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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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