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December 17, 2013

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 HighlightsBasic Immunology & Novel Therapies | ImmunphenotypingPDF Version |Previous Issues

Highlights from Recent Literature

HIF Signaling Combats CD8+ T Cell Exhaustion

A review of Doedens, A.L., et al., Hypoxia-inducible Factors Enhance the Effector Responses of CD8+ T Cells to Persistent Antigen. Nature Immunology. 14, 1173-1182 (2013).PMID: 24076634

Under conditions of chronic antigen exposure, CD8 T cells often enter a state of exhaustion, in which effector functions are markedly decreased. Exhausted T cells are observed in chronic infections such as HIV and hepatitis and contribute to impaired antitumor immunity in both mice and humans. Pharmacologic interventions that reverse T cell exhaustion are of great interest in the treatment of both chronic infections and cancer. In this study, the authors discovered an important role for hypoxia-induced factor (HIF) pathway signaling in preventing CD8 T cell exhaustion.

  • The authors studied a mouse model in which expression of VHL, the gene encoding the negative regulator of HIF signaling, was turned on or off. The authors found that VHL-deficient (HIF active) mice died after infection with lymphocytic choriomeningitis virus (LCMV) but wild type mice survived.
  • Death of infected mice also occurred in wild type mice adoptively transferred with VHL-deficient virus specific CD8+ T cells, suggesting that death after infection was caused by over-active virus specific CTL responses.
  • These pathogenic CD8+ T cells did not express the terminal effector marker KLRG1 during chronic viral infection. Transcriptional profiling showed that VHL deficient CD8+ T cells had increased HIF signaling, increased cytotoxic functions, and did not enter an exhausted state.
  • Hypoxia alone also enhanced and prolonged CD8 T cell effector functions in normal T cells, and this response was dependent on HIF signaling. HIF-activated T cells also had decreased expression of Eomes, a transcription factor upregulated in exhausted T cells.
  • VHL deficient (HIF activated) T cells also provided superior antitumor immune protection in a mouse B16 melanoma model.

Immunomodulatory therapies that enhance T cell activity and reverse T cell exhaustion, including PD-1 and CTLA-4 blockade, are proving to be effective in enhancing antitumor responses in cancer patients. These studies suggest that agents that increase HIF signaling could be promising as new therapies for patients with cancer and chronic infections.

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


Science Comes to the Aid of Emergency Room Physicians

A review of Zaas, A.K., et al., A Host-Based RT-PCR Gene Expression Signature to Identify Acute Respiratory Viral Infection. Science Translational Medicine 5, 203ra126 (2013). PMID: 24048524

Billions of dollars of NIH research funds are spent each year investigating the effects of various infections on the immune system, most commonly in mouse models. Despite this remarkable outlay of resources, today’s emergency room and hospital physicians have only the crudest tools, such as fever, elevation of the white counts, and tests for specific pathogens to discriminate patients with dangerous bacterial infections from those with viral illnesses. In this study, the authors studied immune profiles of patients with viral vs. bacterial infections in order to determine a rapid and effective test to discriminate between these patients.

  • The authors experimentally infected healthy patients with H3N2 or H1N1 influenza and carried out RT-PCR studies of blood to develop a reproducible signature of viral infection.
  • They validated this signature by comparing these results to those obtained from healthy patients and “real world” patients seen in the emergency room with fever arising from bacterial vs. viral illnesses.
  • The authors identified a set of 48 genes that, when expression levels were measured by RT-PCR from blood samples, reliably distinguished patients with viral respiratory illnesses from those with bacterial infections.
  • The test had an excellent sensitivity (87%-100%) for detecting influenza infection that was superior to commercially available viral PCR studies and provided superior ability to both rule in and rule out viral respiratory infections. The assay for viral infection also detected patients with unrelated viral infections, including the common cold rhinovirus.

The authors have demonstrated that profiling of a patient’s immune response is as good or better at detecting viral infection than more specific PCR’s for the individual infectious organisms. This rapid test could be used to effectively discriminate patients with viral infections from those with bacterial infections, allowing the appropriate use of antiviral versus antibiotics therapies. If adopted clinically, this test could be a major advance in rapidly diagnosing and appropriately treating patients with fever.

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


Hide and Seek: Identifying TCRs to Attack Patient Specific Cancer Antigens

A review of Linnemann, C., et al. High Throughput Identification of Antigen-specific TCRs by TCR Gene Capture. Nature Medicine. (2013) 19: 1534-1543. PMID: 24121928

T cells require multiple signals for activation, carefully protecting us from autoimmunity (a self antigen is not sufficient for activation) and preventing reactions against irrelevant antigens (a foreign antigen is not sufficient for activation). These signals include antigen, processed and then presented by antigen presenting cells to be recognized by specific T cell receptors (TCRs), as well as costimulation and cytokine signals. In this study, the group takes as a starting point the exciting recent advances in oncologic therapy that make use of techniques to engineer TCRs against specific tumor antigens. They argue for the importance of generating libraries of TCRs that recognize diverse tumor antigens in the setting of a wide variety of HLA alleles. One can also argue for the value of understanding the population of TCRs in many other clinical scenarios, from autoimmunity to infectious disease.

SUMMARY

  • In order to capture the extremely diverse population of TCRs, the team devised a ‘bait’ system which uses conserved components of the TCRs to ‘fish’ for the variable components of the TCRs. It is important to remember the underlying recombination events that yield TCRs, which include the random selection of inherited ‘V’, ‘D’ and ‘J’ segments for VDJ rearrangement and the addition of random bases between the segments. Thus, there are elements of the TCR that are conserved and these can be used to find the novel regions. In this study, each ‘V’ and ‘J’ element in the section of the genome encoding the TCR was targeted.
  • In practice, genomic DNA is sheared into very short pieces (~500bp) and the above ‘bait’ strategy is used. Once the regions of interest are ‘fished’ out of the massive quantity of short pieces of DNA, they perform deep sequencing on these regions and then use analysis techniques to reconstruct TCRs.
  • To facilitate the process, individual T cells are ‘indexed’ so that they can be sequenced in parallel.
  • Once the DNA is sequenced, it is possible to decode the VDJ rearrangements that took place (i.e. this TCR includes V1 and J4). Beyond that, they could also identify the highly variable complementarity determining region 3 (CDR3) component of the TCRs in silico.
  • To initially validate the process, they examined CMV and HA-2 specific T cell samples and studied the limits of how few cells they need to successfully determine the VDJ rearrangement.
  • To test the clinical efficacy of the technique, they collected blood and tumor infiltrating lymphocyte samples from melanoma patients, isolated T cells with responsiveness to known melanoma antigens and created TCR gene libraries using the TCR capture approach (identifying 147 T cell responses from 61 patients). The genes for 21 TCRs against 11 tumor antigens were identified were used to transduce human peripheral blood lymphocytes, (n = 14), which were then tested for antigen specific cytokine production. Using cell lines that expressed the relevant tumor antigens to test the transduced cells, they found that the TCRs could recognize the tumor antigens, and only one had non-specific activation.
  • Interestingly, each tumor antigen yielded only a few TCRs.
  • The group then moved on to show that they could identify novel TCRs from bulk tumor infiltrating lymphocytes or short-term expansions of single CD8+ T cells in melanomas (i.e. without preselecting T cells for tumor antigen specificity)

