with recent onset type 1 diabetes (TID). Diabetes. 2014;63:A428

Reviewed by Sandra Lord, MD, Benaroya Research Center

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June 15, 2015

Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital

Highlights in Recent Literature | Clinical Immunology Highlights | Immunphenotyping | Clinical TrialsPDF Version Previous Issues

Highlights from Recent Literature

Regulating the Regulators: Shutting Down Regulatory T Cells with GARP-TGFβ1 Antibodies

A review of Julia Cuende et al. Monoclonal antibodies against GARP/TGFβ-1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo. Science Translational Medicine (2015) 7, 274ra18. PMID: 25904740

Regulatory T cells (Treg) play critical roles in initiating and maintaining self tolerance but they can also interfere with tumor immunity and permit the persistence of chronic infections. One of the mechanisms by which Treg suppress other T cells is by presenting inactive TGFβ1 on the cell surface that becomes activated to immunosuppressive active TGFβ1 by unknown mechanisms. The authors demonstrate that the protein GARP plays a key role in TGFβ1 activation and that a monoclonal antibody that blocks this conversion inhibits Treg function.

  • GARP is a transmembrane protein expressed by Treg but not by other T cell subsets and it binds to latent TGFβ1 on the Treg cell surface.
  • The authors generated 31 new mouse and llama-anti human GARP antibodies and cloned these onto the human IgG1 backbone, creating humanized anti-GARP antibodies.
  • Two of these antibodies inhibited the production of active TGFβ1 by Treg, suggesting that GARP is directly involved in production of active TGFβ1 by human Treg.
  • These two inhibitory antibodies both mapped to a similar domain of the molecule, hGARP101-141, a part that is involved with TGFβ1 association on the cell surface.
  • The two inhibitory antibodies blocked Treg mediated suppression in in vitro assays and in a model of human anti-mouse xenogenic graft vs. host disease (GvHD).

By creating and studying antibodies against GARP, a protein that associates with latent TGFβ1 on the surface of Treg, the authors demonstrated that GARP is involved in the activation of TGFβ1 by Treg and that inhibition of this activity abrogates Treg mediated suppression in vitro and in an in vivo human anti-mouse GvHD model. Neutralizing anti-GARP antibodies are therefore a novel new class of Treg inhibitory therapeutics that may be valuable in the treatment of cancers and chronic infections.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


Stem-like CD8T Cells Mediate Long-term Protection After Yellow Fever Vaccination

A review of Silvia A. Fuertes Marraco, et al. Long-lasting stem cell–like memory CD8+ T cells with a naïve-like profile upon yellow fever vaccination. Science Translational Medicine (2015) 7, 282ra48. PMID:25855494

The yellow fever (YF) vaccine is a live attenuated virus that confers lifelong protection in humans. Protection is thought to be mediated by CD8+ T cells but there are no studies on how immunity is maintained long term. The authors studied long term immune responses in vaccinated patients and found that a stem cell-like population of CD8+ memory T cells persisted and mediated immune protection for decades after vaccination.

  • The authors studied a cohort of 41 patients who had received the vaccine between .27 years and 35 years before the current study.
  • T cells specific for yellow fever were detected in 38 of 41 patients. Early after vaccination, effector cells dominated the response, with the frequency of these cells tending to decrease over time.
  • However, a separate population of yellow fever specific naïve-like CD8+ T cells were found to be stably maintained for more than 25 years and were capable of self renewal in vitro.
  • Phenotypic and gene transcription studies demonstrated that these YF-specific long-lasting CD8+ T cells were similar to but distinct from genuine naïve cells from unvaccinated donors and also that they resembled a recently described stem cell-like population of memory T cells (Tscm)

The YF vaccine is one of the few vaccines that induces lifelong immunity in humans and the mechanism behind this long term protection was previously unknown. The authors demonstrate that long-term protection against YF in vaccinated patients was mediated by a unique population of T cells that had some features in common with naïve T cells, although they were clearly distinct, and also in common with a recently described population of stem cell-like memory T cells. These studies are the first to identify these cells in humans, implicate them in long-lived immune responses and demonstrate that they can persist for decades. Further studies of this cell type may lead to better vaccination strategies that confer life-long immune protection.

Reviewed by Rachel A. Clark, MD, PhD, Brigham and Women's Hospital


It's Only Skin Deep?

A review of Watanabe et al. Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells. Science Translational Medicine (2015) 279: 279ra39. PMID: 25787765

To better understand the components of the human cutaneous immune response, this study from the Clark lab extends earlier work on murine skin T cells and human cutaneous T cell lymphoma (CTCL) patients to characterize the populations of skin tropic non-recirculating (resident) memory T cells and recirculating T cells.

SUMMARY

  • To characterize the human cutaneous T cell immune response, they began by optimizing a mouse model of human skin. They began by grafting human foreskin to a healthy mouse, followed by an infusion of human PBMCs. Of note, human foreskin was chosen as a model of T cell depleted skin, based on prior work. Allogeneic T cells were injected and they specifically infiltrated the grafted skin and created a dermatitis. Over time, populations of resident T memory cells (Trm) developed in the skin graft
  • Anti-CD52 (alemtuzumab) administration specifically depleted circulating T cells (in blood) and over time the recirculating population of skin T cells was also depleted, while sparing a subgroup of T cells.
  • They focused on the non-recirculating Trm and compared the T cells remaining after alemtuzumab therapy in human skin grafts in mice and in patients with cutaneous T cell lymphoma. Retained skin Trm in those settings were concordant, with elevated CD69 and a subset coexpressing CD103.
  • Trm were divided into two groups, CD103 positive and negative. To study these carefully, human adult skin was split using enzyme treatment into epidermal and dermal layers and studied the T cell populations in each layer of skin. The number and function of these T cell subsets were then compared. Interestingly, Trm made more cytokine than circulating T cells. In addition, CD103+ Trm had limited proliferation potential though increased cytokine production.
  • While keratinocyte contact was shown to be needed for CD103 expression in T cells, it was shown to be mediated, at least in part, by TGFb release.
  • Circulating skin homing T cells were also divided into two groups: skin tropic central memory T cells (CCR4+ CCR7+ and L-selectin+, or Tcm) and CCR7+/L-selectin negative (or migratory memory cells, Tmm).
    • These cells (Tcm and Tmm) were compared to Tem by CyTOF mass cytometry. Interestingly, cytokine profile of Tmm was between Tem and Tcm. In Tem, Th1, Th17 cytokines were highest and in Tcm, Th2 cytokines were highest.
  • To characterize Tmm in vivo, patients with CTCL were studied. Interestingly, atypical lesions correlated with presence of Tmm.

This group was able to delve deeper than previous studies into the nature and migration patterns of human cutaneous CD4+ T cell responses. They confirmed the presence of four groups of skin tropic human T cells (two recirculating populations and two resident populations). They employed clever tools, including using infant foreskin engrafted onto mice with subsequent PBMC infusion to create inflammation and thus foster the development of the appropriate T cell infiltrate in the graft. In order to fully treat skin diseases including CTCL and psoriasis, we need an increased understanding of the baseline pathways of cutaneous immunity to allow optimization of our therapies. In addition, this knowledge improves our understanding of a fundamental component of our immune system.

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


Interrogating the Pathogenicity of Anti-Myelin T Cells in MS

 A review of Cao et al. “Functional inflammatory profiles distinguish myelin-reactive T cells from patients with multiple sclerosis.” Science Translational Medicine (2015) 287: 287 PMID: 25972006.

While there are anti-myelin T cells in patients with MS, it has been hard to implicate these cells directly to disease pathogenesis as similar numbers of anti-myelin T cells have been found in healthy controls. What has not been possible to examine previously is whether there is a difference between the function of those cell populations in the two different patient groups. In this study, anti-myelin T cells are isolated from patients with MS and healthy controls and compared functionally (both by cytokine production and proliferation) and transcriptionally.

SUMMARY 

  • In order to better characterize the autoreactive T cells in MS patients and healthy controls, T cells were sorted into appropriate subsets (i.e. +/- CCR6+, using the CCR6+ cell population as enriched in Th17 and Tr1 cells) and then cultured in vivo with activating stimuli for two weeks. This yielded libraries of polyclonally stimulated cells. In this study, 13,324 T cell libraries from 23 patients and 22 controls were created.
    • T cells from each library were characterized for their antigenic specificity using myelin peptides and Candida. 
    • T cells from MS patients had greater proliferation and more inflammatory cytokines (IFNg, IL-17 and GM-CSF production) in response to myelin antigens when compared to T cells from healthy controls 
    • Principal components analysis was performed on T cell libraries from 13 MS patients and 13 controls. 
      • CCR6+ memory cells in MS patients had two signatures, one that had higher production of GM-CSF and one with higher production of IL-17 or IFNγ.
      • Interestingly, healthy controls had a population of IL-10 secreting (inhibitory) T cell libraries stimulated by myelin antigen
  • In order to investigate whether complex and varied functional responses were based on assays that polyclonally activated cells (or whether individual cells had various functional profiles), single cell cloning was performed on libraries with the most proliferation.
    • 144 clones were generated from 2 MS patients and 2 healthy controls (all HLA typed and DR4+, which has been shown to be an MS susceptibility locus) by sorting cells using HLA-DR4 tetramers loaded with peptides from MOG and PLP.
    • The clones were stimulated with autologous monocytes with DR4 peptides from MOG and PLP. 
    • Myelin responsive clones from MS patients had higher production of IL-17, GM-CSF and/or IFNγ.
    • Interestingly, 22 of 23 total IL-10 secreting clones isolated came from healthy controls.
  • Finally, RNA sequencing was performed on sorted populations of myelin peptide tetramer positive and negative CCR6+ CD4+ memory T cells from T cell libraries (from 5 MS patients and 4 healthy controls) which showed the greatest amount of proliferation.
    • GO enrichment analysis and GSEA were performed
      • 197 genes were found only in the MS patient tetramer positive samples
      • GSEA analysis revealed 305 gene sets enriched in expression in MS patient tetramer positive cells (versus tetramer negative cells), 
        • 19 were shared with tetramer positive healthy controls
        • 112 were unique to MS patient tetramer positive samples
      • The pathways in the mouse model of MS, experimental autoimmune encephalitis, were compared to the MS patient CCR6+ tetramer positive memory cells, with evidence of overlap with pathways previously characterized in pathogenic murine Th17 cells
      • They also compared to signatures from rat EAE, murine signatures from specific cell subsets (Th1, Th17, Th2) and human Th17 effector memory signatures
        • They found human Th17 effector memory, human Th2 and mouse Th17 enrichment in the patient samples
    • Leading edge analysis connected IL-8, IFNγ, CCL20 and CCL5 in MS patient tetramer positive cells.
      • When samples were clustered, it was noted that the resulting groups were based on disease status or tetramer status. 
    • Pathway analysis was performed and defined possible mechanistic networks that can be used to differentiate MS patient autoreactive T cells, with roles shown for Th17 and T follicular helper cell associated cytokines among others.

This study demonstrates that while autoreactive cells can be found in both affected patients with MS and healthy controls, there are functional differences at the levels of proliferation and cytokine production as well as transcriptional differences, in the autoreactive cells from patients versus healthy controls. The functional differences and transcriptional differences link mechanisms proposed in the major murine model of MS (EAE) as well as previous human studies at least in part to the autoreactive cells identified in the MS patients. In addition, protective signatures are seen in the autoreactive cells in healthy controls, which may help explain the presence of the cells without disease in healthy controls. There are many more studies that will need to be performed to further characterize these rare cells in their native state, but this study furthers our understanding of a longstanding paradox in the field.

Reviewed by Sarah Henrickson, MD, PHD, Children's Hospital of Philadelphia


Immune Effects of a Personalized Melanoma Vaccine 

A review of Carreno, B. M. et al. “A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells”. Science  348, 803–808 (2015). PMID:25837513

Melanoma cells harbor up to hundreds of somatic mutations that provide a putative source of tumor-specific neoantigens for personalized tumor vaccines. Although the induction of T cell immunity against tumor mutation-specific neoantigens has been reported, it remains unknown whether vaccination can augment neoantigen-specific T cell responses. Carreno et al. explore the effect of a personalized vaccine on the repertoire of neoantigen specific T cells. The authors sequenced the exomes of three patients with stage 3C cutaneous melanoma, then created and administered a personalized vaccine composed of seven neoepitope peptides pulsed on to autologous dendritic cells.

  • In all patients, a CD8 T cell-mediated response to at least one neoepitope was detected in pre-vaccination PBMCs.
  • Vaccination augmented pre-existing T cell responses, and also induced new responses in all patients.
  • Isolated ex vivo T cells specific for these neoepitopes were functional, and all but one neoepitope-specific T cell line could distinguish the mutant epitope from the wild-type.
  • Vaccination increased the frequency of pre-existing neoepitope specific T cells, and induced many new clones, increasing the diversity and size of the anti-tumor T cell repertoire.

