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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.


  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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