In this study, TCR gene capture demonstrates the potential to identify the TCRs responding to a wide range of antigens, from tumor antigens (including an individual’s tumor neoantigens) to self-antigens underlying autoimmunity and foreign antigens contained within infections. While the ability to identify the potentially novel protein antigens recognized by these TCRs remains a challenge, this work raises many new questions and the potential to assess the T cell repertoire of a host in rapid fashion in a number of fascinating and clinically important contexts

Reviewed by Sarah Henrickson, MD, PhD Universiy of Pennsylvania 


How Does IL-21R/STAT3 Signaling Regulate Human B Cell Function?

A review of Deenick E.K., et al. Naïve and Memory Human B Cells have Distinct Requirements for STAT3 Activation to Differentiate into Antibody-secreting Plasma Cells. Journal of Experimental Medicine., 210:2739-53, (2013). PMID: 24218138

The role of IL-21R/STAT3 signaling in regulating plasma cell generation from naïve and memory B cells is poorly understood. Previously published studies have revealed that STAT3 but not STAT1 is necessary for IL-10- and IL-21-mediated differentiation of human naïve B cells into plasmablasts. Autosomal-dominant hyper-IgE syndrome (AD-HIES) is caused by a heterozygous mutation in STAT3 that acts in a dominant negative manner, reducing the pool of function STAT3. By comparing AD-HIES patients to healthy controls, Deenich and colleagues reveal the distinct requirements for STAT3 activation to induce plasma cell differentiation in naïve and memory human B cells. The authors examine responses naïve and memory B-cell responses to STAT3-activating cytokine IL-21 and the molecular machinery necessary for plasma cell differentiation in this paper, which provides several intriguing observations:

  • STAT3 mutations impair the response of naïve but not memory B cells to IL-21. The authors cultured naïve and memory B cells isolated from normal donors or STAT3-mutant patients with CD40L and IL-21 and measured Ig secretion. After 10-12 days in stimulating culture, the authors found that STAT3-deficient naïve B cells failed to secrete Ig while their memory B cell counterparts secreted similar levels of IgM, IgG and IgG as healthy controls. In addition, STAT3-mutant naïve B cells fail to proliferate upon stimulation with IL-21.
  • Memory B cell pools in STAT3-deficient patients contain antigen-specific B cells. The authors examined the relative amounts of anti-tetanus IgG produced by B cells from STAT3-mutant donors vs. healthy controls. Naïve B cells from both STAT3-mutants and healthy controls produced very low to undetectable levels of anti-tetanus IgG. Surprisingly, STAT3-mutant memory B cells produced anti-tetanus IgG, albeit at lower levels than healthy controls indicating that STAT3-deficient individuals are capable of generating Ag-specific antibody responses and a stable pool of memory B cells.
  • Plasma cell lineage commitment is unaffected by mutations in STAT3. During differentiation of human B cells into plasma cells, PAX5 is down regulated, which increases Blimp-1 and XBP-1 expression. The authors examined expression of PAX5, PRDM1 (Blimp-1), and XBP-1 in naïve and memory B cells in STAT3-deficient and healthy donors. They found that STAT3-deficient naïve B cells fail to up regulate Blimp-1 and XBP-1 upon stimulation with IL-21. In contrast, memory B cells do not have this defect.
  • Memory cells exhibit greater sensitivity to differentiation-effects of IL-21 and IL-10. To determine differences between naïve and memory B cell responses to IL-10 and IL-21, the authors cultured naïve and memory B cells with CD40L and increasing concentrations of IL-10 or IL-21 and measured expression of XBP-1 and Blimp-1. The authors found that memory B cells have greater sensitivity to both IL-10 and IL-21.

These data suggest that naïve and memory B cells have distinct requirements for STAT3 activation during differentiation into plasma cells. While STAT3 is not required for generation of antigen-specific memory, naïve B cells fail to differentiate into plasma cells upon stimulation with IL-21. By targeting the STAT3-deficient memory populations, it may be possible to improve antibody-responses in AD-HIES patients.

Reviewed by Si-Han V. Hai and Michelle L. Hermiston, MD, PhD, University of California San Francisco


Role of Myeloid Suppressor Cells in Development Of Myelodysplastic Syndrome

A review of Chen X., et al., Induction of Myelodysplasia by Myeloid-derived Suppressor Cells. Journal of Clinical Investigation 123:4595-4611 (2013). PMID:24216507

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders resulting in disordered hematopoiesis and progressive pancytopenia with irreversible loss of blood-forming cells. The mechanistic basis for MDS is incompletely understood. Direct genetic and epigenetic mutations in hematopoietic stem cells as well as an altered bone marrow immune microenvironment are thought to both contribute to disease pathogenesis. Indeed, although not curative, broad immunosuppression is one therapeutic approach. The paper by Chen, Wei, and colleagues provides new insights into the role of immune dysregulation in MDS pathogenesis. Focusing on the innate immune system, they evaluate the composition and function of the myeloid compartment in primary bone marrow cells obtained from patients with MDS, non-MDS cancer patients (4 breast and 4 lymphoma), and age-matched healthy controls. There are several intriguing findings from this work.