Carreno et al. provide a valuable paradigm for the discussion of cancer neoepitopes; they classify pre-existing neoantigens as “dominant”, and vaccine-induced neoantigens as “subdominant”. The abundance of these subdominant neoantigens highlights the importance of vaccination in an immunotherapy regimen, as T cells targeting these epitopes are unlikely to arise from immunomodulatory treatment (anti-CTLA4, anti-PD1, etc.) alone. The authors have shown that personalized cancer vaccines are able to induce a strong anti-tumor immune response. The pipeline for the creation of these vaccines is currently too expensive and time consuming for widespread use; however falling sequencing costs and improved in silico methods for antigen identification are bringing personalized vaccine therapy closer to fruition.

Reviewed by Alexander Hopkins, Johns Hopkins University, Cellular and Molecular Medicine Program and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center.


Identification of Human T Cell Receptors with Optimal Affinity to Cancer Antigens Using Antigen-negative Humanized Mice

 A review of  Obenaus, M. et al. “Identification of human T cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice.” Nature Biotechnology 33, 402–407 (2015). PMID: 25774714

High affinity tumor-reactive T cells that bind to unmutated tumor-associated antigens (TAAs) are subject to deletion via central tolerance in the thymus and thus are rarely found in the periphery. Those that do escape deletion are often of low affinity and are susceptible to peripheral tolerance mechanisms. Obenaus et al. use antigen negative, HLA-A2 and human T cell receptor (TCR) transgenic mice to isolate CD8+ T cells specific for the unmutated tumor antigens MAGE-A1 and NY-ESO. These T cells have improved cytolytic activity and interferon (IFN) γ production when compared to MAGE-A1- and NY-ESO-reactive CD8+ T cells isolated from human donors. Furthermore, these TCRs derived from mice exhibit no alloreactivity or functional antigen cross-reactivity, contributing to their potential utility in future adoptive cell therapies.

  • ABabDII mice, transgenic for human TCR-αβ gene loci and chimeric HLA-A2, were immunized with peptide corresponding to the HLA-A2 restricted epitope MAGE-A1278, which differs from the murine homolog by 6 out of 9 amino acids. CD8+ T cells reactive to MAGE-A1278 HLA-A2 multimers were detected after a boost 300 days later, indicating a functional memory response. RACE-PCR amplification identified 3 TCR clones, reflecting a single expanded TCR clone from each of 3 mice. Transduction of human T cells with these TCRs (T1367, T1405, and T1705) showed cytolytic activity against and IFNγ production in response only to HLA-A2+, MAGE-A1+ cells.
  • T1367 was tested against 114 known HLA-A2 restricted peptides and showed no off-target cross-reactivity. Alanine substitution identified the MAGE-A1 epitope recognized by T1367 and other peptides with similar motifs were identified in the human proteome and tested for T1367 recognition. Although a peptide derived from the SAMD9 gene could be recognized by T1367, it was only recognized at very high peptide concentrations and not on T cells, which naturally express SMAD9, demonstrating a lack of off-target toxicity.
  • When CD8+ T cells were transduced with MAGE-A1-specific TCRs isolated from human donors (hT27 and hT89) compared to the murine-derived TCRs, the CD8+ T cells expressing murine-derived TCRs showed greater cytolytic activity and IFNγ production. The human-derived TCRs were only stimulated by high concentrations of peptide loaded onto the T2 target cell line but only hT27 could recognize MAGE-A1+ cancer cells, whereas all murine-derived TCRs were stimulated by both the MAGE-A1+ cancer cells and T2 cells loaded with substantially lower concentrations of MAGE-A1278. When mice bearing large MAGE-A1+ tumors were treated with either ht27- or T1367-transduced T cells, only the T1367-transduced T cells were capable of inducing tumor regression.
  • Similar results were seen when ABabDII mice were immunized against NY-ESO: the murine-derived TCRs exhibited improved functionality when compared to TCRs isolated from human donors. The murine-derived TCRs produced more IFNγ and were capable of responding to lower antigen concentrations than human-derived TCRs.

Isolation of tumor antigen-specific T cells from patients has yielded low avidity T cells due to mechanisms of central and peripheral tolerance, which limits the available repertoire of tumor-reactive T cells. Obenaus et al. demonstrate that antigen-negative hosts expressing cognate human TCR-αβ gene loci and chimeric HLA molecules provide a system for identifying and expanding tumor-reactive T cells with increased functional avidities capable of inducing an anti-tumor response.

Reviewed by Heather Kinkead, Johns Hopkins University, Cellular and Molecular Medicine Program and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

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Highlights From Clinical Immunology, the Official Journal of FOCIS

B Cells and GCs in EAE

A review of Batoulis H. et al. “Central nervous system infiltrates are characterized by features of ongoing B cell-related immune activity in MP4-induced experimental autoimmune encephalomyelitis.” Clinical Immunology. (2015) 158, 47–58 PMID: 25796192

B cell aggregates and perhaps tertiary lymphoid organs (TLOs) form in the meninges of multiple sclerosis (MS) patients, and these have been correlated with more aggressive disease. Nonetheless, the contribution of B cells, antibodies, and TLOs to MS is poorly understood. Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of mice that is widely used as model of human MS. EAE is induced by immunization with myelin proteins and strong adjuvant, but the disease is usually helper T cell dependent. The authors have previously described a B cell-dependent variation of the EAE model, induced by immunization of C57BL/6 mice with MP4, a fusion protein of myelin basic protein (MBP) and parts of proteolipid protein (PLP). In this report, the authors goal was to determine the function and disease impact of B ell infiltrates in the CNS in MP4-EAE. There main findings were:

  • Using immunohistochemistry, cells in cerebellar B cell aggregates were CXCL13 and CD10 positive, but negative for makers of B1 or regulatory B cells.
  • Cerebellar germline transcripts indicative of IgG2b and IgG3 class switching ere found in the majority of mice with MP4-EAE at 50 and 70 days after onset.
  • NexGen sequencing of cNDA from cerebellum and spleen of MP4-EAE mice indicated restricted VH usage and abnormally long, positively charged CDR3 loops, which have been associated with autoimmune disease.
  • Elispot analyses indicated antigen-specific B cell responses spread from the immunizing antigens to other myelin and neuronal antigens.

The authors state that these data are consistent with the development of tertiary lymphoid organs with active germinal centers that contribute to chronic B cell-dependent EAE. Further direct analysis of B cells within aggregates in their model, using laser capture, will be required to better characterize the TLO-like structures. The relationship between their MP4-EAE model, and T cell dependent EAE or human MS, where TLOs are in meninges and not in the cerebellum, remains to be clarified.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital


TIM3 on NKs in HCV Infection

A review of Golden-Mason L. et al. “Hepatitis C viral infection is associated with activated cytolytic natural killer cells expressing high levels of T cell immunoglobulin- and mucin-domain-containing molecule-3.”Clinical Immunology (2015) 156, 1–8. PMID 25797693

Chronic viral infections, including hepatitis C virus (HCV) infection, are associated with exhausted T cells which express high levels of various inhibitory molecules, and are unresponsive to viral antigens. Among these inhibitory molecules is T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3). Previous studies have shown highTim-3 expression on human natural killer (NK) cells, but the expression and function of NK Tim-3 in chronic viral infections is not well characterized. In this study, the authors examined Tim-3 expression on NK cells in chronic hepatitis-C virus (HCV)-infected patients. They examined blood NK cells from 37 chronically HCV-infected subjects and 20 uninfected controls, using FACS, qRT-PCR, and functional assays. The findings include the following:

  • Tim-3 expression was increased on activated NK cells in patients with chronic HCV.
  • The high NK levels of Tim-3 was not reversed by effective antiviral therapy with IFN alpha and ribavirin.
  • IFN-alpha treatment causes a greater increase in NK TRAIL expression in Tim-3 high cells than Tim-3 low cells.
  • Tim-3 high cells in the INF-alpha treated patients showed greater lymphokine-activated killing activity, more viral control, and more degranulation compared toTim-3 low NK cells, although cytokine production was the same.

These results show that high Tim-3 expression on NKs in the setting of chronic HCV infection is not a marker of functional exhaustion, but rather of activation and enhanced cytotoxic activity. This is consistent with other published data, but in contrast to studies showing that NK Tim-3 marks exhausted NK cells in HBV infection. The authors advise that more studies on Tim-3 expression and function on different cell types and in different infections are needed to better understand how stimulating and blocking Tim-3 reagents might be considered therapeutically.

Reviewed by: Andrew Lichtman, MD, PhD, Brigham and Women’s Hospital

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Human Immunophenotyping Update

Immunophenotyping of Human NK Cells 

Catherine A. Blish, MD, PhD, Stanford University School of Medicine 

Natural killer (NK) cells are innate lymphocytes that can rapidly respond to tumor or infected cells by killing (cytolysis) or by secreting cytokines. Unlike adaptive T and B lymphocytes that somatically rearrange antigen-specific receptors, NK cells express a variety of germline-encoded activating and inhibitory receptors on their cell surface. These receptors include the killer immunoglobulin-like receptors (KIR), C-type-lectin-like receptors (for example, NKG2A, NKG2C, and NKG2D), leukocyte immunoglobulin-like receptor subfamily b member 1 (LILRB1, also known as ILT-2 and CD85j), natural cytotoxicity receptors (NCRs, including NKp30, NKp44, NKp46, and NKp80), and signaling lymphocyte activation molecule (SLAM) family receptors (for example, 2B4, NTB-A). NK cells also express a variety of adhesion molecules such as CD2 and DNAM-1 that influence their function. These receptors allow NK cells to recognize ‘altered self’ on virus-infected, malignant or stressed cells.

NK cells rely on combinatorial signaling from this diverse array of receptors (1). The inhibitory receptors, which include inhibitory KIR (e.g., KIR3DL1, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5), LILRB1, and NKG2A, are specific for self-HLA class I molecules, and provide an “all clear” or “back off” signal to the NK cell. Engagement of these receptors by HLA dampens the NK cell response. However, engagement of activating receptors, which recognize a variety of stress-related molecules and pathogen- or tumor-derived ligands, activates the NK cells to kill or secrete cytokines by indicating that the cell is a threat. Since the ultimate outcome of the interaction between an NK cell and a target cell is determined by the combinatorial effects of these inhibitory and activating signals, NK cell phenotype and function are closely and uniquely linked.

This vast array of receptors has presented a challenge for the immunophenotyping of NK cells. There has been increasing recognition that the NK cell receptor repertoire, as determined by the expression patterns of this array of receptors, is incredibly complex (2,3). Further complicating this matter is the challenge of determining which cells are actually NK cells, because unlike a B or a T cell, there is no single receptor that defines the subset. In the classic definition, NK cells are broken down into two subsets based on expression of CD56 and CD16 (the FcγRIII). In the peripheral blood, the vast majority (~90%) of NK cells express CD56 at low levels and also express CD16. These CD56dimCD16+ NK cells are considered mature NK cells that are highly effective in cytolysis. The more rare CD16brightCD16- NK cells are thought to be relatively immature NK cells that are specialized for cytokine secretion (4). However, this classification ignores many subsets, including intermediate populations such as rare CD56brightCD16+ NK cells, or the CD56-CD16+ NK cells that are particularly prominent during chronic viral infections (5). Thus, NK cells are best defined as much as what they are not (not B cells, T cells, monocytes, or dendritic cells) as by what they are (lymphocytes with cytolytic activity or cytokine secretion that express CD56 and/or CD16). Finally, within these populations there are a vast array of different subsets based on expression of other NK cell receptors, whose functional significance we are only beginning to unravel. In fact, based on combinatorial expression of 28 NK cell receptors, Horowitz et al. estimated that each individual has between 6,000-30,000 unique NK cell phenotypes, and more than 100,000 subsets in a small population of 22 individuals (3).

With this framework in mind, the first step in immunophenotyping is to carefully identify the NK cell subset. While the most common approach to do this is to identify CD3-CD56+ NK cells, as discussed above, this approach fails to identify CD56- NK cells which are present at varying levels in human subjects. In fact, CD56 expression alone is not particularly specific to NK cells (6), as it is expressed on activated T cells and NKT cells. Of all the NK cell markers, NKp46 is the most specific to NK cells, but like CD56, it is not expressed on all NK cells (6). Thus, the best approach to identify NK cells is to perform serial negative gating.

In standard fluroresence cytometry, a simple approach is to gate first on lymphocytes by forward and side scatter. Depending on the number of channels available, either a single “dump” channel or sequential negative gating should be performed on CD3, CD19 and/or CD20, and CD14 and/or CD33. Then NK cells can be identified as the remaining cells that express CD56 and/or CD16. As monocytes, in particular, can express CD14 and/or CD33 at low levels, this approach can result in some monocytes within the NK cell gate. This can be avoided by adding a stain for CD7, as NK cells and T cells, but not monocytes, express CD7 (7). Thus, NK cells are CD3-CD19-CD20-CD33-CD14-CD7+ cells that express either CD56 or CD16. In mass cytometry, or CyTOF, more channels are available; however, there is no forward or side scatter to help identify lymphocytes vs. monocytes. Thus, in addition to the scheme recommended above, additional gating to avoid CD56-HLA-DRbright cells and LILRB1bright cells will help to further decrease the number of monocytes inadvertently included in the NK cell gate (6).

