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Translational Immunology Update©
A quarterly publication of FOCiS
March 15, 2013

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

Highlights in Recent Literature | Clinical Immunology Highlights | Immunphenotyping | Basic Immunology | Clinical Trials | Previous Issues

Highlights from Recent Literature

CD8 T Cell Avidity is Dependent on T Regulatory Cells

A review of Pace L., et al. Regulatory T Cells Increase the Avidity of Primary CD8+ T Cell Responses and Promote Memory. Science 338; 532-36, 2012. PMID: 23112334

T regulatory cells (Tregs) are necessary to prevent aberrant immune activation that can lead to autoimmunity. Interestingly however, Tregs do not appear to prevent immune activation in response to pathogens. In fact, in two separate reports, Tregs have been shown to contribute to the clearance of L. major and herpes simplex virus (Belkaid et al., 2002; Lund et al., 2008). The precise mechanisms as to how Tregs contribute to the priming of the immune response remain unclear. Here, Pace et al. utilize FoxP3 DTR mice, in which FoxP3+ cells express the human diphtheria toxin receptor, to eliminate Tregs via an injection of diphtheria toxin. They administered the toxin before administering a foreign antigen which activates CD8 T cells, and employed MHC-multimers to measure the frequency of antigen reactive T cells during the response. Their key findings were:

  • A low percentage of high affinity Db-Uty (male) specific CD8+ T cells were replaced by a relatively high percentage of low affinity Db-Uty specific CD8+ T cells upon depletion of Tregs from female recipients that received an i.p. injection of male splenocytes.
  • Under conditions more similar to a physiological infection, high multimer-binding Ova-antigen specific OT-I cells exist at a higher frequency following infection with Listeria monocytogenes that express ova (LM-OVA). However, when Tregs are depleted prior to infection, the low multimer-binding OT-I cells increase in frequency.
  • When Tregs are depleted from mice at the time of infection with LM-OVA, and mice are rechallenged with LM-OVA 50 days later, there are significantly more bacteria in the spleen and liver as well as a lower frequency of antigen specific CD8 T cells, suggesting a diminished memory response to LM-OVA in the absence of Tregs.

Taken together, these results paradoxically highlight the importance for Tregs in the immune response to non-self antigens and also in the formation of memory to pathogens. Not only do low-affinity clones expand in the absence of Tregs, but the avidity of the response also decreases, highlighting the importance of Tregs in promoting efficient pathogen clearance. This study also presents another potential mechanism by which Tregs might prevent autoimmunity by inhibiting the accumulation of the low-affinity and potential self-crossreactive CD8 T cell clones that arise in the periphery due to constant exposure to self-antigen.

Reviewed by Christopher Borges and Laurence Turka, MD, Beth Israel Deaconess Medical Center and Harvard Medical School.


Bacterial Flagellin Present in House Dust Extracts (HDEs) Promote Asthma

A review of Wilson R.H., et al. The Toll-like Receptor 5 Ligand Flagellin Promotes Asthma by Priming Allergic Responses to Indoor Allergens. Nature Medicine 181705; 1705-11, 2012. PMID: 23064463

Regulatory T cells (Tregs) are critical for suppressing harmful autoimmune reactions. In chronic inflammatory diseases such as multiple sclerosis, Tregs are often present but are incapable of suppressing the activity of pathogenic effector T cells. The authors studied the mechanism of Treg-resistance in patients with multiple sclerosis and found that it was critically dependent on IL-6 signaling.

  • Using assays of T cell suppression, the authors demonstrated that effector T cells in patients with ongoing flares of relapsing-remitting multiple sclerosis (RRMS) were resistant to Treg-mediated suppression. Effector T cells from RRMS patients in remission and from normal individuals were suppressed normally by Tregs.
  • Previous studies have shown that IL-6 signaling and phosphorylation of STAT3 in effector T cells can contribute to effector T cell resistance to suppression.
  • Effector T cells in RRMS patients with active disease showed increased IL-6 receptor alpha expression and elevated IL-6 signaling as shown by increased levels of phosphorylated STAT3 (pSTAT3), as compared to patients with quiescent disease and healthy controls. Increased levels of pSTAT3 and IL-6 receptor expression correlated with resistance to suppression in in vitro suppression assays.
  • Blockade of STAT3 phosphorylation in vitro in effector T cells restored the ability of these cells to be suppressed by Tregs.