  • LIN-HLA-DR-CD33+ myeloid derived suppressor cells (MDSC) are expanded in primary bone marrow specimens of MDS patients (median 35% of bone marrow mononuclear cells versus <5% in controls). Mutation analysis demonstrated that the MDSC were not derived from the malignant MDS cells.
  • LIN-HLA-DR-CD33+ produced excessive levels of the immunosuppressive cytokines IL10 and TGF-β as well as high levels of nitric oxide and arginase relative to LIN-HLA-DR-CD33+ from healthy controls or patients with other types of cancer.
  • Functional studies demonstrated that MDSC cells from MDS patients had increased suppressive function relative to controls. Co-culture experiments demonstrated they could inhibit proliferation of autologous T cells. T cell proliferation and cytokine production could be restored with depletion of MDSC prior to co-culture. Burst-forming unit-erythroid (BFU-E) and GM-CFU colony formation was also lower in co-cultures with MDSC-supplemented versus MDSC-depleted samples.
  • MDS-MDSC cells also had increased cytotoxic effector function with increased levels of granzyme B polarization and granule release when cultured with autologous erythroid precursors. This resulted in increased apoptosis of target erythroid precursors, revealing a potential direct mechanism contributing to progressive loss of these cells in MDS.
  • Using a series of elegant transfection cell models, the authors demonstrate that the Siglec 3 receptor CD33, which is elevated in MDS-MDSC, can directly promote MDSC suppressive function.
  • Using a high-throughput mass-spectrometric approach, the authors identified the S100A9 as a putative ligand for CD33. Functional studies confirmed a direct interaction between CD33 and S100A9. Interestingly, S100A9 levels were increased in MDS bone marrow relative to controls. S1009 treatment of MDS-MDSC resulted in phosphorylation of CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), recruitment of the inhibitory phosphatse SHP-1, and production of IL10 and TGF-β. Depletion of S100A9 attenuated IL-10 and TGF-β and restored BFU-E and GM-CFU colony formation. In addition, overexpression of S100A9 in transgenic mice resulted in bone marrow dysplasia reminiscent of human MDS.

Taken together, this work highlights a potential role for MDSC in MDS that is mediated, at least in part, by the S100A9/CD33 pathway. Several important questions remain: What triggers the initial expansion of MDSC in MDS patients? Are they necessary for disease initiation, maintenance/progression, or both? Can this pathway be targeted therapeutically? While the authors postulate that sustained activation of innate immune signaling in the bone marrow microenvironment creates a permissive inflammatory milieu that allows for development of MDS, additional studies will be required.

Reviewed by Si-Han V. Hai and Michelle L. Hermiston, MD, PhD, University of California, San Francisco


Localized Immunomodulation with CTLA-4 Blocking Antibody: Same Antitumor Effect Without The Side Effects

A review of Fransen M.F., et al. Controlled Local Delivery of CTLA-4 Blocking Antibody Induces CD8+ T Cell–dependent Tumor Eradication and Decreases Risk of Toxic Side Effects. Clinical Cancer Research. 2013;19(19):5381-5389. PMID: 23788581

Systemic delivery of CTLA-4 blocking antibodies induces antitumor immune responses in some preclinical models and patients, and has been approved for the treatment of advanced melanoma by the FDA. However, because systemic treatment with CTLA-4 blocking antibodies lowers the activation threshold for all T cells, and not just the tumor-reactive T cells, clinical use of CTLA-4 blocking antibodies is hampered by dose-limiting autoimmune and inflammatory side effects. In this study, Marieke Fransen and colleagues demonstrate that it may be possible to achieve the antitumor effect without causing adverse side effects by administering CTLA-4 blocking antibodies locally rather than systemically.

  • The authors first compared the antitumor efficacy of systemic versus local administration of CTLA-4 blocking antibody against subcutaneously implanted murine colon carcinoma MC-38 tumors expressing Ovalbumin (MC-38-OVA). They demonstrate that a single local injection of 50 μg of antibody in Montanide ISA-51 was just as effective as treatment with two systemic doses of 200 μg of antibody. They also showed that localized treatment with CTLA-4 blocking antibodies induced antitumor responses in mice bearing MC-38 tumors that do not express OVA, and mice bearing more aggressive OVA-expressing EG7 thymoma tumors.
  • Low dose treatment with 50 μg of antibody was shown to only be effective when delivered in the tumor-draining area, and not when given systemically, or in the contralateral flank of tumor-bearing mice.
  • Similar to systemic treatment, localized treatment with CTLA-4 blocking antibody was shown to enhance systemic tumor-specific T cell responses, and as a result, was capable of controlling distant tumors.
  • By depleting CD4+ and CD8+ T cells in treated mice, the authors demonstrated that CD8+ T cells, and not CD4+ T cells, were responsible for the antitumor effect in their model, indicating that CTLA-4 blocking antibodies can operate directly on CD8+ T cells.
  • Analysis of serum from treated mice showed that antibody levels were more than 1,000-fold lower in mice treated with local doses of antibody compared to mice treated with systemic doses. Levels of the liver enzymes ALT and AST were also lower in locally-treated mice suggesting that local slow-release administration of the antibody decreases the induction of adverse effects.

Although systemic treatment with CTLA-4 blocking antibodies can induce significant antitumor effects in some patients, and has been FDA approved for the treatment of advanced melanoma, this treatment can also cause severe autoimmune and inflammatory side effects. This study provides a simple, yet novel delivery system for distributing CTLA-4 blocking antibody in the tumor-draining area. In preclinical models, use of this delivery approach resulted in antitumor responses that were comparable to systemic antibody treatment, but were associated with 1,000-fold lower serum levels of antibody. These data indicate that use of this delivery approach may make it possible to achieve the same antitumor effects in patients without inducing unwanted side effects. This strategy could also be useful for other immunomodulatory agents being developed for cancer treatment.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center


Different Adjuvants have Contrasting Effects on Antigen-specific T Effector and T Regulatory Cells

A review of Perret R. et al. Adjuvants that Improve the Ratio of Antigen-specific Effector to Regulatory T Cells Enhance Tumor Immunity. Cancer Res. 2013;73(22):6597-608. PMID: 24048821 

The balance between tumor-antigen-specific effector T cells (Teffs) and regulatory T cells (Tregs) plays an important role in regulating antitumor immunity. Tumor-specific vaccines aim to selectively activate and expand Teffs to skew this balance in order to promote the induction of protective antitumor responses. Immunization with tumor antigen peptides is one vaccination approach that can stimulate tumor antigen-specific T cells. Because vaccination with peptide alone is not effective, and results in expansion of both Teffs and Tregs, numerous adjuvants are being developed to enhance the antitumor activity of peptide-based vaccines. However, prior to this study by Rachel Perret and colleagues, few studies have directly compared the impact of various adjuvants on the balance between antigen-specific Teffs and Tregs.