Once NK cells have been identified, antibodies to a variety of NK receptors or functions can be used. Beziat and colleagues have recently reported a detailed flow cytometry method to identify KIR gene expression patterns in peripheral blood mononuclear cells (8). CyTOF, with it ability to identify up to 42 parameters simultaneously, is currentlyt the most comprehensive platform to identify expression patterns of multiple classes of NK cell receptors, including KIRs, C-type lectin-like receptors, LILRB1, natural cytotoxicity receptors, and adhesion molecules (3,6). The specific receptors queried should be tailored to the question.

With the new attention to the potential of harnessing NK cells in immunotherapeutic approaches(9,10), it is becoming increasingly apparent that we need to identify which subsets of NK cells should be targeted. For this to be effective, stringent efforts to identify NK cells by flow cytometry, with particular attention to avoiding monocyte inclusion in NK cell gates, will be critical. Given the vast array of surface receptors expressed by NK cells, highly parametric flow cytometry techniques, including new use of highly intense fluorescent dyes (11), or the use of the highly parametric mass cytometry platform (12), will be central to these efforts.

References:

  1. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, Yokoyama WM, Ugolini S. Innate or Adaptive Immunity? The Example of Natural Killer Cells. Science. 2011 Jan 6;331(6013):44–9.
  2. Di Santo JP. Functionally distinct NK-cell subsets: Developmental origins and biological implications. Eur. J. Immunol. [Internet]. 2008 Nov;38(11):2948–51. Retrieved from: http://doi.wiley.com/10.1002/eji.200838830
  3. Horowitz A, Strauss-Albee DM, Leipold M, Kubo J, Nemat-Gorgani N, Dogan OC, Dekker CL, Mackey S, Maecker H, Swan GE, M DM, Norman PJ, Guethlein LA, Desai M, Parham P, Blish CA. Genetic and Environmental Determinants of Human NK Cell Diversity Revealed by Mass Cytometry. Sci Transl Med. 2013 Oct 23;5(208):208ra145–5.
  4. Freud AG, Caligiuri MA. Human natural killer cell development. Immunol Rev. 2006 Dec 1;214(1):56–72.
  5. Björkström NK, Ljunggren H-G, Sandberg JK. CD56 negative NK cells: origin, function, and role in chronic viral disease. Trends Immunol. 2010 Nov 1;31(11):401–6.
  6. Strauss-Albee DM, Horowitz A, Parham P, Blish CA. Coordinated regulation of NK receptor expression in the maturing human immune system. J Immunol. 2014 Nov 15;193(10):4871–9. PMCID: PMC4225175
  7. Milush JM, Long BR, Snyder-Cappione JE, Cappione AJ, York VA, Ndhlovu LC, Lanier LL, Michaelsson J, Nixon DF. Functionally distinct subsets of human NK cells and monocyte/DC-like cells identified by coexpression of CD56, CD7, and CD4. Blood. 2009 Nov 26;114(23):4823–31.
  8. Béziat V, Traherne J, Malmberg J-A, Ivarsson MA, Björkström NK, Retière C, Ljunggren H-G, Michaëlsson J, Trowsdale J, Malmberg K-J. Tracing dynamic expansion of human NK-cell subsets by high-resolution analysis of KIR repertoires and cellular differentiation. Eur. J. Immunol. 2014 May 7;44(7):2192–6.
  9. Keener AB. Natural killers:. Nat Med. Nature Publishing Group; 2015 Mar 1;21(3):207–8.
  10. Childs RW, Carlsten M. Therapeutic approaches to enhance natural killer cell cytotoxicity against cancer: the force awakens. Nature Publishing Group. Nature Publishing Group; 2015 May 22;:1–12.
  11. Kling J. Cytometry: Measure for measure. Nature. 2015 Feb 19;518(7539):439–43.
  12. Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK. A deep profiler's guide to cytometry. Trends Immunol. 2012 Jul 1;33(7):323–32. PMCID: PMC3383392

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Selected Recent Clinical Trial Results

Randomized Trial of Peanut Consumption in Infants at Risk for Peanut Allergy

Clinical Trial: George Du Toit, Graham Roberts, Peter Sayre, et.al. “Randomized Trial of Peanut Consumption in Infants at Risk for Peanut Allergy.” New England Journal of Medicine 2015;372(9):803-813

Disease: Peanut allergy 

Intervention: Consumption of peanuts or avoidance of peanuts until age 5 in infants with eczema and/or egg allergy.

Study design:

  • 640 infants with history of severe eczema and/or egg allergy in UK ages 4-11 months (mean 7.8 months) randomized to consume or avoid peanuts until age 60 months
  • Infants with strongly positive skin prick test to peanuts were excluded.
  • Infants were placed into two cohorts: Those with negative (n=542) or weakly positive (n=98 ) skin prick test.
  • Open-label design
  • All participants had a baseline feeding challenge and were instructed to avoid peanuts (regardless of their randomization assignment) if their feeding challenge was positive, but were included in the ITT analysis. 
  • Consumption group instructed to consume at least 6 g of peanut protein/week (equivalent of about 24 peanuts) until age 60 months
  • Primary outcome was the proportion of participants with peanut allergy at 60 months as measured by an oral food challenge. 

Results:

  • In the cohort with a negative baseline skin prick test, 13.7% of avoidance group and 1.9% of consumption group were allergic to peanuts, an 86.1% relative reduction (P<0.001)
  • In the cohort with a positive baseline skin prick test, 35.3% of avoidance group and 10.6% of the consumption group were allergic to peanuts, an 70.0% risk reduction (P=0.004)

Why the Trial is of Interest to the Broader FOCIS community:

The prevalence of peanut allergies in Western countries has doubled in the past 10 years and represents a significant lifestyle and financial burden to individuals and families (1). The mainstay of treatment has been avoidance of peanuts when possible, along with preparation for and treatment of accidental exposures. However, there has been limited evidence to guide prevention of peanut allergy. In 2000, the American Academy of Pediatrics (AAP) published guidelines advising parents to delay the introduction of peanuts to children at high risk of allergies until age 3. In 2008, the AAP revised its guidelines, stating that there was no evidence that delayed introduction of particular foods beyond 4-6 months (the current age at which parents are advised to introduce solid foods) would prevent allergies. The 2008 AAP revision was supported by increasing observational data suggesting that early and sustained oral exposure to peanut protein could reduce the risk of clinical peanut allergy. In particular, a 2008 study conducted by the same investigators as the reviewed study compared Jewish children raised in Israel, where peanut protein is introduced at a median age of 7 months, to Jewish children raised in the UK, where peanut consumption in infancy is discouraged. It was found that school age children in the UK have 10-fold higher rate of peanut allergy than school age children in Israel (2). The reviewed study, the Learning Early About Peanut Allergy (LEAP) Study was launched from the 2008 study and is the first randomized trial to demonstrate that earlier and sustained exposure to peanut protein may prevent peanut allergies in at-risk individuals.
The results of the LEAP study demonstrate that early oral exposure to allergens results in immune tolerance even amongst the cohort of children who were already demonstrating some peanut sensitivity (those with a weakly positive skin prick test). Moreover, the LEAP-On study (Persistence of Oral Tolerance to Peanut) will address the important question whether individuals will remain allergy-free after prolonged cessation of oral peanut exposure. In contrast to this terrific result in allergy, no clear successes have been yet reported using oral antigen therapy in autoimmune disease. An interesting question is whether mimicking this timing, delivery method, and dosing in autoimmunity would result tolerance to autoimmune disease as well.

References:

  1. Prescott SL, Pawankar R, Allen KJ, et al. World Allergy Org J 2013;6:21.
  2. Du Toit G, Katz Y, Sasieni P, et al. J Allergy Clinic Immunol 2008;122:984-91.

Submitted by Sandra Lord, MD, Benaroya Research Institute
Edited by Carla J. Greenbaum, MD, Benaroya Research Institute


Effects Of High-dose Oral Insulin on Immune Responses in Children at High Risk for Type 1 Diabetes: The Pre-point Randomized Clinical Trial

Clinical Trial: Ezio Bonifacio, Anette-G Ziegler, Georgeanna Klingensmith, et. al, Effects of High-Dose Oral Insulin on Immune Responses in Children at High Risk for Type 1 Diabetes: the Pre-POINT Randomized Clinical Trial. JAMA, 2015;313(15):1541-1549

Disease: Islet autoimmunity/Type 1 diabetes (TID)

Drug: Oral insulin

Study design:

  • Double-blind, placebo-controlled, dose-escalation, phase 1/2 clinical pilot study in Germany, Austria, UK, US.
  • 25 autoantibody (insulin, GAD65 and IA-2) negative children ages 2-7 years with a family history of type 1 diabetes and HLA class II susceptibility genotypes.
  • Randomized to receive placebo (n=10) or oral insulin (n=15) once daily for 3-18 months. Oral insulin subjects were randomized to one of 5 different dosing blocks, each block with 3 subjects. Dosing schedules were such that each dosing cohort (2.5 mg, 7.5 mg, 33.5 mg, 67.5 mg) had 6 subjects each. This is depicted in table 1:

Table 1:

ClinicalTrialsTable

  • Outcome measures:
    • Primary: Positive response to any one of the following: serum IgG low-affinity binding to insulin; salivary IgA binding to insulin; CD4+ T cell proliferative responses to insulin
    • Secondary: measurement of pro-regulatory FOXP3 gene signature and pro-inflammatory IFNγ signature in T cells with a response to insulin.

Results:

  • Immune responses: Only one subject (at dose 22.5) developed a Salivary IgA response. In the high dose group (67.5 mg), 3/6 developed IgG insulin antibodies and 2 others developed antigen specific CD4+ T cells detected; thus 5/6 subjects in this group had an immune effect of receiving therapy. This was in contrast to 2/10 in placebo group, and 1-2/6 in each of the other dosing groups (p=.02 for trend).
  • Comparing the gene signatures of antigen specific cells (to insulin and proinsulin) pooled from these antibody negative subjects treated with oral insulin to the signatures of antigen specific cells pooled from antibody positive subjects from the BABYDIET study suggested a more pro-regulatory gene signature in those receiving oral antigen therapy.
  • No adverse events.

Why the Trial is of Interest to the Broader FOCIS Community:

The Pre-POINT study was a dose-finding and feasibility study in anticipation of the phase 2 POINT study to determine if oral insulin can delay or prevent TID in those at high risk based on HLA haplotype and family history. The study aimed to correlate specific oral insulin dosing in this islet antibody negative group with desirable and expected immune responses. The results support the plan to use the higher dose in the fully powered POINT trial. Nonetheless, this pilot study highlights the difficulties in making such judgements; few responses were seen overall and whether these are salutatory will depend on the definitive study.


The POINT study and other oral tolerance studies aim to determine whether early, repeated exposure to self-antigens will promote immune tolerance. This concept has gained traction in clinical allergy, where trials have demonstrated that such exposure can prevent allergy (1), but trials have been less convincing in autoimmunity. In TID, previous trials of antigen therapy (including nasal insulin, parenteral insulin, low-dose oral insulin, GAD65, Diapep277) in both the at-risk and new-onset populations have not met their primary endpoints (2-7). The difficulties in conducting such studies and their long duration emphasize the need to select the “right” dose. Two other studies are underway using insulin as antigen therapy in antibody positive relatives; INIT II is testing nasal insulin 440 IU weekly, and Diabetes TrialNet is testing oral insulin at a dose of 7.5 mg daily. The TrialNet study is the follow-on to the Diabetes Prevention Trial oral insulin study in which post-hoc analysis suggested a benefit (3). Selection of dose in these studies anticeeded the technologies used in the Pre-POINT study. While trial results from the TrialNet study will not be available for another year, samples will be assessed for mechanistic studies akin to those used in Pre-POINT to determine whether the dose chosen had an immune effect in this population. In addition to choosing dose, choosing when to administer oral antigen therapy in the course of the disease is another question. The POINT study will enroll antibody negative, genetically at-risk children. In this group, antibodies generally develop very early: 64% of those who develop T1D before puberty will have antibodies by age 2 and 95% by age 5 (8-9). Since the mean age of those in the Pre-Point study was 5 years old, it is possible that these children had an underlying regulatory tendency. The TrialNet and INIT II study enrolls relatives with 2 or more antibodies. These individuals have “islet autoimmunity”. While only ~40% are likely to develop T1D in 5 years, recent data emphasizes that essentially all of these individuals will eventually progress to clinical disease. Much will be learned from comparing trial results and mechanistic assays in using oral antigen therapy to treat before or after antibodies are present.