Taken together, these results paradoxically highlight the importance for Tregs in the immune response to non-self antigens and also in the formation of memory to pathogens. Not only do low-affinity clones expand in the absence of Tregs, but the avidity of the response also decreases, highlighting the importance of Tregs in promoting efficient pathogen clearance. This study also presents another potential mechanism by which Tregs might prevent autoimmunity by inhibiting the accumulation of the low-affinity and potential self-crossreactive CD8 T cell clones that arise in the periphery due to constant exposure to self-antigen.

Reviewed by Christopher Borges and Laurence Turka, MD, Beth Israel Deaconess Medical Center and Harvard Medical School.


Double Trouble: IL-17 Producing Tregs Promote Colon Cancer and Impair Immune Responses

A review of Blatner, N.R., et al. Human Colon Cancer Expression of RORt Marks a Pathogenic Regulatory T Cell Subset in Human Colon Cancer. Science Translational Medicine 4, 164ra159 (2012). PMID: 23241743

Immunosuppressive regulatory T cells (Tregs) play critical roles in suppressing autoimmunity. Tregs are expanded in and/or recruited to most human cancers, where they either suppress anti-cancer immunity or, paradoxically, protect the host by suppressing pro-carcinogenic inflammation. By studying colon cancer in humans and in a mouse models, the authors found that a subset of RORγt -expressing Tregs promoted colon cancer while at the same time suppressing antitumor immunity.

  • Human colon cancers contained a distinct population of RORγt -expressing Tregs that retained suppressive capacities and yet appeared to be pro-inflammatory. These Tregs were not observed in normal donors.
  • Tregs with similar characteristics were present in the tumors of mouse models of hereditary polyposis. Ablation of the RORγt gene in FOXP3+ Tregs restored the anti-inflammatory effects of these cells, suppressed inflammation, improved anti-polyp immune responses and reduced the number of pre-cancerous polyps in these mice.
  • Deficiency of the IL-17A gene in these mice reduced the formation of precancerous polyps but these mice still had RORγt -expressing Tregs. Although fewer polyps were present, the remaining precancerous lesions were more likely to progress to invasive colon cancer in the absence of IL-17. This suggests IL-17 promotes polyp formation but may protect against progression to invasive cancer.

The authors observed that distinct Treg subsets are present in human colon cancer with pro-inflammatory and anti-inflammatory phenotypes and functions. RORγt -expressing Tregs were pro-inflammatory, supported the development of pre-cancerous polyps, and reduced anti-polyp immune responses. IL-17 produced by these Tregs increased the formation of precancerous polyps but paradoxically impaired progression to invasive cancer. In summary, the authors find Tregs within cancer can be surprisingly diverse and that some of Tregs can directly promote carcinogenesis as well as suppressing anticancer responses.

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


IL-6 Mediates Effector T Cell Resistance to Tregs in Multiple Sclerosis

A review of Schneider A.,et al. In Active Relapsing-remitting Multiple Sclerosis, Effector T Cell Resistance to Adaptive Tregs Involves IL-6–mediated Signaling. Science Translational Medicine 5, 170ra15 (2013). PMID: 23363979

Regulatory T cells (Tregs) are critical for suppressing harmful autoimmune reactions. In chronic inflammatory diseases such as multiple sclerosis, Tregs are often present but are incapable of suppressing the activity of pathogenic effector T cells. The authors studied the mechanism of Treg-resistance in patients with multiple sclerosis and found that it was critically dependent on IL-6 signaling.

  • Using assays of T cell suppression, the authors demonstrated that effector T cells in patients with ongoing flares of relapsing-remitting multiple sclerosis (RRMS) were resistant to Treg-mediated suppression. Effector T cells from RRMS patients in remission and from normal individuals were suppressed normally by Tregs.
  • Previous studies have shown that IL-6 signaling and phosphorylation of STAT3 in effector T cells can contribute to effector T cell resistance to suppression.
  • Effector T cells in RRMS patients with active disease showed increased IL-6 receptor alpha expression and elevated IL-6 signaling as shown by increased levels of phosphorylated STAT3 (pSTAT3), as compared to patients with quiescent disease and healthy controls. Increased levels of pSTAT3 and IL-6 receptor expression correlated with resistance to suppression in in vitro suppression assays.
  • Blockade of STAT3 phosphorylation in vitro in effector T cells restored the ability of these cells to be suppressed by Tregs.