  • The authors first established a model to evaluate the effects of vaccination on antigen-specific CD4+ and CD8+ Teffs and CD4+ Tregs by transferring Ovalbumin (OVA) specific CD8+ OT-1 T cells and CD4+ FOXP3-GFP OT-II T cells into immuncompetent mice.
  • Using this model, they compared immunization with OVA257-264 and OVA323-339 peptides with and without various adjuvants. Adjuvants tested included the TLR-2 agonist Pam3Cys, TLR3 agonist Poly(I:C), TLR-4 agonist LPS, TLR-5 agonist Flagellin, TLR-7/8 agonist imiquimod, TLR-9 agonist CpG, and Quil A saponin.
  • Analysis of OVA-specific Teffs and Tregs in inguinal vaccine-draining lymph nodes and spleens 7 days following immunization showed that Poly(I:C) and CpG induced significantly more expansion of OT-1 CD8+ Teffs compared to the other adjuvants. OT-II CD4+ Teffs were expanded similarly by all adjuvants, but expanded significantly less to imiquimod and Quil A. All adjuvants, except for Quil A, reduced the proportion of Tregs among OT-II cells compared to immunization with peptide alone.
  • Comparison of ratios of OVA-specific Teffs to Tregs in spleens and draining lymph nodes demonstrated that Poly(I:C) and CpG significantly enhanced CD8+ Teff:Treg ratios compared to immunization with OVA peptides alone, whereas CpG, Poly(I:C) and Pam3Cys significantly enhanced OVA-specific CD4+ Teff:Treg ratios.
  • Due to their contrasting immunologic effects, the authors chose to further analyze the antitumor effects of Poly(I:C), CpG, imiquimod and Quil A in mice bearing B16 melanoma tumors expressing OVA (B16-OVA). Analysis of tumor infiltrating lymphocytes 7 days following immunization demonstrated that significantly more CD8+ OT-1 Teffs infiltrated tumors following vaccination with Poly(I:C) and CpG compared to the other adjuvants tested. Because too few OT-II cells were found infiltrating tumors, OVA-specific Teff:Treg ratios in the tumor could not be evaluated.
  •  OVA-specific Teff:Treg numbers and ratios in tumor-draining lymph nodes in tumor-bearing mice resembled those observed in vaccine-draining lymph nodes in tumor-free mice, and correlated with the degree of CD8+ OT-1 Teff tumor infiltration and tumor protection.
  • When mice were vaccinated one week prior to B16-OVA challenge, and boosted 10 days after tumor challenge, mice treated with peptides plus CpG were completely protected, and Poly(I:C) resulted in a delay in tumor growth, whereas the other adjuvants had no effect on tumor growth.
  • Cytotoxic analysis of CD8+ OT-1 Teffs 7 days following immunization showed that CpG and Poly(I:C) induced the highest level of OVA-specific cytotoxic function.
  • Therapeutic vaccination with OVA peptide plus Poly(I:C) and CpG were also shown to induce enhanced tumor-protection in mice bearing 10-day old EG7 thymomas, whereas use of imiquimod or Quil A had no tumor-protective effect.
  • The authors also confirmed their results in a self-antigen melanoma system by quantitating GP100-specific Pmel CD8+ Teffs and Trp-1-specific Trp-1 CD4+ Teffs and Tregs following immunization with peptides plus the various adjuvants. In addition, they showed that immunization with Poly(I:C) and CpG induced significantly more polyfunctional CD8+ and CD4+ Teffs than imiquimod or Quil A.
  • Immunization with peptides plus Poly(I:C) or CpG were also shown to induce the greatest cytotoxic and antitumor effects in the self-antigen melanoma model.
  • Analysis of cytokine production in vaccine-draining lymph nodes 24 hrs following vaccination showed that Poly(I:C) and CpG induced significantly more production of the proinflammatory type I cytokines IFN-ß, IL-12, IFN-γ and IL-6 compared to imiquimod and Quil A.

This study provides a comprehensive analysis of the effects of a broad panel of adjuvants on tumor antigen-specific Teffs and Tregs, and the induction of antitumor immunity. The authors demonstrated that, compared to imiquimod and Quil A, Poly(I:C) and CpG induce more potent antitumor immunity that is associated with the preferential expansion of antigen-specific Teffs over Tregs resulting in increased Teff:Treg ratios, and the induction of polyfunctional antigen-specific Teff responses, both of which have been shown in previous studies to correlate with improved antitumor immunity. Moreover, this study shows that not all adjuvants are equal, and suggests that Poly(I:C) and CpG are the most ideal adjuvants currently available for peptide vaccines for cancer.

Reviewed by Elizabeth Jaffee, MD, Johns Hopkins Institute for Clinical and Translational Research and Eric Lutz, PhD, Johns Hopkins University, Sidney Kimmel Cancer Center

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Highlights From Clinical Immunology, the Official Journal of FOCIS

Histopathology in Relapsing and Remitting EAE

A review of Soellner IA. et al. Differential Aspects of Immune Cell Infiltration and Neurodegeneration in Acute and Relapse Experimental Autoimmune Encephalomyelitis Clinical Immunology 149, 519–529, 2013       PMID: 24239839

Relapsing–remitting multiple sclerosis is the most common clinical subtype of human MS, and is modeled by proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. The authors of this paper have previously shown that myelin oligodendrocyte glycoprotein (MOG)-induced EAE, which displays a chronic disease phenotype, is characterized by early acute inflammation leading to chronic neuro-degeneration, and that axonal damage was present early and remained constant but myelin damage increased chronically. In this paper, the authors characterized and quantified CNS inflammation, extent of demyelination, and axon damage in acute and relapse phase of PLP-induced EAE, with the goal of better understanding mechanisms by which relapsing-remitting MS transitions to chronic-progressive disease. The major finding were:

  • The predominant cell type found in acute and relapse EAE within the CNS infiltrates were CD4+-cells, however there were differences in other cell types between the two phases, namely, a reduction of CD8+ T cells and macrophages and an increase in B in the relapse phase.
  • There was significantly less edema in the relapse phase. Decreased in relapse EAE.
  • In acute EAE, there was extensive demyelination taking place, while in the relapse phase, there was less myelin pathology, with both demyelinated nerve fibers and axons in the process of demyelination.
  • The number of dead axons also increased significantly in the relapsed phase, and there were more damaged axons without inflammation of myelin sheaths, than in the acute phase.
  • Damaged axons without myelin sheath pathology were increased significantly in relapse phase compared to initial acute phase.

Overall, the results are consistent with previous studies in showing that clinical severity of acute EAE correlates with the degree of spinal inflammation while late neurological disease correlates with loss of axons. The authors argue that their findings support the hypothesis that nerve fiber destruction in late EAE reflects neuro-degeneration independent from ongoing inflammation and myelin damage. Based on EM findings in this study, they suggest early mitochondrial damage is one factor that leads to neuro-degeneration.

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


Immunotherapy with Human Allogeneic CD8REG

A review of Horwitz, DA., et al. Therapeutic Polyclonal Human CD8+ CD25+Fox3+ TNFR2+ PD-L1+ regulatory cells induced ex-vivo. Clinical Immunology 149, 450–463, 2013. PMID: 24211847

Most of the effort on therapeutic use of Treg has focused on isolating CD4+ CD25+ Foxp3+ Tregs from blood of patients with autoimmune diseases or allografts, expanding them in vitro, and transferring them back into the patient. Horwitz and colleagues cite problems with expansion of these classical Treg from limited numbers isolated from blood and limited efficacy on pathogenic memory CD4+ effector cells as the rationale for characterizing the therapeutic potential of CD8+ regulatory T cells (CD8reg). In this data-rich paper, the authors describe the in vitro generation and in vivo suppressive activity of human CD8+ T regs that phenotypically resemble exhausted CD8+ CTL or mouse donor-derived CD8+ Foxp3+ Tregs that are found in recipient mice after transplantation.