References:

  1. George Du Toit, et.al. N Engl J Med 2015;372(9):803-8133
  2. Skyler JS et al. Diabetes Care 2005 May: 28(5): 1068-76
  3. Diabetes Prevention Trial-Type 1 Diabetes Study Group: Effects of insulin in relatives of patients with type 1 diabetes mellitus. N Engl J Med 345: 1685–1691, 2002
  4. Wherrett DK et al. Lancet. 2011 Jul 23;378(9788):319-27
  5. Näntö-Salonen K et al. Lancet. 2008 Nov 15;372(9651):1746-55
  6. Schloot NC et al. Clin Immunol. 2013 Dec;149(3):307-16
  7. Ziegler AG et al. JAMA. 2013. Jun 19;309(23):2473-9
  8. Parikka V et al. Diabetologia. 2012 Jul;55(7):1926-36

Submitted by Sandra Lord, MD, Benaroya Research Institute
Edited by Carla J. Greenbaum, MD, Benaroya Research Institute


Mongersen, an Oral Smad7 Antisense Oligonucleotide, and Crohn’s Disease

Clinical Trial: Monteleone G., Neurath M., Ardizzone, et al. Monbersen, an Oral SMAD7 Antisense Oligonucleotide, and Crohn’s Disease. New England Journal of Medicine 2015:372:1104-13

Disease: Active Crohn’s Disease (CD)

Drug: Mongersen, an oral SMAD7 Antisense oligonucleotide. SMAD7 acts as an inhibitor of the immunosuppressive cytokine TGF-β1. Mongerson targets ileal and colonic SMAD7, via an anti-sense mechanism that facilitates SMAD7 messenger RNA degradation.

Study design:

  • 166 randomized, ages 18-75 years, moderate-severe active CD (as defined by Crohn’s Disease Activity Index/CDAI score of 220-400 on a scale 0-600)
  • 2 weeks of treatment, evaluated at days 15, 28, 84
  • 1:1:1:1 randomization: placebo, 10, 40, 160 mg doses
  • Primary outcome: clinical remission at day 15 (CDAI score < 150) and maintenance of remission for at least 2 weeks
  • Secondary outcome: clinical response (reduction of CDAI sore of ≥ 100 points) at 4 weeks.

Results:

  • Primary outcome, clinical remission: 65% in 160 mg group; 55% in 40 mg; 12% in 10 mg; 10% in placebo (P<0.001)
  • Secondary outcome, clinical response: 72% in 160 mg (P<0.001); 58% in 40 mg (P<0.001); 37% in 10 mg (P=0.04); 17% in placebo
  • 102/166 (61.4%) with elevated C reactive protein (CRP) at screening. Response rates similar among patients with elevated vs normal baseline CRP.
  • No correlation between normalization of CRP levels and improvement of CDAI score
  • No identified safety issues

Why the Trial is of Interest to the Broader FOCIS Community:

Mongersen belongs to a promising new category of therapy for Crohn’s Disease (CD) and other diseases; namely, antisense oligonucleotide therapy. Antisense oligonucleotides bind to messenger RNA, preventing gene translation and effectively turning the gene off. Antisense therapies have been studied as potential drugs for infections, cancers, inflammatory disorders, and genetic disorders. To date, mongersen is the third antisense drug to be approved by the FDA. Fomivirsen is approved for treatment of cytomegalovirus retinitis, and mipomersen for treatment of homozygous familial hypercholesterolemia.

There is an unmet need for additional therapies for CD, as one-third of patients with CD do not respond to currently available therapies, including anti-TNFα antibodies (infliximab, adalimumab, certolizumab pegol), anti-integrin antibodies (vedolizumab, natalizumab) and immunomodulating drugs (azathioprine, 6-mercaptopurine, methotrexate). In addition, the efficacy of TNFα therapies decreases over time, there is no evidence that the TNFα therapies reduce overall disease progression, and there is a risk of infection and malignancy with long term usage. In CD, TGF-β1 intracellular signaling is blocked by the SMAD7 protein (1;2). TGF-β1 has several anti-inflammatory/pro-regulatory effects, including: inhibition of effector T cell proliferation and differentiation, induction of regulatory T cells, reduced macrophage activation and reduced dendritic-cell maturation (3). Mongersen is a SMAD7 antisense oligonucelotide which facilitates mRNA degradation and decreases SMAD7 to increase TGF-b signaling. It provides specific targeting to the terminal ileum and right colon (the most commonly affected areas in CD) via a pH-dependent coating of the tablet. In this phase 2 trial, participants treated with mongersen experienced 2 week remission rates of 65% and 55% with the 2 highest doses, compared to 10% in the placebo group. By comparison, reported remission rates seen in the large trials of TNFα inhibitors in moderate-severe CD range are 32.5-36% (4;5), and remission rates seen with vedolizumab (an LPAM1 antibody, which selectively blocks gut integrin activity) are 14.5-39% (6). Furthermore, despite only 2 weeks of treatment, the majority of participants (62% and 67% in the 40 mg and 160 mg groups respectively) who were in remission after 2 weeks remained in remission after 12 weeks of follow-up. This contrasts with the rapid relapse typically seen after withdrawal of other anti-inflammatory agents.

There are some important caveats to consider. There was limited evaluation of objective markers of disease activity. Namely, there were no required endoscopic evaluations, either at entry or endpoint; therefore, it’s not known whether improvements in CDAI scores correspond to mucosal healing. The CDAI is somewhat subjective: of the 8 criteria, 3 are patient-reported. Moreover, fecal calprotectin, a stool test for colonic inflammation, was not measured. CRP was measured, but interestingly, there was no correlation between clinical response and normalization of CRP. As the investigators suggest, there may be lag between clinical response and normalization of CRP. In addition, treatment duration and follow up (2 weeks and 12 weeks, respectively) were relatively brief, which should impact any conclusions that are made about safety and efficacy. Although there were no significant safety issues identified, longer-term data is needed. There was one instance of intestinal obstruction, which is a known complication of CD, but given that TGFβ1 can promote fibrosis (7;8), this might be of special interest.

Mongersen’s specific targeting to the terminal ileum and right colon is both a strength and a limitation; although it does not appear to have systemic side effects, it also may not be effective for extra-intestinal manifestations of CD or for perianal disease, the latter of which affects about half of patients with CD.

Despite these caveats, mongersen may represent an entirely new, safe, and effective category of Crohn’s Disease therapy. Antisense therapies have been studied for potential treatment of infections and genetic disorders. To date, mongersen is the third antisense drug to be approved by the FDA. Fomivirsen is approved for treatment of cytomegalovirus retinitis, and mipomersen for treatment of homozygous familial hypercholesterolemia. Follow up studies are needed, to include both longer duration of follow up and additional objective measures of disease activity.

References:

  1. Boirivant M, et al. Gastroenterology 2006;131:1786-1798
  2. Monteleone G, et al. J Clin Invest 2001;108:601-609
  3. Boirivant M et al. Gastroenterology 2006;131:1786-1798
  4. Colombel JF et al. N Engl J Med 2010;362:1383-1395
  5. Hanauer SB, et al. Gastroenterology 2006;130:323-333
  6. Sandborn WJ et al. N Engl J Med 2013;369:711-721
  7. Fichtner-Feigl S et al. Nat Med 2006;12:99-106
  8. Medina C et al. J Pathol 2011;224:461-472

Submitted by Sandra Lord, MD, Benaroya Research Institute
Edited by Carla J. Greenbaum, MD, Benaroya Research Institute

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October 1, 2015

Editor: Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital
Editorial Board: Abul K. Abbas, MD, University of California, San Francisco | Carla J. Greenbaum, MD, Benaroya Research Institute | Andrew H. Lichtman, MD, PhD, Brigham & Women's Hospital

Highlights in Recent Literature | Clinical Immunology Highlights | Immunphenotyping | Basic ImmunologyClinical TrialsPDF VersionPrevious Issues

Highlights from Recent Literature

Dendritic Cell Immunotherapy is Safe and Effective in HLA-associated Rheumatoid Arthritis

A review of Helen Benham, et al. Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype–positive rheumatoid arthritis patients. Sci. Transl. Med. 7, 290ra87 (2015).PMID: 26041704

Dendritic cells (DC) are a subset of antigen presenting cells that are critical in controlling T cell responses. Immature DC can tolerize T cells to specific antigens whereas mature dendritic cells tend to produce strong inflammatory responses. Mouse studies have demonstrated that immunomodulatory DC exposed to autoantigens can suppress experimental arthritis in an antigen specific manner. In this work, the authors describe the first human trials of an immunomodulatory DC therapy in rheumatoid arthritis.

  • For this form of immunotherapy, the autoantigen needs to have been identified. The authors studied patients with a particular subtype of rheumatoid arthritis (RA). RA patients with particular HLA-DRβ 70-74 alleles frequently have disease specific auto-antibodies specific for citrullinated peptide antigens (ACPA or anti-CCP).
  • The authors modified the patient’s own DC with a nuclear factor κB (NF-κB) inhibitor, a modification that prevented DC from maturing, and then exposed these cells to four separate citrullinated peptide antigens. A single dose of these immature, antigen pulsed DC were then injected into the skin of patients. 18 patients were treated and 16 untreated RA patients were included as controls.
  • The injection was well-tolerated and resulted in reduced numbers of effector T cells and an increased regulatory/effector T cell ratio. Serum levels of IL-15, IL29, CX3CL1 and CXCL11 also declined. In treated patients, in vitro T cell production of IL-6 was decreased in response to stimulation with a citrullinated peptide antigen.
  • Patients with quiescent disease did not flare with this therapy and patients with active disease significantly improved within one month of treatment.

In this manuscript, the authors describe a first-in-human phase I study of the safety and efficacy of a novel DC based immunomodulatory therapy. Results from this initial trial are promising. Patients tolerated the therapy well and a single injection resulted in measurable decreases in inflammation indices as well as clinical improvement in patients with active disease. The goal of this type of therapy is to establish stable tolerance to autoantigens in the hope of suppressing inflammatory diseases long-term. Further studies are needed to determine how long improvement will last and if additional injections can further suppress the disease. If successful, this approach could be utilized in other autoimmune diseases where the autoantigens have been clearly defined.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


Genetic Variants Driving Increased NFκB Signaling are Present in a Subset of Multiple Sclerosis Patients

A review of William J. Housley, et al. Genetic variants associated with autoimmunity drive NFkB signaling and responses to inflammatory stimuli. Sci. Transl. Med. 7, 291ra93. 2015. PMID: 26062845

The nuclear factor κB (NF-κB) transcription factor is a major regulator of inflammatory pathways. Some of the susceptibility genes for patients with autoimmune disorders map to the variants in the NF-κB pathway or to binding sites for NF-κB. In this manuscript, the authors studied patients with multiple sclerosis (MS) whose susceptibility genes localized to NF-κB binding sites.

  • The authors studied MS patients with two distinct susceptibility gene variants, one located proximal to NFκB1 and one located in an intron of TNFRSF1A (TNFR1).
  • In both cases, the variants were associated with increased NF-κB signaling after stimulation of patient cells in vitro with TNFα
  • Both variants had increased degradation of the inhibitor of NF-κB α (iκBα), an important negative regulator of NF-κB and translocation of p65 NFκB into the nucleus.
  • One variant caused up to a 20-fold increase in the expression of the NFκB protein itself, and decreased levels of three separate proteins that negatively regulate NFκB inflammatory pathways.
  • Naïve CD4 T cells from patients with MS were found to have enhanced activation of NFκB signaling at baseline.

This study demonstrates that two genetic variants associated with MS result in increased NFκB signaling. This signaling decreased the inflammatory threshold in CD4 T cells and may have made these patients more susceptible to autoimmune diseases. Because autoimmunity in these patients may be driven by inappropriate activation of the NFκB signaling pathway, drugs that target this pathway may be useful in treating this subset of patients. This work suggests that screening for NFκB activating genetic variants may identify patients that could improve with NFκB or cytokine blocking therapies.

Reviewed by Rachael A. Clark, MD, PhD, Brigham and Women's Hospital


Susceptibility to Herpes Simplex Encephalitis in the Setting of IRF3 Deficiency

A review of Andersen et al.  Functional IRF3 deficiency in a patient with herpes simplex encephalitis. J Exp Med 212:1371-9. 2015. PMID: 26216125

The discovery of single gene mutations underlying susceptibility to unusual or severe infections has provided a myriad of new mechanisms underlying primary immunodeficiency. Herpes simplex encephalitis has been closely studied and a number of mutations have been identified in anti-viral pathways, including TLR3 and many of its downstream factors leading to type I interferon signaling. This study focuses on a patient with a personal history of HSE and her father and identifies a novel genetic susceptibility to HSE.

SUMMARY

  • The proband and her father had whole exome sequencing performed and a heterozygous mutation in IRF3 was identified both in the patient and her (asymptomatic) father. Polyphen, SIFT and CADD predicted a likely deleterious effect of the mutation computationally. They specifically assessed the genes previously implicated in HSE and no mutations were noted in that gene list.
  • The patient’s IRF3 protein level was found to be normal
  • Pathogen associated molecular patterns (PAMPs), including poly !:C, were used to stimulate patient cells, with a decrease in type 1 IFN production from patient cells. 
  • Given that the specific susceptibility in this otherwise healthy patient was to HSE, PBMCs from the patient and controls were infected with HSV-1, HSV-2 (as well as HHV8 and IAV) and it was noted that type I IFN production was significantly reduced to HSV-1 infection with some reduction to other human herpes viruses but intact IAV responses. 
  • Using IRF3-deficient HEK293T cells they were able to show that the patient IRF3 mutation is not phosphorylated upon stimulation (with virus or upstream pathway members) and does not dimerize.
  • With regards to mechanism, they were able to demonstrate normalization of type I IFN function in patient fibroblasts with introduction of WT IRF3, and support a haploinsufficiency model rather than a dominant negative model for the effect of the mutation. 
  • They link their single patient molecular analysis to the updated Koch’s postulates proposed in a recent paper by Cassanova et al., which describes criteria for linking a single gene mutation in a single patient to a phenotype: the novel genotype must not occur in patients without the phenotype, the genetic variant has to have a deleterious effect on the gene product and the link between the genetic variant and the phenotype has to be recapitulated in a cellular or animal model.

