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March 23, 2015

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

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

Highlights from Recent Literature

Modulation of CD4+ T Cell Metabolism Improves Lupus

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

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

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

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

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


Inhibition of the mTOR Pathway Improves Immune Function in Older Individuals

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

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

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

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

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


Nature or Nuture? T Cell Differentiation?

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

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

SUMMARY

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

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

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


Divergence: Twin Study into Immune Variation

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

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

SUMMARY

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

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

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


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

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

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

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

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

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


Plasmablasts Induce Tfh Differentiation via IL-6 Production

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

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

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

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

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


IL-9 in Peanut Allergy

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

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

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

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

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


Biopolymer Implants Enhance the Efficacy of Adoptive T Cell Therapy

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

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

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

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

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


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

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

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

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

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

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

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Highlights From Clinical Immunology, the Official Journal of FOCIS

Double Negative Epigenetics

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

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

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

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

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


No Tears for a Sick Mouse?

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

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

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

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

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Human Immunophenotyping Update

Publishing Flow Cytometry Data

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

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

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


                                                  Table 1. Reagents Used                           Table 2. Cytometer Configuration

rsz 3tables

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

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

Picture1

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

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

References

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

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

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Developments in Basic Immunology and Novel Therapies

B Cell Depletion Therapy for Autoimmune and Inflammatory Diseases

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

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

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

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

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

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

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

References:

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

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

Rixuximab Versus Azathioprine for Maintenance in ANCA-associated Vasculitis

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

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

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

Study design:

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

Results:

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

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

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

References:

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

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

Reviewed by Sandra Lord, MD, Benaroya Research Center


Brodalumab, An Anti-IL17RA Monoclonal Antibody in Psoriatic Arthritis

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

Disease: Psoriatic Arthritis

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

Study design:

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

Results:

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

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

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

References:

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

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

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

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

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

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

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

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

Reviewed by Sandra Lord, MD, Benaroya Research Institute


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

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

Disease: Type 1 diabetes

Drug:

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

Study design:

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

Results:

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

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

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

References:

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

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

Reviewed by Sandra Lord, MD, Benaroya Research Center

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