  • Human CD8reg were generated from blood derived naïve CD8+ cells by stimulation with anti-CD3/28 coated beads, IL-2, with or without TGF-β.
  • The in vivo suppressive activity of these T cells was tested in a xenogeneic graft vs. host model in which human PBMC were transferred in into lightly irradiated NOD SCID IL-2Rγc deficient mice, which results in rapid engraftment of human T cells and death by GVHD in two weeks.
  • Transfer of allogeneic CDr8eg generated with or without TGFβ quadrupled survival time in the GVHD model. When the PBMC and the CD8reg were from the same donor (autologous) there was no protection.
  • The CD8reg expressed IL-2Rαβ chains, TGF-β dependent Foxp3, TNFR2, PD-L1, PD-1, and Tim3. In vitro, FOXP3 expression was enhanced by TGF-β, and sustained by IL-2.
  • The in vivo protective activity of the CDreg was dependent on IL-10.
  • The CD8regs lack cytotoxic activity against allogeneic T cells.

Based on their findings, the authors propose that polyclonal CD8regs generated with anti-CD3/28 beads will preferentially target allogeneic T cells when transferred into hosts with autoimmune disease, GVHD or allograft rejection. They suggest that the transferred allogeneic cells CD8regs would not be rejected because they would suppress the lymphocytes they come into contact with. The findings on this paper are similar in several ways to another recent report that showed transfer of human CD8+ regulatory inhibit GVHD when transferred into humanized mice without causing generalized immunosuppression (Zheng J,Sci. Transl. Med. 5,2013). A limitation described by the authors of the CD4+ dependent xeno-GVHD model is that it does not test efficacy of the CD8reg on pathogenic class I MHC restricted pathologies. The precise mechanisms of in vivo suppression remain unclear, and testing the efficacy of human allogeneic CD8reg in models of autoimmunity will be challenging.

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

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Developments in Basic Immunology and Novel Therapies

The Yin and Yang of IL-2

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

The historical perspective
Interleukin-2 (IL-2) has a storied history in the annals of Immunology. It is the first cytokine to be purified and characterized, and shown to stimulate the proliferation of T cells, the activity that gave rise to its early name T cell growth factor. It is also produced mainly by T cells, and thus functions as an autocrine growth factor. The discovery of IL-2 launched the concept that the immune system was regulated by soluble, secreted factors; this concept was revolutionary when first proposed and is, of course, now considered self-evident. IL-2 was also the first cytokine whose gene was cloned, and the first whose cellular receptor was identified and characterized. The IL-2 receptor (IL-2R) is considered the founding member of the “type 1 cytokine receptor” family. It consists of three chains, all of which are required for high-affinity ligand binding. The α chain is unique to IL-2 and the β and γ chains are shared with other receptors. The receptor signals by a Jak-Stat pathway as well as pathways involving Ras-MAP kinase and Akt.

In addition to serving as a proliferative signal for T cells, IL-2 also stimulates the differentiation of naïve T cells into effector and memory cells. There is great interest in defining the actions of IL-2 on follicular helper T cells and, therefore, on germinal center B cell reactions, and on innate lymphoid cells.

The established T cell stimulating activity of IL-2 provided the impetus for the clinical applications of this cytokine for boosting immune responses in patients with advanced cancers. This led to another first – IL-2 was the first cytokine, and, in fact, probably the first immunologically active biological agent, given to patients. The high incidence of serious adverse events, likely related to the production of other cytokines induced by high doses of IL-2, has seriously limited the use of this agent in cancer patients. The immune stimulatory activity of IL-2 was the basis of a much later clinical trial combining low doses of the cytokine with anti-retroviral drugs to boost immune responses in HIV-infected patients. Although IL-2 significantly increased CD4 T-cell counts, it showed no clinical benefit over anti-retroviral drugs alone. These disappointing clinical trial results have led to uncertainty about systemic IL-2 therapy to enhance immune responses in patients.

Revisions to the history: The unexpected actions of IL-2
Dogmas about the functions of IL-2 were shaken by the unexpected finding that germline deletion of the IL-2 gene led not to immune deficiency, as might have been predicted, but to severe lymphoproliferation, dominated by T cells, and systemic autoimmunity. The autoimmunity is manifested primarily by colitis or hemolytic anemia in different mouse strains. This was soon followed by observations that knockout of the IL-2Rα or β chain led to a similar lymphoproliferative and autoimmune disorder. (Knockout or mutations of the IL-2Rγ chain result in a severe combined immunodeficiency [SCID] phenotype because this chain, also called the common γ [γc] chain, is a component of the receptor for the essential lymphopoietic cytokine IL-7.) The excessive proliferation and expansion of T cells in the absence of the prototypic T cell growth factor was an obvious paradox. The solution to this conundrum came with the finding that IL-2 is necessary for the development and maintenance of Foxp3+ regulatory T cells (Treg). It was known from the earliest discovery of Treg that these cells express high levels of CD25, the IL-2Rα chain, so it was not a surprise that they also respond to the cytokine. Mice lacking IL-2 have a profound deficiency of Treg and, as a result, uncontrolled activation of conventional (non-regulatory) T cells. It is still not known which cytokines or other stimuli that drive this massive T cell proliferation in the absence of IL-2 or IL-2R signaling. Nevertheless, the conclusion of the knockout mouse studies is that the essential, or non-redundant, function of IL-2 is to limit immune responses by acting on Treg, and its role in stimulating immune responses is redundant, and can be replaced by other stimuli.

Retrospective analyses of patients treated with IL-2 have shown a predicted increase in the numbers and suppressive activity of blood Foxp3+ T cells. Very few patients have been described with mutations in CD25. These patients typically present with immune deficiency and infections, as well as features of autoimmunity. In two of the patients who have been studied, Foxp3+ T cells are present but appear to be functionally defective.

The role of IL-2 in the life of Treg
The realization that IL-2 is essential for Treg has led to a vast number of studies asking how the cytokine generates and maintains these cells. Despite some minor contradictions in the literature, the bulk of the evidence, mostly from mouse models, suggests that IL-2 has multiple actions that collectively enhance Treg numbers and functions.