This study yields a novel link between a key anti-viral innate immune molecule (IRF3) and HSE and is consistent with the fascinating literature surrounding genetic susceptibility to HSE. The points raised by the referenced recent paper by Cassanova et al. are a very useful metric for assessing new single mutations linked to susceptibility to human disease and this study compares their own evidence to this standard. While they do show a deficiency in type I IFN (both to HSV-1 and upstream innate pathway members) with patient cells and can rescue the type I IFN response with WT IRF3, it is concerning that the father is asymptomatic with the same heterozygous mutation (which, though novel, is very rare). Andersen et al. comment that mutation is not fully penetrant. This leaves open the possibility that the mutation in IRF3 is not causative, though the battery of evidence that they present is intriguing. In vivo studies of this mutation, perhaps using a CRISPER-Cas mouse model of the patient mutation and HSV-1 infection would be fascinating. The methodology of explicitly applying the newly suggested model for assessing the stringency of the connection between a genetic variant and a clinical phenotype is a good model for future studies of this nature.

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia


T Cell Differentiation Skewing and Asthma

A review of Choy et al.  Th2 and Th17 inflammatory pathways are reciprocally regulated in asthma. Sci Transl Med  7: 301ra129. 2015. PMID: 26290411

Once considered a monomorphic disorder, asthma has become very nuanced, with a wide range of studies examining variably defined and categorized phenotypes of asthmatics. Our understanding that asthma is an umbrella diagnosis that encompasses a number of subtypes with distinct mechanisms is exciting, both to better understand those mechanisms and for the potential to better target therapeutics. Where once a one-size-fits-all model of asthma management was widely accepted, there is now a growing appreciation that we are moving rapidly towards more personalized therapy in this disorder.

In this study, Choy et al evaluated endobronchial tissue gene expression signatures for Th2 and Th17 immune function to better understand the relative contributions of these two cell types.

SUMMARY

  • They began by using a Th2 (IL-13) and Th17 (IL-17a) cytokine stimuli to define resulting gene expression profiles in bronchial epithelial cells. A previously generated Th2 gene list was confirmed and a gene list focused on neutrophil recruitment was optimized by upregulation upon IL-17a and TNFa treatment. 
    • Of note, IL-13 suppressed IL-17a induced transcripts
  • Previously microarrayed endobronchial biopsies were then assessed for Th2 and/or Th17 signature expression
    • While most samples were negative for IL17a or IL13, when either was present it was correlated with its respective signature
    • There was a negative correlation between the Th2 and Th17 signatures
    • Patients were classified into Th2 high, Th17 high or Th2-Th17 low.
  • Though the Th17 signature is focused on neutrophil chemotaxis-related chemokines, they found that PBMCs from patients with Th2 and Th17 groups had eosinophilia, and neither had a statistically significant enrichment of neutrophils.
    • Interestingly, the Th17 signature was only seen in moderate or severe asthmatics
  • In a house dust mite (HDM) model of murine asthma, blockade of IL-4 and/or IL-13 led to a Th17 skewing, as did steroid treatment. Finally, treatment of a HDM murine asthma model with both anti-Th2 (anti-IL13) and anti-Th17 (anti-IL-17) showed both clinical improvement and did not show the Th17 skewing seen when only Th2 cytokines were blocked.

This group was able to use local, endobronchial, tissue gene expression to define small gene signatures for two important immunophenotypes associated with asthma (and many other conditions): IL-13 (called Th2) and IL-17a (called Th17). While different genes could be suggested as components of these signatures, the fact that these signatures completely segregated into two groups of patients (and one group with neither) is intriguing. The link between corticosteroids and induction of IL-17 induced genes will lead to further studies. The idea of dual blockade, anticipating the effect of removing a counter-regulatory force when only one of the two is blocked therapeutically, is an interesting strategy. It is important to note that different subsets of innate lymphoid cells (ILCs) are important sources of IL-13 and IL-17a in the lung in both humans and mice, so linking the responses of this tissue to those cytokines cannot be exclusively linked to T cell skewing. This focus of this article on identifying small sets of genes that could theoretically be used to prospectively identify patients for therapeutic trials (and later for therapy itself), along with elucidating inhibition of some of the core pathways in play, is both pragmatic and productive, with many subsequent experiments that can be anticipated in the near future.

Reviewed by Sarah Henrickson, MD, PhD, Children's Hospital of Philadelphia 


Cell Penetrating Peptides Deliver Cancer Antigens

A review of Derouazi, M. et al. Novel Cell-Penetrating Peptide-Based Vaccine Induces Robust CD4+ and CD8+ T Cell-mediated Antitumor Immunity. Cancer Res. 75:3020-31. 2015. PMID:26116496

Peptide vaccination holds enormous potential as an immunotherapy because of its low side effect potential and simplicity of administration. Murine studies and early clinical trials, however, have indicated that peptide vaccination will likely require combination with other immunomodulatory treatments to be clinically useful. Derouazi et al describe another approach, in which peptide antigens are attached to a virus-derived cell penetrating peptide (CPP), which delivers the antigen to the antigen presentation pathways of dendritic cells. The authors show that:

  • CPPs easily cross into the cytosol. Even when bound to bulky reporter proteins, 80% of the dendritic cells were transduced when exposed to only 900nM concentrations of the CPP-reporter construct.
  • Vaccination with a CPP-OVA construct and an adjuvant (a TLR3 agonist) induced a robust CD8 T cell response in mice. With a 4 vaccine treatment regimen, this response was still strong after 6 months. 
  • Multiple peptides can be delivered to cells in a single construct, and both MHC class I and II can be targeted because CPPs are able to cross the membrane directly into the cytosol or through endocytic vesicles. 
  • In an orthotopic glioma model, vaccination with a CPP conjugated to 5 antigens doubled the percentage of CD8 cells found in the tumor and increased their cytokine production, while simultaneously lowering the abundance of Tregs and MDSCs. 
  • Vaccination with a CPP-antigen construct improved survival in 4 different murine tumor models. It was particularly successful in the glioma model, in which 7/8 mice treated showed complete tumor regression at the conclusion of the experiment.

Derouazi et al. describe a novel approach to the growing field of anti-cancer peptide vaccination. By conjugating the antigens to viral peptides evolved to access DCs, robust CD4 and CD8 responses can be generated to any viable epitope. The authors demonstrate the potential of this new approach in a variety of cancer models, with astonishing success. Further investigation will show if any tumor antigen can be successfully delivered to induce these dramatic immune effects, but this new technique provides a promising new avenue for enhancing the potency of peptide-based cancer vaccines.

Reviewed by Alexander Hopkins, Johns Hopkins University, Cellular and Molecular Medicine Program and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center


Nanoparticle-carrying T Cells for Cancer Chemotherapy

A review of Huang, B. et al. Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells. Science Translational Medicine 7:291. 2015.PMID: 26062846

Disseminated lymphoma reservoirs are found in lymphoid tissues, including lymph nodes, where chemotherapeutic agents do not effectively accumulate. Furthermore, poor pharmacokinetics of potent drugs require large doses that result in off-target toxicity without maximizing drug efficacy. Nanoparticles can improve drug half-life, resulting in better accumulation at tumor sites, but are still dependent on leaky vasculature associated with tumors. Lymphocytes naturally circulate through lymphoid tissue and thus polyclonal, non-tumor-antigen T cells could be effective targeting molecules for lymphoma reservoirs that cannot be reached by normal chemotherapeutic infusion. Huang et al. have already shown that nanoparticles can be covalently tethered to T cell membranes without hindering their function. Using a malignant B cell line derived from Eμ-myc Arf-/- mice (Eμ-myc cells) to model Burkitt’s lymphoma, they show that nanoparticles containing the active form of the topoisomerase I inhibitor irinotecan, SN-38, can be conjugated to lymphocytes, which then circulate to lymphoma reservoirs in lymph nodes and result in tumor lysis.

  • In vitro culture of Eμ-myc cells with SN-38 resulted in near complete tumor cell lysis at 10 ng/mL. However, when SN-38 was administered intravenously at 10 mg/kg, less than 0.05% could be detected in the lymph nodes. When SN-38 was encapsulated in controlled-release lipid nanoparticles (SN-38 NCs), up to 100 ng/g tissue could be detected in the serum, liver, and spleen; however, the concentration found in the lymph nodes was 10-fold lower.
  • T cell trafficking to lymph nodes is dependent on CD62L and CCR7. During ex vivo expansion of bulk T cells using IL-2, CD62L expression was down-regulated via mTOR signaling. The addition of rapamycin preserved CD62L expression on the surface of both CD4+ and CD8+ T cells, as well maintaining expression of additional homing receptors (CXCR4 and integrins α4β7, β1, and β2), thus mimicking homing receptor expression found on circulating T cells and resulting in efficient trafficking to lymphoid tissues. When treated with 10 ng/mL SN-38, the dose found to effectively eradicate Eμ-myc cells, minimal loss of T cell viability was observed. 
  • In vitro culture of nanocapsules conjugated to T cells (NC-T) resulted in selective killing of tumor cells at ratios as low as 1 NC-T cell to 20 tumor cells, while minimal T cell death was observed. When compared to SN-38 administered in nanocapsules, adoptively transferred NC-T cells resulted in accumulation of 63-fold greater levels of SN-38 in tissues at 20 hours post-administration, and substantial levels of drug could be detected at 4 days post-administration.
  • Adoptive transfer of NC-T cells at ratios estimated to result in 1 NC-T to 5 tumor cells in lymph nodes (within clinically accepted doses for adoptive T cell therapies) significantly improved survival of tumor-bearing mice by a median of 12 days when compared to mice treated with IL-2/rapamycin expanded T cells, free SN-38, or encapsulated SN-38. Moreover, no toxicity resulting from NC-T therapy was observed, as measured by animal weight and liver enzyme levels.

Active chemotherapeutic targeting strategies aim to increase drug concentrations at the site of tumor while reducing off-target toxicities. While encapsulating drugs in nanoparticles does result in better pharmacokinetics, accumulation in tumors is dependent on the leaky tumor vasculature that occurs during abnormal angiogenesis and nanoparticles may not fully disseminate throughout the tumor. Nanoparticles may also become trapped in the perivascular space, where they are ineffective at causing tumor cell death. By harnessing natural T cell trafficking mechanisms, Huang et al. were able to show organ-specific targeting of a highly-potent drug with poor pharmacokinetics, with no observed off-target toxicities. While this study did not focus on tumor-specific targeting, this approach could likely be applied to cancers where tumor-specific T cells can be isolated.

Reviewed by Heather Kinkead, Johns Hopkins University, Cellular and Molecular Medicine Program and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

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Reversing Lupus by Altering T Cell Metabolism

A review of Yin Y, S-C Choi, Z Xu, DJ Perry, H Seay, BP Croker, ES Sobel, TM Brusko, L Morel. Normalization of CD4+ T cell metabolism reverses lupus. Sci. Transl. Med 7: 274ra18 (2015). PMID:25673763

Recent studies have highlighted a role for both glycolysis and mitochondrial metabolism in regulation of immune cell function. However, whether these metabolic programs are deregulated in the autoimmune disease systemic lupus erythematosus (SLE) is less clear. Prior work by Morel and colleagues had shown that the murine SLE susceptibility loci Sle1c2 mapped to decreased expression of Esrrg, which regulates mitochondrial oxidative phosphorylation. In their current work, Yin, Morel, and colleagues test the hypothesis that altered cellular metabolism contributes to SLE pathogenesis through T cell activation and that this can be reduced with metabolic modulators. They address this issue through a detailed analysis of CD4+ T cell metabolism in two distinct mouse models of SLE as well as in primary human SLE patient samples. There are several interesting findings from this work.

  1. Both glycolysis and mitochondrial oxygen consumption were elevated in naïve CD4+ T cells from SLE prone mice relative to healthy controls. This is observed prior to disease onset and amplified with age and disease onset. Enhanced metabolic activity correlated with increased activity of mTORC1 and could be inhibited with the TORC1 inhibitor rapamycin.
  2. In vitro treatment with the mitochondrial electron transport chain complex I inhibitor metformin or the glucose metabolism inhibitor 2-deoxy-D-dlucose (2DG) normalized interferon gamma production. Interestingly, metformin was more effective in the early stages of T cell activation while 2DG was more effective during the proliferative phase, suggesting that distinct metabolic pathways predominate during different phases of T cell activation.
  3. Mice from two different SLE models and wild-type controls were treated with vehicle or metformin and 2DG in combination after the onset of SLE. Metabolic modulation resulted in reversal of the SLE phenotype including a decrease in splenic size, T cell activation, and production of autoantibodies. Importantly, blood glucose levels, body weight, and total serum IgM and IgG were unaffected by the treatment in either control or SLE mice.
  4. Consistent with the in vitro studies, combination therapy with metformin and 2DG was superior to treatment with either agent alone. This further supports distinct roles for glycolysis and mitochondrial metabolism in SLE pathogenesis.
  5. Analysis of metabolism in CD4+ T cell subsets from SLE patients and healthy controls recapitulated the findings in mice with elevated glycolysis and oxidative metabolism in patients versus controls. This could be reversed in vitro with exposure to metformin.