Active and effective regulatory T cell function is critical for maintaining self tolerance. The authors find that effector T cells become resistant to Treg suppression during flares of MS and that this resistance is likely mediated by IL-6 signaling. These studies suggest that inhibiting IL-6 signaling in effector T cells may have the potential to enhance tolerance, suppress disease flares and slow disease progression in multiple sclerosis and possibly other autoimmune diseases as well.

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


Immune Crosstalk and Off Target Immunity

A review of Su, et al. Virus Specific CD4+Memory-Phenotype T Cells are Abundant in Unexposed Adults. Immunity (2013) S1074-7613(13). PMID: 23395677

In order to train the immune system to fend off pathogens, we design antigen specific vaccines to stimulate the activation of naïve B and T cells in order to create a pool of memory B and T cells that will remain at the ready to protect against any attempt by that pathogen to infiltrate our physical and biological defenses. However, there has been recent mouse model work showing that there can be memory-phenotype T cells in a host in the absence of exposure to the relevant antigens. In this paper, Su et al investigate the relationship between antigen exposure and memory cell populations in human T cell populations. Interestingly, they were able to delve deeper into the role of non-cognate antigen exposure by comparing rates of antigen specific memory T cells in the absence of viral exposure in adults and neonates (using cord blood). Potential mechanisms for the generation of antigen specific memory without evidence of viral infection are explored.

  • Su et al. characterize antigen specific T cell frequencies for unexposed viral antigens and self antigens (implicated in oncologic and autoimmune processes) in 26 healthy adults using an enrichment technique with class II MHC tetramers bearing the selected peptide antigens.
    • They compared the frequency of anti-viral T cells in individuals who had and had not been exposed to a given antigen (for example, seropositive vs. negative for HSV) and found that antigen exposure did not always predict a larger population of antigen specific T cells.
    • Interestingly, there was also not a significant difference between the frequencies of antigen specific T cells that recognize self antigens versus non-exposed viral antigens.
    • In addition, they noted that anti-self antigen T cells were more frequent in females versus males, consistent with the known gender bias in autoimmunity incidence.
  • When they examined the nature of the antigen-specific T cells, they noted that many T cells that recognized unexposed viral antigens were in fact memory phenotype cells.
    • To show that these memory phenotype cells were functionally memory cells they assayed the ability of tetramer positive cells to produce cytokines more quickly than naïve and tested known gene expression profiles associated with naïve and memory T cells. Finally, there was evidence of increased clonality in the memory phenotype versus the naïve phenotype cells.
  • To examine the potential role of non-cognate activation, they compared adult samples to umbilical cord blood samples. While there were a similar frequency T cells specific for non-antigen exposed pathogen and self-antigen specific T cells they did not show similar frequency of memory phenotype cells at birth in comparison to the adults studied in the first part of the study.
  • The chemokine CCL22 was identified as a direct miR-34a target. Importantly, there was an inverse relationship between CCL22 and miR-34a expression in HBV+ HCC.
  • CCL22 binds CCR4, a chemokine receptor expressed on regulatory T (Treg) cells. In an elegant cell culture system, inhibition of miR-34a in HCC cells resulted in increased CCL22 and robust migration of Tregs.
  • Importantly, a neutralizing CCL22 antibody blocked Treg migration.
  • To study the possible source of antigen specific memory phenotype T cells in the absence of relevant pathogen exposure in adults, they created 24 HIV specific T cell clones from three donors and then stimulated them with HIV-1 antigens and with similar peptide sequences from other microorganisms, finding that more than 20% of the clones produced cytokines in response to similar peptides from a wide range of bacteria, algae and plants.
  • In other words, anti-HIV-1 T cells can be stimulated by activating the immune system with many, varied microorganisms in our environment.
  • Finally, they vaccinated two volunteers that had not had influenza vaccines in > 5 years, which resulted in influenza antibody titers and the development of cross-reactivity to other microorganisms.