  • IL-2 is a survival factor for Treg in the periphery, and in its absence Treg lose their competitive fitness and undergo death by apoptosis.
  • IL-2 promotes the function of Treg by maximizing expression of key effector molecules such as CTLA-4.
  • IL-2 maintains the stability of Treg, in part by inducing demethylation of the Treg-specific demethylated region in the Foxp3 gene locus.
  • IL-2 may promote the development of Treg in the thymus, in response to recognition of self antigens.

One of the puzzles in this area is that IL-2 functions mainly as an autocrine growth factor, so the same T cells produce and respond to the cytokine, but Treg do not produce IL-2. It is believed that small amounts of IL-2 produced by conventional T cells during normal responses to environmental antigens may serve to maintain the peripheral pool of functional Treg. Thus, one under-appreciated role of immune responses to foreign antigens may be to indirectly maintain tolerance (unresponsiveness) to self antigens!

The therapeutic potential of IL-2: Prospects and challenges
As the ability of IL-2 to maintain Treg has become increasingly established and accepted, the possibility of using the cytokine to suppress pathologic immune responses has been raised. Two proof-of-concept studies were published in 2011 showing, in small numbers of patients, that treatment with low-doses of recombinant IL-2 had a beneficial effect in steroid-resistant graft-vs-host disease (GVHD) and in hepatitis virus-associated vasculitis. Based on these encouraging results, many more studies are now in the planning and execution stages, in type 1 diabetes, transplant rejection, and GVHD.

The continuing challenge in the therapeutic application of IL-2 is that it does serve dual opposing roles – it stimulates and inhibits immune responses, by acting on different T cell populations. A central question then becomes how to selectively manipulate its activity so it does what is desired. So far many of the clinical studies have relied on dosing, based on the idea that activation of effector and memory T cells requires transient exposure to high concentrations of IL-2 whereas maintenance of Treg requires prolonged exposure to low concentrations. It is, however, difficult to develop precise quantitative criteria for the appropriate concentration or dose of any cytokine needed for diverse actions. Several other approaches are being tried.

  • A very interesting possibility was raised by the finding that IL-2 could be targeted to Treg or effector T cells by making complexes with different anti-IL-2 antibodies. This phenomenon has been demonstrated in mice and humans. The antibodies may conceal different receptor-binding sites on the cytokine, thus leaving it available to bind to CD25 (and thus preferentially to Treg) or to the IL-2Rβγ receptors (thus focusing the cytokine preferentially on CD8+ T cells and NK cells).
  • Based on these results, attempts have been made to structurally modify IL-2 to target it to different receptors. A version of the cytokine has been synthesized that targets it to CD8+ T cells (and, probably, NK cells). So far, there is no modified IL-2 that preserves its activity on Treg only.
  • Another approach is to combine IL-2 with drugs that block different immune responses. The first clinical trial of IL-2 with rapamycin, predicted to inhibit effector T cells and preserve Treg, was done in type 1 diabetes but was terminated because the effector response was not adequately inhibited. As newer kinase inhibitors are developed to inhibit different lymphocyte populations, this type of combination therapy clearly has potential, especially if it becomes possible to identify differences in the signaling pathways used by effector T cells and Treg.

Summary and conclusions
The first cytokine to be characterized continues to reveal surprises. Perhaps the most impressive aspects of the story of IL-2 are how basic research in the biology of the cytokine has overturned dogma and provided unexpected new insights, and how these insights are being translated into therapies. IL-2 remains the paradigm for an immune modulator with often opposing actions, raising great possibilities as well as substantial challenges.

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Human Immunophenotyping Update

Imaging Flow Cytometry: Picture This!

J. Philip McCoy, Jr., PhD, NHLBI and the Center for Human Immunology, National Institute of Health

A number of variants of traditional flow cytometry, such as CyTOF and Luminex, have recently emerged as exciting new technologies that extend the utility of cytometry for clinical research. Imaging flow cytometry, a technology in which immunophenotyping data can be acquired in concert with capturing images of every cell analyzed, should also be recognized as one of these revolutionary new cytometric endeavors. Since separate images are captured for every fluorochrome on each cell, which can be merged into a multicolor image, it is possible to a myriad of analyses with these data. The main advantage of imaging flow cytometry is that it is now possible to visualize the pattern of staining of every marker examined on every cell in a sample. Such visualization permits one to examine features with relative ease as one is obtaining routine immunophenotyping data. Furthermore, as one can obtain these data on hundreds of cells per second, it is practical to avoid the pitfalls that one may encounter using confocal microscopy such as insufficient cell numbers for rigorous statistical calculations or the subjectivity of selecting a particular field of view for study. Using imaging flow cytometry data from thousands of cells can be collected in only a few minutes without any potential bias in how these cells were selected for analysis.

So other than satisfying your curiosity about how your cells look, how can you apply this technology to your research? Adding morphology to your flow cytometry permits answering some rather obvious questions, such as is the staining capped or polarized? Thaunat and colleagues (1) demonstrated asymmetric segregation of polarized antigens on B lymphocyte division that correlated with the ability of progeny to present antigen and activate T cells. Visualization of cellular protrusions or podopods, for example, may permit one to ask which antigens are present on these structures. Nobile and colleagues used this approach to visualize cell protrusions in lymphocytes induced by HIV-1 Nef (2). It is also possible to discriminate nuclear from cytoplasmic staining, hence nuclear translocation of transcription factors such as NF-κB is readily detectable (3). This technology is also capable to performing polychromatic immunophenotyping with roughly 6 to 8 different fluorochromes. Furthermore, as images are captured of each color separately; it is possible to determine the extent of antigen co-localization by staining cells simultaneously with two fluorochrome-labeled antibodies to those antigens. One can also examine cellular functions such as apoptosis and autophagy on cell subsets defined by multiple markers, and a recent publication has demonstrated an imaging flow cytometry techniques for measuring average telomere length using labels for repetitive DNA sequences in the human telomeric repeat (4).

Even more applications can be devised which take even more advantage of being able to capture images of the immunophenotyping. For example, phagocytosis of fluorescent particles can not only be detected, but also the number of phagocytosed particles can be counted (5). Imaging flow cytometry can also be used to directly visualize immunological synapses between T cells and antigen presenting cells (APCs) and the redistributions of antigens as these synapses form (6). Similarly, one can directly image target-effector conjugates as exemplified by Pascal et al (7). Erythrocyte morphology has been studied using imaging flow cytometry in studies of sickle cell anemia (8) and in Plasmodium faciparum (9).