Taken together, these results strongly support a role for deregulation of glycolysis and mitochondrial metabolism in SLE and suggest that these could be points for therapeutic intervention. Given that agents that alter metabolism are already approved, translating these findings into randomized clinical trials should be relatively straightforward. While focusing on CD4+ T cells is logical given their central role in providing T-cell help to autoreactive B cells, it will be interesting to see whether deregulation of these same pathways is important in B cells and/or myeloid lineage cells in SLE. Basic understanding of the relative roles of metabolic deregulation in SLE will be important for design of biology correlative studies in trials aimed at restoring metabolic balance in these patients.

Reviewed by Michelle L. Hermiston, MD, PhD, University of California, San Francisco

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Identifying a Genetic Basis for Life-Threatening Infection with Influenza

A review of Ciancanelli MJ, et al. Life-threatening influenza and impaired amplification in human IRF7 deficiency. Science, 24 April 2015. PMID:25814066

The response to the influenza virus can range from mild to life threatening. The mechanistic basis for these variable responses of human patients to common viral infections has been poorly understood. Interferon (IFN) production and amplification is an essential part of the anti-viral immune response, and low or loss of IFN production can lead to severe infection or even death of the host. Ciancanelli and colleagues hypothesized that severe infection in an otherwise healthy child might could be due to a underlying genetic defect. They thus sequenced the genome of a previously healthy child hospitalized with H1N1 influenza and acute respiratory distress syndrome (ARDS). This paper demonstrates several interesting findings.

  1. Sequencing of the patient's genome revealed two inherited mutations, a previously unreported missense mutation (F410V) and a low frequency nonsense mutation (Q421X) in the transcription factor interferon regulatory factor (IRF) 7.
  2. Functional studies indicated both mutations resulted in a loss-of-function phenotype, but by different mechanisms. IRF7Q421X could localize to the nucleus without phosphorylation, while IRF7F410V could not localize to the nucleus in spite of being phosphorylated. Neither mutation exhibited a dominant-negative phenotype with IFR7 homo- or IRF3 heterodimers.
  3. At baseline, the patient's innate immunity gene profiles were down regulated. Upregulation of the IRF-7 targets, type I and III IFNs, were defective in response to stimulation. In contrast, interferon stimulatory genes (ISG) and IL-6 production were normal, likely due to pathway redundancy by IFN-β.
  4. Plasmacytoid dendritic cells constitutively express IRF7, and thus are major producers of type I interferons. The patient's PBMCs and pDCs however produced no IFN-α2 after infection with influenza H1N1 or with herpes-simplex virus-1. This was attributed to the Q421X mutation, as the Q421X-carrier parent PBMCs produced similar results. Time courses showed a transient upregulation of ISGs by IFN-β that was IRF7-independent). However, sustained upregulation was abolished by the mutations correlating with increased viral titers and suggesting a requirement for IRF7 in controlling viral load.
  5. The patient's fibroblasts were immortalized and showed the same decreased IRF7 mRNA expression post infection as the isolated primary immune cells, resulting in increased H1N1 and H5N1 viral loads, indicating it isn't a strain specific response. This phenotype could be rescued by exogenous treatment with IFN-α2b, dsRNA stimulation (IRF3-dependent), or exogenous IFR7 expression.

Taken together, this paper has several interesting insights into IFR7-dependent anti-viral responses. Perhaps more importantly, it adds to the growing body of evidence that atypically severe infections to common pathogens may have a genetic basis, at least in children and young adults. With the advent of affordable and rapid whole genome sequencing, defining the genetic basis for these diseases may enhance our understanding of the immune response to human disease and reveal potentially actionable targets for therapeutic intervention.

Reviewed by Melissa A. Ruck and Michelle L. Hermiston, MD, PhD, University of California, San Francisco

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Highlights From Clinical Immunology, the Official Journal of FOCIS

TB and HIV

A review of: B Siddiqui, S. et al. Tuberculosis specific responses following therapy for TB: Impact of HIV co-infection. Clinical Immunology. 159: 1–12. 2015. PMID: 25889622.

CD4+ Th1 responses are essential for immune control of mycobacterium tuberculosis (MTB) infection. Not surprisingly, HIV infection impairs protective immune responses to MTB, and it is possible that MTB infection exacerbates HIV disease. The combined influences of HIV and MTB coinfection on the immune system are very complex and poorly understood. In this study, the authors looked at TB specific T cell responses in people in Mali with TB infection alone or with both MTB and HIV infection. Four groups were enrolled in the study including a) 20 Healthy HIV negative and tuberculin skin test negative (HIV- TST-) subjects, (b) 19 HIV positive and TST negative subjects (HIV+), (c) 36 active pulmonary tuberculosis (HIV- MTB+) subjects ; and (d) 26 HIV+ and pulmonary tuberculosis (HIV+ MTB+) subjects. Sputum was collected from TST+ patients to identify patients with active pulmonary MTB, and HIV infection was confirmed by ELISA and Western blot. All patients were started on MTB therapy after enrollment. Some patients were already on HAART before enrollment, and some started after. The median baseline CD4+ T cell count of the HIV infected subjects was 151 cells/mm3. The subjects were followed at 4, 13 and 52 week intervals. Analyses included T cell activation markers, in vitro T cell responses to MTB, and plasma cytokines. Some of the main findings were:

  • The absolute numbers and percentages of CD4+ T cells were significantly lower in HIV+ TB+ subjects compared to single-infected MTB+ subjects.
  • Absolute counts and percentages of CD8+ T cell counts were significantly higher in the HIV+ MTB+ subjects compared to MTB+ subjects throughout the study period.
  • Both HIV+ and HIV- MTB-infected groups experienced an increase in the absolute number and percentage of activated CD4+ T cells in peripheral blood upon initiation of MTB treatment.
  • Absolute numbers and percentages of γδT cells were significantly lower in HIV+ TB+ subjects when compared to TB+ with at all time points.
  • The percentages of activated T cells (HLADR and CD38 expression by CD4+ and CD8+ T cells) was persistently higher in HIV+ MTB+ vs HIV++ MTB- subjects.
  • Serum levels of IFNγ and TNF levels were comparable in HIV+ TB+ vs HIV+ TB- subjects 
  • The absolute number of PPD responding cells was lower in HIV+ MTB+ subjects vs. MTB+ subjects, but the % responding T cells (IFNγ+, and proliferating cells), were similar in both groups.

Overall this study shows that MTB/HIV co-infected have persistent immune activation before and after starting treatment for MTB. Furthermore MTB-specific T cell response are maintained in co-infected individuals. Given the lower CD4+ counts in the dual infected patents, this study corroborates the idea that T cell activation in response to MTB exacerbates progression of HIV disease. One significant drawback to this study is that some patients were receiving HAART therapy at onset, and some were started on HAART at different times after initiation of the study, making the HIV+ group quite heterogeneous. The authors point out the importance of supporting sophisticated research on this topic in resource limited countries with extensive HIV/TB morbidity and mortality.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital


The Seedy 4 Side of Memory Stem Cells

A review of Takeshita M. et al. Polarization diversity of human CD4+ stem cell memory T cells. Clinical Immunology 159:107–117. 2015.PMID 25931384.

Recently, a new twist on T cell memory has emerged with the identification of a human memory T cell population which has features of stem cells, including enhanced self-renewal and multipotentiality, and the ability to differentiate into central memory, effector memory and effector T cells (Gattinoni L et al. Nat Med. 2011 PMID: 21926977). Although these memory stem cells share many markers of naïve T cells (e.g. CCR7, CD45RA, CD62L) they are distinguished by expression of CD95, IL-2Rβ, CXCR3, and LFA-1, and by their abilities of rapid cytokine production and reconstitution of memory responses. There has been interest in this subset with respect to vaccine design and tumor immunity. Most of the previous work on these memory stem cells has focused on CD8+ T cells. Takeshita et al. report on characteristics of human CD4+ memory stem cells. Using human blood cells and flow sorting, they isolated and interrogated different developmental stages of CD4+ memory T cells, including naïve (Tn), stem cell memory (Tscm), central memory (Tcm) and effector memory (Tem). Th1, Th2, Th17, and Tfh polarization was analyzed in each of the memory subsets, using microarray analysis, RT-PCR, and cytokine protein analyses in unstimulated cells and ex vivo Th subset differentiation cultures. Some of the major findings include:

  • Tscm cells can be placed at a distinct developmental stage between Tn and Tcm.
  • Each CD4+ memory developmental stage shows a distinct pattern of Th subset polarization, with acquisition of multiple polarities during differentiation. 
  • The ability to change subset polarity, i.e. plasticity, differed between the developmental stages.
  • CD4+ Tscm proliferated and produced cytokines at similar levels as Tcm.
  • Th17 were derived from CCR6+ Tscm, in addition to Tcm and Tem fractions, but not Tn.

Overall, the results of these analysis provide new information on the degree of Th subset polarization and multipootentiality of different subsets of memory T cells. The authors conclude that CD4+ memory T cell differentiation progresses as Tn → Tscm → Tcm → Tem. They show that Tscm look like Tcm and Tn cells, and provide parameters by which to distinguish them in future studies.

Reviewed by Andrew H. Lichtman, MD, PhD, Brigham and Women’s Hospital

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Human Immunophenotyping Update

Increasing the Number of Flow Cytometry Parameters

Holden T. Maecker, PhD, Stanford University School of Medicine

We are now a few years into the mass cytometry revolution, i.e., the explosive growth of the CyTOF platform (Ornatsky et al. 2010; Bendall et al. 2012) as an alternative to fluorescence flow cytometry. Compared to conventional flow cytometry, CyTOF offers many more parameters (currently over 40) with little or no spillover between detector channels. This is achieved by the replacement of fluorescent labels with heavy metal ion tags, and readout by time-of-flight mass spectrometry. Despite some trade-offs in sensitivity, collection speed, and cell recovery, this promises to be the most radical change in multiparameter flow cytometry technology to date.

However, there has been another, quieter revolution going on these last several years as well, involving the increase in available fluorescence flow cytometry dyes. At its core, this revolution began with the discovery of so-called π-conjugated polymers, which have tunable fluorescence properties based on their chemical structure, as well as repeated optical subunits, resulting in unparalleled versatility and brightness (Chattopadhyay et al. 2012).

The first reagents built with π-conjugated polymers were excitable with violet lasers (approximately 405 nm emission) and sold under the trade name “Brilliant Violet” (BV). Beginning with BV421, a series of these dyes and tandems built from them (Table 1) created highly desirable alternatives to quantum dot semiconductors (Qdots) (Chattopadhyay et al. 2006). Qdots represented the only other class of fluorochromes to offer multiple violet-excited options that make full use of a violet laser. But two historical issues associated with Qdots, aggregation and cross-laser excitation, are of much less concern with the Brilliant Violet dyes. Together with their intrinsically high signal-to-noise ratios and relatively low spillover characteristics, the BV dyes have quickly become favorites of flow cytometrists. Some of these dyes, in particular BV421, can rival PE in terms of brightness. And finding another fluorochrome as bright as PE is kind of like finding another planet the size of Jupiter in our solar system.

But the revolution does not stop there. By tuning the excitation characteristics of these dyes, Brilliant Blue (BB) and Brilliant UV (BUV) classes of fluorochromes have also been created (Table 1). BB515 is a brighter alternative to FITC. BUV dyes (Stall et al. 2013) range in emission peak from 395 to 805 nm, creating a whole family of dyes for those in possession of cytometers with UV lasers. No doubt many retrofits are being done now to take advantage of these BUV reagents.

What is the impact to those who are casual users of flow cytometry? Basically, the effect has been to bring 14+ color flow cytometry into the realm of “easy” as opposed to “expert”. The only requirement is a cytometer with a properly-equipped set of violet laser detectors, and preferably also UV-laser detectors. Even without a UV laser, 13-14 colors become quite doable; with a UV laser, it is more like 17+ colors. And while panel design is still an important consideration, the individual spillovers involved are much less daunting than in the days before BV and BUV dyes. Also, for experiments involving perhaps only 6-8 colors (including sorting experiments), the new dyes allow for more flexibility and greater distribution of reagents across lasers. This in turn leads to less overall spillover, a greater ability to include dump channels and ancillary markers, and thus higher sort purity.

Surely there are some caveats to the use of these new dyes. The main one is that there is a propensity for multiple polymer-based conjugates to form aggregates, causing variable compensation issues. This is mostly avoided by use of a staining buffer designed for these reagents, which blocks such heteroclitic interactions. Lyophilization of multiple polymer-based conjugates in a single cocktail may still be an issue, however.

With so much happening in the fluorescence world, one can ask: Which is the better investment, a high-end fluorescence flow cytometer or a CyTOF? The answer clearly depends upon your priorities. Fluorescence still cannot match the number of parameters of CyTOF, and it does involve considerable compensation, even with the new dyes. But it is considerably faster and cheaper to run, with less cell loss. And at least some channels, like PE, APC, and BV421, offer greater sensitivity than is possible by CyTOF. While the latest version of the CyTOF platform (Helios) has made modest gains in cell recovery, sensitivity, and collection speed, it still doesn’t match fluorescence flow cytometry for these properties. But if you want to analyze 40 or more parameters in parallel, without compensation, then you need CyTOF.

Table 1. New flow cytometry dyes based on tunable fluorescence polymers

MaeckerTable1

References:

  1. Bendall, S.C., Nolan, G.P., Roederer, M. and Chattopadhyay, P.K. 2012. A deep profiler’s guide to cytometry. Trends in Immunology 33(7), pp. 323–332.
  2. Chattopadhyay, P.K., Gaylord, B., Palmer, A., Jiang, N., Raven, M.A., Lewis, G., Reuter, M.A., Nur-ur Rahman, A.K., Price, D.A., Betts, M.R. and Roederer, M. 2012. Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments. Cytometry A 81(6), pp. 456–466.
  3. Chattopadhyay, P.K., Price, D.A., Harper, T.F., Betts, M.R., Yu, J., Gostick, E., Perfetto, S.P., Goepfert, P., Koup, R.A., De Rosa, S.C., Bruchez, M.P. and Roederer, M. 2006. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12(8), pp. 972–977.
  4. Ornatsky, O., Bandura, D., Baranov, V., Nitz, M., Winnik, M.A. and Tanner, S. 2010. Highly multiparametric analysis by mass cytometry. J Immunol Methods 361(1-2), pp. 1–20.
  5. Stall, Gaylord, A., Liang, B., Uckert, Y., Leonard, F., Li, B., Tran, H., Bartholomew, L., Chen, G., Luan, Y., Widmann, F. and Stephanie 2013. Brilliant UltraVioletTM Dyes: A new set of high sensitivity fluorescence reagents for multicolor flow cytometry with a UV laser (P3301). The Journal of Immunology. Available at: http://www.jimmunol.org/cgi/content/meeting_abstract/190/1_MeetingAbstracts/211.8.

 

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Development in Basic Immunology and Novel Therapies 

Resident Memory T Cells: Relevance to Human Inflammatory Disease

Rachael A Clark, MD, PhD, Brigham and Women’s Hospital, Harvard Medical School

Epithelial barrier tissues such as the skin, gut, lung and female reproductive tract separate the body from the environment and are the most common sites of pathogen exposure. There are distinct populations of memory T cells in the circulation that migrate preferentially to each of these epithelial barrier tissues. It was previously assumed that the T cells responsible for protecting barrier tissues from infection remained in the blood until infections developed. Upon infection, T cells were thought to migrate into the affected tissue, clear the infection and then either undergo apoptosis or migrate back into the bloodstream. However, studies in both humans and mice have now demonstrated that a population of nonrecirculating tissue resident memory T cells (TRM) are generated by infections and remain in tissues long-term thereafter, providing rapid on-site immune protection against known pathogens.

There are few if any T cells in neonatal human skin but by the time we reach adulthood, there are 1 million memory T cells residing in every square centimeter of human skin. The skin surface of a healthy adult human being contains 20 billion T cells, nearly twice as many as are present in the entire circulation. About half of these T cells are now recognized to be nonrecirculating resident memory cells. Elegant mouse models have clarified how so many T cells end up in skin. Local skin infections with vaccinia and HSV led to the development of TRM that remained long term in the skin and provided potent protection against reinfection, even in the complete absence of circulating T cells. The highest number of protective TRM were found at the site of prior infection but highly protective TRM were also distributed to all areas of the skin surface, leading to global protection against the pathogen. Moreover, skin infection also led to significant seeding of the lung and gut with protective TRM, leading to partial protection of at least two other epithelial barrier tissues. This helps to explain the success of smallpox vaccination, which is delivered by skin scarification but leads to pulmonary protection against smallpox, which is an airborne pathogen. In other words, this cross dissemination of TRM from skin infection may underlie the significant pulmonary protection afforded to smallpox vaccinated patients.

Mouse studies in the lung, gut and reproductive tract have demonstrated that similar populations of TRM also develop in these tissues following local infections. Moreover, TRM persist long-term, do not recirculate and persist in the absence of antigens. TRM have a distinct transcriptional signature that differentiates them from circulating T cells. These studies were critical in demonstrating that TRM are a unique, stable and distinct differentiation state. In general, these studies find that TRM can provide rapid and effective protection against known pathogens in epithelial barrier tissues and that circulating T cells provide a slower but more flexible response. Circulating T cells migrate through peripheral lymph nodes and can therefore detect new infections at tissue sites that are not yet seeded by TRM. The immune responses initiated by circulating T cells, although slower to get started, eventually also lead to seeding of the affected peripheral tissue with TRM.
A recent study demonstrated that skin infection, contact dermatitis and delayed type hypersensitivity responses all led to the generation of roughly equal numbers of recirculating T cells and skin TRM. This observation puts to rest the previous theories that the fate of a memory T cell depends on its antigen affinity or other stochastic factors. The equal generation of both recirculating and resident memory cells appears to be a fundamental mechanism by which the immune system elegantly and equally distributes immunity both to the circulation and to peripheral tissues such as the skin.

Parallel studies in humans have demonstrated the importance of TRM in human immunity. Patients with leukemic cutaneous T cell lymphoma (CTCL) treated with alemtuzumab, a medication which depletes all circulating and recirculating T cells but spares skin resident T cells, demonstrated that a population of nonrecirculating highly protective TRM existed in human skin that protected against infection even in the complete absence of circulating T cells. Likewise, HSV specific T cells persisted long-term in HSV exposed individuals, proliferated locally in response to early HSV reactivation and were capable of killing virus producing keratinocytes and suppressing viral reactivation when these T cells were present in adequate numbers within the skin.

Although TRM play an important role in protecting barrier tissues from infection, these cells can also undergo malignant transformation or become autoreactive, thereby becoming part of the problem instead of part of the solution. Two human inflammatory diseases, psoriasis and mycosis fungoides-a subtype of cutaneous T cell lymphoma-have now been identified as being caused by TRM. Given how accessible the skin is to study, it is perhaps not surprising that both are inflammatory skin diseases. These disorders are both characterized by inflammatory skin lesions that appear to completely resolve with therapy but then often recur in the same anatomic locations once therapy is discontinued. Studies in both psoriasis and mycosis fungoides have demonstrated that TRM are present in both active and clinically resolved skin lesions. This highlights the fact that most conventional therapies probably do not eradicate pathogenic T cells but instead simply suppress the activation of these cells. Withdrawal of active therapy then allows T cells to reactivate and re-initiate inflammatory lesions at the same sites. Although no other human autoimmune and inflammatory conditions have been definitively identified as being mediated by TRM, several human diseases have clinical characteristics that suggest TRM may play a pathogenic role. For example, one characteristic of TRM mediated skin lesions is a well-defined area of inflammation with an abrupt cutoff leading to normal tissue. This type of well-defined inflammatory lesion is observed in Crohn’s disease, where inflammatory lesions, so-called “skip lesions” alternate with normal appearing mucosa. Another characteristic of TRM mediated diseases is the tendency of inflammation to recur at previously affected sites. Patients with rheumatoid arthritis have only a certain subset of joints affected by their disease and the particular joints affected are different in each individual. However, when rheumatoid arthritis flares, it flares reliably in previously affected joints. This clinical behavior suggests that TRM may remain resident in affected joints when the disease is quiescent and that reactivation of these cells leads to the flaring of disease. Future studies are needed to isolate and study the phenotype of T cells from the affected tissues of patients with autoimmune and inflammatory diseases in order to definitively determine which human diseases have a TRM mediated component.

TRM have a unique biology. They persist long-term in peripheral tissues in the absence of antigen but maintain readiness to rapidly respond when antigen is re-encountered. The signals that induce T cells to undergo differentiation into TRM are not yet characterized. More importantly, the signaling pathways that allow these TRM to remain in a state of readiness for long periods of time in the absence of antigen are completely uncharacterized. Identification of these signals is a high priority because this information could lead to the development of two new classes of therapeutics. Drugs that enhance the formation of TRM could be useful in improving vaccination strategies and antitumor immune responses. In contrast, therapeutics that block TRM survival signals could be useful in chronic inflammatory diseases where these cells play a pathogenic role. In fact, drugs that kill TRM potentially represent a cure for what are otherwise chronic recurrent inflammatory diseases.
In summary, TRM are highly protective, nonrecirculating long-lived memory T cells that populate epithelial barrier tissues and evolutionarily, allowed immunity to build up in the barrier tissues where pathogen re-exposures were most likely. However, the very biology that makes TRM so effective in fighting infections makes them difficult to control when these T cells become autoreactive or otherwise contribute to inflammatory diseases. Future studies are needed to identify which human autoimmune and inflammatory diseases have a TRM component and a better understanding of TRM biology is needed to develop novel therapeutics that specifically target these cells.

References:

  1. Clark RA. Resident memory T cells in human health and disease. Science Translational Medicine 2015;7:269rv1.
  2. Park CO, Kupper TS. The emerging role of resident memory T cells in protective immunity and inflammatory disease. Nature medicine 2015;21:688-97.

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Selected Recent Clinical Trial Results

Submitted by Sandra Lord, MD, Benaroya Research Institute
Edited by Carla J. Greenbaum, MD, Benaroya Research Institute

A Phase 2 Trial of Guselkumab Versus Adalimumab for Plaque Psoriasis

Clinical Trial: Kenneth B Gordon, Kristina Callis Duffin, Robert Bissonnette, et al. A Phase 2 Trial of Guselkumab versus Adalimumab for Plaque Psoriasis, N Engl J Med 2015;373:136-44

Disease: Plaque Psoriasis

Intervention: Guselkumab (anti- IL-23 human monoclonal antibody) or Adalimumab (anti-TNF-α human monoclonal antibody) or placebo

Study design:

  • Phase 2, dose-ranging, randomized, double-blind, placebo-controlled active-comparator trial
  • 293 patients with moderate-severe plaque psoriasis treated for 40 weeks and follow up through 52 weeks.
  • Baseline clinical characteristics in all groups were similar
  • Random assignment to 1 of 3 treatment groups: guselkumab administered subcutaneously (n=208) in variable doses and dosing interval (5 mg at weeks 0, 4, then every 12 weeks, 15 mg every 8 weeks, 50 mg at weeks 0, 4, then every 12 weeks, 100 mg every 8 weeks, or 200 mg at weeks 0, 4, then every 12 weeks), placebo (n=42) through week 16 then crossover to 100 mg guselkumab, or standard dose adalimumab (n=43)
  • Primary endpoint: Physician’s Global Assessment (PGA) score of 0 or 1 at week 16. 
  • Secondary endpoints: 
    • Psoriasis Area and Severity Index (PASI) score with ≥75% improvement from baseline to 16 weeks
    • Decrease in Dermatology Life Quality Index (DLQI) score (which indicates improved QOL) at 16 weeks. 
    • Proportion of patients with PGA score of 0 or 1 at week 40

Results:

  • At week 16, proportion of patients with PGA score 0 or 1 was significantly higher in each guselkumab group than in placebo (P ≤ 0.002 for all comparisons) and higher in the 50, 100 and 200 mg guselkumab group than adalimumab group (P<0.05).
  • At week 40, proportion of patients with PGA score of 0 or 1 remained higher in 50, 100 and 200 mg guselkumab group than adalimumab group (P<0.05)
  • A modest loss of efficacy was seen in the guselkumab groups whose dosing interval was every 12 weeks rather than every 8 weeks. 
  • At week 16 (end of placebo controlled period,) AEs, SAEs and infections were similar in guselkumab, placebo, and adalimumab groups. 3 major adverse cardiovascular events seen in guselkumab groups, including one death from MI. Study agent was discontinued in 15% of the guselkumab group vs 26% of the adalimumab group.