Naturally, new technologies are not perfect in every way. Some shortcomings of the current generation of imaging flow cytometers include the lack of ability to sort – thus any cells identified by this technology cannot be purified for downstream analyses such as genomic studies. Imaging glow cytometry also has a much lower throughput and number of colors that can be run simultaneously than traditional flow cytometry, and the highest magnification is 60x. Finally, the sheer size of the data files limits the number of events that can be acquired in a single file. This is due to the fact that images are captured for every cell in every color, hence thousands of images are present in a single data file, yielding data files that can require 500MB for storage. Even with these caveats, imaging flow cytometry is an interesting technique that presents new opportunities for addressing difficult research questions.

References

1. Thaunat O, Granja AG, Barral P, Filby A, Montaner B, Collinson L, Martinez-Martin N, Harwood NE, Bruckbauer A, Batista FD. Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity. Science. 2012 Jan 27;335(6067):475-9. doi: 10.1126/science.1214100.

2. Nobile C, Rudnicka D, Hasan M, Aulner N, Porrot F, Machu C, Renaud O, Prévost MC, Hivroz C, Schwartz O, Sol-Foulon N. HIV-1 Nef inhibits ruffles, induces filopodia, and modulates migration of infected lymphocytes. J Virol. 2010 Mar;84(5):2282-93. doi: 10.1128/JVI.02230-09.

3. George TC, Fanning SL, Fitzgerald-Bocarsly P, Medeiros RB, Highfill S, Shimizu Y, Hall BE, Frost K, Basiji D, Ortyn WE, Morrissey PJ, Lynch DH.Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J Immunol Methods. 2006 Apr 20;311(1-2):117-29.

4. Ma H, Reyes-Gutierrez P, Pederson T. Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors. Proc Natl Acad Sci U S A. 2013 Dec 9. [Epub ahead of print].

5. Ackerman ME, Moldt B, Wyatt RT, Dugast AS, McAndrew E, Tsoukas S, Jost S, Berger CT, Sciaranghella G, Liu Q, Irvine DJ, Burton DR, Alter G. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J Immunol Methods. 2011 Mar 7;366(1-2):8-19. doi: 10.1016/j.jim.2010.12.016.

6. Hoffmann S, Hosseini BH, Hecker M, Louban I, Bulbuc N, Garbi N, Wabnitz GH, Samstag Y, Spatz JP, Hämmerling GJ. Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells. Immunol Lett. 2011 Apr 30;136(1):13-20. doi: 10.1016/j.imlet.2010.11.005.

7. Pascal V, Laffleur B, Debin A, Cuvillier A, van Egmond M, Drocourt D, Imbertie L, Pangault C, Tarte K, Tiraby G, Cogné M. Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells. Haematologica. 2012 Nov;97(11):1686-94. doi: 10.3324/haematol.2011.061408.

8. Fertrin KY, van Beers EJ, Samsel L, Mendelsohn LG, Saiyed R, Nichols JS, Hepp DA, Brantner CA, Daniels MP, McCoy JP, Kato GJ. Imaging Flow Cytometry Documents Incomplete Resistance of F-Cells To Hypoxia-Induced Sickling In Blood Samples From Patients With Sickle Cell Anemia. ASH, 2013.

9. Safeukui I, Buffet PA, Perrot S, Sauvanet A, Aussilhou B, Dokmak S, Couvelard A, Hatem DC, Mohandas N, David PH, Mercereau-Puijalon O, Milon G. Surface area loss and increased sphericity account for the splenic entrapment of subpopulations of Plasmodium falciparum ring-infected erythrocytes. PLoS One. 2013;8(3):e60150. doi: 10.1371/journal.pone.0060150.

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Selected Recent Clinical Trial Results

Review by: Elisa Boden, MD, Benaroya Research Institute

Phase 1/2 Study of Transdermal Delivery of Myelin Peptides in Relapsing-Remitting Multiple Sclerosis

Clinical Trial:
Walczak, A. et al. JAMA Neurol. 2013; 70(9): 1105-1109
PMID: 23817921

Disease: relapsing-remitting multiple sclerosis

Drug:
• Myelin peptides (MBP85-99, PLP139-151, MOG35-55) applied transdermally via skin patch

Study:

  • Phase 1/2, single center, randomized, double-blind, placebo-controlled study of transdermal delivery of myelin peptides in subjects with relapsing-remitting multiple sclerosis
  • 30 patients were randomized to placebo or 2 doses of myelin peptides delivered transdermally for one year; placebo (n=10), 1 mg myelin peptides (n=16), 10 mg myelin peptides (n=4)
  • MRI was performed at screening and every 3 months
  • The primary endpoints were safety and efficacy measured as the number of active gadolinium positive lesions on MRI per patient per scan
  • Secondary endpoint measures were mean volume of gadolinium positive lesions and cumulative number of new T2 lesions at months 3, 6, 9, and 12 and T2 lesion and T1 lesion volume change from baseline to 12 months
  • Secondary clinical endpoints included annual relapse rate, proportion of relapse-free subjects, proportion of subjects with 3 months of confirmed disability worsening on the Expanded Disability Status Scale (EDSS) at month 12.

Results:

  • There were some differences in baseline characteristics of subjects randomized to placebo versus myelin patches with a higher proportion of female subjects, shorter duration of disease and slightly higher baseline EDSS scores among subjects receiving placebo.
  • There were no serious adverse events documented in the study. The most common side effect of myelin peptide patches was a local reaction in the area of the skin patch.
  • The primary efficacy endpoint was met for the 1 mg dose with a 66.5% reduction in the number of gadolinium positive lesions per patient per scan (p= .02)
  • In terms of secondary endpoints, there was a trend toward reduction in the volume of gadolinium positive lesions per patient and the cumulative number of new T2 lesions per patient among patients receiving the 1 mg dose. There was a statistically significant reduction in the mean T1 and T2 lesion volume at 12 months among patients receiving the 1 mg dose of myelin peptides.
  • There were no differences in primary or secondary outcome measures for MRI between placebo and the 10 mg myelin peptide dose, although numbers in this group were small.
  • Subjects receiving both the 1 mg and 10 mg myelin peptide patch had statistically significant reductions annual relapse rates compared to placebo. In the 1 mg myelin patch group, there was also significant increase in the number of relapse-free subjects compared to placebo. There was a trend toward fewer patients with disability progression measured by EDSS in subjects receiving myelin peptide patches.

Why this trial is of interest to the broader FOCIS community:
The majority of therapeutic agents for the treatment of autoimmune diseases are globally immunosuppressive and leave patients vulnerable to serious infections and lymphoproliferative disorders. Therapies capable of inducing antigen-specific tolerance hold great promise as safer alternatives to current therapeutics for autoimmune disease. Specific immunotherapy (SIT) is such a therapeutic strategy that has been used clinically for many years to induce antigen-specific tolerance in allergic diseases. This method delivers repeated subcutaneous doses of antigens or peptides which ultimately induces antigenic tolerance that lasts beyond the duration of therapy. Although the mechanisms that lead to tolerance have not fully been elucidated, evidence suggests induction of regulatory T cells and/or immune deviation from Th2 to Th1 response play an important role1.