Why the Trial is of Interest to the Broader FOCIS community:

In recent years, treatments of both plaque psoriasis and psoriatic arthritis have improved substantially, related to the increased understanding of the immune dysregulation of psoriasis, which is characterized by upregulation of type 1 helper (Th1) cells and the type 17 helper (Th17) cell signaling pathway. Agents that block IL-12 (and induction of Th1 cells,) IL-23 (which is required for maintenance of the Th17 phenotype), and IL-17 (which has proinflammatory effects in skin) can successfully treat psoriasis and/or psoriatic arthritis. The evolution of psoriasis therapy illustrates the benefits of more targeted systemic therapy; namely, greater efficacy and fewer risks and side effects. The earliest biologics used in psoriasis were the TNFα blockers etanercept, adalimumab and infliximab, which broadly block systemic inflammation. Therapy became more targeted with sekukinumab and ixekizumab, which block IL-17, and ustekinumab, which blocks the p40 subunit common to both IL-12 and IL-23. Guselkumab specifically targets IL-23. As reviewed in the paper, IL-23 blockade leads to loss of the Th17 phenotype and decrease in IL-17 level. Interestingly, neutrophils in psoriatic lesions have been shown to release IL-17 (1), and IL-23 has been shown to regulate the production of IL-17 in neutrophils (2), recent findings that suggest an additional possible benefit to IL-23 blockade in psoriasis. In the reviewed study, the investigators compared different doses and dosing intervals of guselkumab to placebo or to adalimumab. The superior efficacy of guselkumab over adalimumab (a TNFα blocker) suggests the relative importance of the IL-23 pathway in psoriasis pathphysiology, at least in comparison to broad blockade of systemic inflammation. There are no head to head comparisons of guselkumab to ustekinumab (IL-12/IL-23 blockade) or guselkumab to sekukinumab/ustekinumab (IL-17 blockade), which would provide an interesting comparision of targeted agents.
It’s unclear how durable the efficacy of guselkumab will be, or whether uncommon adverse effects may become evident with longer durations of therapy.

References:

  1. Lin AM, Rubin CJ, Khandpur R et al. Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis, J Immunol 2011;187:490-500
  2. Taylor PR, Roy S, Leal SM Jr, et al. Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2, Nat Immunol 2014;15:143-51

Apremilast for Behcet’s Syndrome—A Phase 2, Placebo-controlled Study

Clinical Trial: Gulen Hatemi, Melike Melikoglu, Recep Tunc, et al. Apremilast for Behcet’s Syndrome—A Phase 2, Placebo-Controlled Study, N Engl J Med 2015;372:1510-8.

Disease: Behcet’s Syndrome

Drug: Apremilast, an oral phosphodiesterase-4 inhibitor

Study design:

  • Phase 2 multicenter (3 in US, 3 in Turkey) double blinded placebo-controlled trial
  • 111 patients with Behcet’s syndrome with ≥ 2 oral ulcers 
  • Randomly assigned into 2 arms: 30 mg aprelimast BID or placebo for 12 weeks followed by 12 week extension during which placebo group switched to aprelimast. Follow up through week 28.
  • Primary endpoint: number of ulcers at week 12
  • Secondary endpoint: ulcer pain, number of genital ulcers, and QOL
  • Significant reduction in number of ulcers (0.5 vs 2.1, P<0.001) in apremilast group compared to placebo at week 12. Reduction evident by week 2 and sustained through week 24
  • Greater decline in pain as measured by a 0-100 scale (-44.7 vs -16.0, P<0.001) in aprelimast group. Baseline score was 54.3/51.7 in apremilast/placebo groups
  • Salutatory effect on clinical outcomes also seen when placebo group received apremilast at 12 weeks
  • Withdrawal of drug led to recurrence of ulcers
  • Nausea, vomiting, diarrhea more common in aprelimast group

Why the Trial is of Interest to the Broader FOCIS Community:

Behcet’s Syndrome is a rare condition characterized by oral ulcers and any of several systemic manifestations including genital ulcers, ocular disease, skin lesions, gastrointestinal involvement, neurologic disease, vascular disease, or arthritis. It appears to be a type of vasculitis, although the pathophysiology remains poorly understood. The prevalence is highest among populations living along the former Silk Road from the Middle East to Japan. The common clinical feature is recurrent painful oral ulcers, although the most specific manifestation is genital ulcers. First line therapy for mucocutaneous lesions is colchcine and/or topical corticosteroids, and for lesions that are resistant to colchicine, a number of other systemic agents have been used, including azathioprine, interferon-α, thalidomide, and TNF-α antagonists, all with limited or unclear efficacy. The greatest unmet clinical need in Behcet’s is for an effective therapy for painful mucocutaneous ulcers. Aprelimast is an oral small molecule inhibitor of phosphodiesterase-4, which leads to decreases in pro-inflammatory cytokines such as TNF-α, IL-23, IFN-γ, and increases in anti-inflammatory cytokines such as IL-10, and therefore, is a fairly broad anti-inflammatory agent. It has been FDA approved for use in plaque psoriasis (1,2) and psoriatic arthritis (3), but its efficacy for these conditions is not as high as compared to the efficacy of the IL-17/IL-23 agents.

The treatment of Behcet’s is limited by the lack of high quality data on effective therapies. Clinicians often choose therapies based on anectodal information or use therapies empirically and off-label. Although colchicine is a benign treatment, its efficacy is unclear (4-6). There has been one small RCT showing good efficacy of the TNF-α blocker etanercept for Behcet’s Syndrome (7). The investigators in the reviewed study used Cohen’s effect size to make indirect comparisons between treatment with colchicine and treatment with aprelimast. Effect size is a name given to a family of indices that measure the magnitude of a treatment effect. Unlike significance tests, these indices are independent of sample size. Cohen’s effect size (d) is estimated by the difference between the mean change in variable, divided by the standard deviation of that variable (8). An effect size (d) of 0.2 is considered to be “small,” 0.5 is “medium,” and 0.8 is “large.” In this paper, the authors calculated the effect size using data reported in other studies for colchicine with the data found in the current study using aprelimast. The effect sizes were estimated at 0.27 for colchicine and 1.62 for aprelimast. Similarly, they compared the odds ratios of freedom from oral ulcers with aprelimast vs placebo to the odds ratio with etanercept vs placebo from other data. The OR were 6.1 (95% CI 2.7-13.9) and 15.6 (95% CI 1.7-139.7) for etanercept and aprelimast, respectively. Such calculations may be of interest, but without head to head comparison studies, should be interpreted cautiously.

The results of this study must also be interpreted in light of its limitations, including the small number of participants, the relatively short follow-up period, and the ethnic homogeneity of enrollees (103 enrollees from Turkey; 8 from US). It is notable that within 2 weeks of discontinuing aprelimast therapy, both the number of ulcers and pain scores returned almost to baseline; providing additional evidence of drug effectiveness. However, it also indicates that long term therapy with aprelimast will be necessary; currently, one year of therapy with aprelimast costs ~$22,000.

References:

  1. Papp K, Kaufmann R, Thaci D, et al. Efficacy and safety of apremilast in subjects with moderate to severe plaque psoriasis: results from a phase II, multicenter, randomized, double-bliind, placebo-controlled, parallel-group, dose-comparison study. J Eur Acad Dermatol Venereol 2013;27(3):e376-e383
  2. Papp K, Cather J, Rosoph L, et al. Efficacy of apremilast in the treatment of moderate to severe psoriasis: a randomized controlled trial. Lancet 2012;380:738-46
  3. Schett G, Wollenhaupt J, Papp K, et al. Oral apremilast in the treatment of active psoriatic arthritis: results of a multicenter, randomized, double-blind, placebo-controlled study, Arthritis Rheum 2013;64:3156-67
  4. Aktulga E, Altac M, Muftuoglu A, et al. A double blind study of cochicine in Behcet’s disease, Haematologica 1980;65:399-402
  5. Yurdakul S, Mat C, Tuzun Y, et al. A double-blind trial of colchicine in Behcet’s syndrome Arthritis Rheum, 2001;44:2686-92
  6. Davatchi F, Sadeghi AB, Tehrani BA, et al. Colchicine versus placebo in Behcet’s disease: randomized, double-blind, controlled crossover trial, Mod Rheumatol 2009;19:542-9
  7. Melikoglu M, Fresko I, Mat C, et al Short-term trial of etanercept in Behcet's disease: a double blind, placebo controlled study. J Rheumatol 2005;32:98-105.
  8. Cohen, J. (1988). Statistical power analysis for the behavioral sciences (2nd ed.). Hillsdale, NJ: Lawrence Earlbaum Associates.

High-dose Immunosuppressive Therapy and Autologous Hematopoietic Cell Transplantation for Relapsing-remitting Multiple Sclerosis (Halt-Ms): A 3 Year Interim Report

Clinical Trial: Richard Nash, George Hutton, Michael Racke, et al. High-dose Immunosuppressive Therapy and Autologous Hematopoietic Cell Transplantation for Relapsing-Remiting Multiple Sclerosis (HALT-MS), JAMA Neurol, 2015; 72(2):159-169

Disease: Relapsing-remitting Multiple Sclerosis

Drug: CD34+ autologous peripheral stem cells selected, followed by high dose therapy with carmustine, etoposide, cytarabine, melphalan, and ATG, followed by autologous hematopoietic cell transplant

Study design:

  • Multi-center single-arm trial
  • 25 patients (age 18-60) with relapsing-remitting multiple sclerosis (RRMS) with continued loss of neurological function despite receiving disease-modifyng therapy during the 18 months prior to enrollment
  • Mean duration of disease 4.9 years
  • Primary end point: event-free survival; survival without death or disease acitivty with one of the following outcomes: 1. Confirmed loss of neurological function, 2. Clinical relapse, 3. New lesions observed on magnetic resonance imaging. 

Results:

  • Overall event-free survival 78.4% at 3 years
  • Of those with event free survival, 90.3% were without progression, 86.3% without clinical relapse, and 100% without new MRI lesions at 3 years.
  • Grade 3/4 AE’s were mostly toxic hematologic and gastrointestinal side effects expected from high dose immunosuppression and AHCT. 
  • 2 suicide attempts
  • 2 deaths: one related to MS progression and one secondary to asthma.

Why the Trial is of Interest to the Broader FOCIS Community:

Durable remission from disease activity remains unreachable for many patients with RRMS. Trials of disease modifying therapies for RRMS suggest that clinical and radiological disease free survival rates at 2 years range between 14-37% (1,2). Furthermore, discontinuation of therapy leads to relapse within months; hence patients remain on therapies with potentially serious adverse effects for years. The deleterious effects of MS relate to both early and ongoing inflammation and secondary neuronal damage. AHCT has been studied as a possible therapy for MS for 20 years (3), as a means to remove the damaging immune cells and “reset” the immune system. From 1995-2001, a trial in Greece transplanted 35 patients with “aggressive MS” as defined by more advanced disability and MRI changes. Continued monitoring of early trial participants suggests that patients with more active CNS inflammation (as defined by inflammatory lesions on MRI) at the time of transplant have had better outcomes than patients without active inflammation. Among subjects with active CNS lesions at time of transplant, 44% had progression-free survival at 15 years, compared to 10% of subjects without CNS lesions at time of transplant (4). It has been suggested, therefore, that if AHCT is going to be used as a therapy in RRMS, it should be used early in the course of disease, when inflammation is most active. Why this should be the case is unknown. As the investigators point out, the remissions soon after transplant in this study were likely the result of lymphoablation, but longer term remissions may result from the persistent immune effects of transplant. The CD4+ T cell receptor clones present prior to transplant were not detected after transplant, which may be consistent with an immune reset. However, the CD8 repertoire before and after transplant was unchanged. The reviewed study is an interim 3 year report of a study with a planned 5 year evaluation period. More will be known after the 5 year observation period regarding durability of remissions.
Although event-free survival rates at 3 years are impressive, there remains uncertainty about the role of HSCT in treating RRMS or other autoimmune diseases. Safety in HSCT has improved, but it remains a therapy with both toxic side effects and the risk of serious adverse events, including death. Given these risks, impressive early efficacy as reported in this study and others (5-8) will likely need to be paired with durable results with due consideration as to appropriate patient selection.

References:

  1. Havrdova E, Galetta S, Hutchinson M, et al. Effect of natalizumab on clinical and radiological disease activity in multiple sclerosis: a retrospective analysis of the Natalizumab Safety and Efficacy in Relapsing-Remitting Multiple Sclerosis (AFFIRM) study, Lancet Neurol 2009;8(3):254-260
  2. Coles AJ, Twyman CL, Arnold DL et al. CARE-MS II Investigators, Alemtuzumab for patients with relapsing multiple sclerosis after disease-modifying therapy: a randomized controlled phase 3 trial, Lancet 2012;380(9856):1829-1839
  3. Fassas A, Anagnostopoulos A, Kazis A et al. Peripheral blood stem cell transplantation in the treatment of progressive multiple sclerosis: first results of a pilot study, Bone Marrow Transplant 1997;20(8):631-638
  4. Fassas A, Kimiskidis VK, Sakellari I, et al. Long-term results of stem cell transplantation for MS: a single-center experience, Neurology, 2011;76(12):1066-1070
  5. Hawkey, CJ, Stem cells as treatment in inflammatory bowel disease, Dig Dis. 2012;30 Suppl 3:134-9. 
  6. van Laar JM, Farge D, Sont JK, et al. Autologous hematopoietic stem cell transplantation vs intravenous pulse cyclophosphamide in diffuse cutaneous systemic sclerosis: a randomized clinical trial., JAMA. 2014 Jun 25;311(24):2490-8.
  7. D'Addio F, Valderrama Vasquez A, Ben Nasr M, et al. Autologous nonmyeloablative hematopoietic stem cell transplantation in new-onset type 1 diabetes: a multicenter analysis. Diabetes. 2014 Sep;63(9):3041-6.
  8. Burt RK1, Traynor A, Statkute L, Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus, JAMA. 2006 Feb 1;295(5):527-35.

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