In the current study, SIT has been adapted for use in the treatment of multiple sclerosis by dermal delivery of peptide epitopes implicated in the immune response that drives multiple sclerosis. Although the study included a small number of patients, significant reductions in new brain lesions were identified in subjects on therapy which correlated with a reduction in symptomatic relapses. In a previously published study, the authors demonstrated that subjects receiving transdermal peptide therapy had reduced myelin-specific proliferative responses and increased IL-10 production in peripheral blood lymphocytes, providing evidence of immunoregulatory modulation by dermal peptide therapy2.

Interestingly, while the 1 mg peptide dose had statistically significant improvements in primary and secondary outcomes, the higher 10 mg peptide dose did not appear to be as effective. This may simply have been a result of the very small sample size in the 10 mg dose group. However, this may be reflective of a dose-dependency to the tolerogenic response, which has been described in tolerance to orally delivered antigens3.

While the results from this study are encouraging and dermal peptide therapy appears safe, the study population is very small and differences in the baseline characteristics subjects may have favored a positive outcome for the treatment group. Notably, while oral delivery of myelin has been demonstrated to prevent and treat animal models of multiple sclerosis, a phase III study of orally administered bovine myelin did not show clinical benefit in patients with multiple sclerosis3. Should significant clinical benefits of dermal myelin peptides hold up in a larger cohort, it may be instructive to study how dermal and oral delivery of antigens result in differential immune responses.


References
1 Moldaver D, et al. Allergy. 2011; 66 (784-91)
2 Jurynczyk M, et al. Ann Neurol. 2010; 68 (5): 593-601.
3 Faria AM et al. Immunol. Rev., 206 (2005): 232-259.


Phase 2 Trial of Anti-IP-10 Antibody for Ulcerative Colitis

Clinical Trial:
An overview of Mayer L. et al., Anti-IP-10 antibody (BMS-936557) for Ulcerative Colitis: A Phase II Randomised Study. Gut 2013;0:1-9. PMID: 23461895

Disease: Ulcerative Colitis

Drug: BMS-936557 (formerly MDX-1100) is a fully human monoclonal antibody that targets the chemokine interferon-γ-inducible protein-10 (IP-10), also referred to as CXCL10.

Study Design:

  • 8-week, Phase II, double-blind, multicenter, randomized study to evaluate safety and efficacy of BMS-936557 in patients with moderately-to-severe active ulcerative colitis (UC).
  • Patients were randomly assigned to receive 10 mg/kg to 1000 mg of BMS-936557 (n=55) or matching placebo (n=54) intravenously every other week for 8 weeks. Eligible patients were ≥18 years old with an active UC flare within 2 weeks of enrollment while on stable treatment with standard, non-biologic therapy.
  • The primary efficacy endpoint was the rate of clinical response 2 weeks after the last study treatment. Clinical response was defined on the basis of standardized scores which include patient reported symptoms such as stool frequency and rectal bleeding.

Results:

  • The primary and secondary efficacy endpoints were not met in this study. The proportion of patients who achieved clinical response was not significantly different in the BMS-936557-treated group (52.7%) versus placebo-treated group (35.2%; p=0.083). However, patients who failed to adequately record symptoms were considered non-responders. That means that all such patients (n=7) were analyzed as if the drug was not helpful. This “imputation model” is an accepted statistical approach to deal with missing data points. If instead of imputing these subjects’ results as being negative the trial was analyzed (post-hoc) using the data that actually was available, the clinical response rates were significantly higher in the active treatment group.
  • The investigators also explored (post-hoc) the relationship of the drug levels [(trough concentrations (Cmin)] with clinical response rate. Sixteen patients with the highest Cmin values were observed to have a significantly higher clinical response rate (87.5%) versus the placebo group (p<0.001). Also, a significantly greater proportion of patients with BMS-936557 Cmin ≥ 100 µg/ml were determined to be in histological remission compared with placebo (73% vs. 41%, p=0.004), exhibiting a marked reduction in inflammatory infiltrates and decrease in erosion, ulceration and crypt destruction.
  • There were no related significant adverse events or deaths during this study. The BMS-936557-treated group had a numerically higher frequency of infections compared with placebo. This was not the case in a previous Phase 2 study of BMS-936557 in patients with rheumatoid arthritis. The safety profile was the same for patients in the Cmin ≥ 100 µg/ml subgroup.

Why the trial is of interest to the broader FOCIS community:
Chemokines and chemokine receptors are central to the inflammatory process and therefore attractive therapeutic targets. This trial illustrates a potential advantage of targeting chemokines as opposed to their receptors in specific autoimmune diseases. Binding of the chemokine IP-10 to its receptor, CXCR3, induces differentiation and recruitment of Th1 and Th17 cells responsible for inflammation and tissue destruction. In addition, IP-10 has been shown to operate independently of its receptor, as a direct inhibitor of epithelial and endothelial cell proliferation, 1 and as a contributor to bone destruction (by upregulating RANKL)2 and apoptosis of pancreatic β-cells.3

Why then did this trial have a negative outcome? This trial illustrates how significantly study design can impact efficacy outcomes. Failure to meet the primary endpoint might be attributed to insufficient compliance on the part of participants to complete the electronic diary of symptoms. Thus, this trial not only had a negative outcome, it is a failed trial in that we are still unclear as to whether or not this therapy is worth pursuing. Though use of imputation attributing missing data as negative is an appropriate pre-specified analysis plan; this trial also illustrates that the choice of analytic approach may often dramatically affect the “results” of a study. 

In addition, the relationship between drugs levels and outcome suggests that a higher dose might have resulted in a different outcome. Because this was a post-hoc analysis, even this premise would need additional testing. There is still reason to believe that, at the right dose, an anti-IP-10 antibody may be effective in UC because of both inhibition of Th1 cell differentiation and trafficking, and promotion of crypt epithelia cell proliferation and regeneration. Based on both this study in UC and the previously published Phase 2 study in rheumatoid arthritis – which showed modest but significant response–4 additional dose-ranging studies of BMS-936557may be informative.

References:

  1. Campanella et al., PLoS ONE 2010: 5(9): e12700.
  2. Kwak et al., Arthritis Rheum 2008:58:1332-42.
  3. Schulthess FT et al., Cell Metab 2009:9:125-39.
  4. Yellin M et al., Arthritis Rheum 2012:64(6): 1730-1739.

Reviewed by Steven Lamola, MD Benaroya Research Institute

 

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