This paper carefully investigates the nature of cross reactivity in human CD4 T cells. The demonstration that adults who have not been exposed to a given pathogen can have a significant population of antigen specific T cells with a memory phenotype, as well as the capacity for speedy functional responses and gene expression profiles consistent with memory cell populations is fascinating. The delineation of the mechanism of the generation of these cells is the most interesting question, coming down to the two most likely mechanisms—homeostatic proliferation versus exposure to environmental crossreactive antigens. With many groups focusing on the details of human immunology, with an upsurge in focus at the single cell level as well as on comparisons between mouse model data and human investigations, data is being accumulated at an incredible rate. This paper investigates the correlates in human T cells of intriguing results in mouse data. Here, while adults have antigen specific populations of memory phenotype T cells in the absence of exposure to the specific pathogens, the same antigen-specific T cells (in the absence of relevant antigen exposure) in newborns do not have memory phenotypes, which suggests a role of a lifetime of exposure to infectious and environmental antigens in the repertoire of both antigen-specific and crossreactive T cells. In addition, T cells enriched based on a specific viral antigen specificity are shown to have TCRs with the capacity to bind peptides from a wide variety of organisms. Finally, when healthy humans were vaccinated with influenza vaccine, they developed a population of T cells whose TCRs could bind peptides from other microorganisms. Of course, there are always questions that remain to be answered—does the ability of a TCR to bind to related peptides in an MHC complex prove that the hosts can effectively defend themselves against those pathogens? If so, is the ability to generate T cells that can respond to pathogens to which the host has not been exposed of benefit to humans (in that we are protected against pathogens we have not experienced) or is it dangerous (in that we are now likely in possession of T cells that recognize both pathogens we have conquered as well as possibly self antigens we’d rather remain tolerant towards) or does the balance of those considerations come out even, at least at the population level? Given the importance of these questions and the diversity of human repertoires, it will require large cohorts to investigate these questions, with a focus on the tantalizing possible “off target” effects of both vaccination and infectious disease with the possible long term correlations including protection from infectious disease, the development of autoimmunity and, possibly, even oncologic transformation.

Reviewed by Sarah Henrickson, MD, PhD, Boston Children’s Hospital and Harvard Medical School.


Of Mouse and Man

A review of Seok, et al. Genomic Responses in Mouse Models Poorly Mimic Human Inflammatory Diseases. Proceedings of the National Academy Sciences. 2013. 110:3507-12. PMID: 23401516

While mice are arguably the key animal model in immunology, elucidating links between murine and human physiology and pathology remains a topic of active investigation, especially given the importance of animal model data in clinical trials and in choosing candidates and questions for logistically complex human studies. While many differences in the two systems have been noted, these could be argued to result from differences in both underlying biology and/or differences in the experimental paradigms (in many cases, likely both could be implicated). This paper takes on the task of comparing inflammatory conditions between murine models and human disease, finding many differences.

  • Seok et al aimed to compare murine and human responses to various systemic inflammatory conditions, including trauma, burn injury and endotoxin exposure by studying gene expression from PBMCs. They showed impressive similarity between human burn and trauma, with some similarity of both to human endotoxin exposure. In comparison, there were poor correlations between gene expression of orthologs in human and murine models of a given condition, and poor correlations among the three murine models.
  • Seok et al also compared the timing of transcriptional changes across human and murine clinical scenarios.
  • Perhaps as expected, given the short duration of murine lifespans, while the initial post-inflammatory transcriptional changes occurred on similar timescales between human and murine systems, the duration of recovery was months in humans and hours to days in mice.
  • In addition, Seok et al investigated the pathways and individual genes underlying these inflammatory models (human and murine), comparing each to human burns. In each case, human injury more closely resembled human burns than any mouse model, including murine burns.

As a community, it is fundamental to our use of murine models in our pursuit of deeper understanding of human disease that we can find significant parallels in physiology, pathophysiology and response to therapeutic interventions between humans and the model system of choice. It is intriguing that they have investigated the details of complex, inflammatory conditions in this study, rather than simply focusing on cells at rest. However, while this study is fascinating, it is important to note, as the authors have, that in comparing disease states, there are not only differences in the inherent biology of the organisms (both at a species level and the amount of difference seen between individuals from inbred mouse strains and humans) but also fundamental differences between a critically ill human and an induced model of critical illness in a mouse. This study carefully focuses on some of the key differences, including the appropriate timepoints to choose for analysis, given the differences in lifespan of the two organisms. In addition, the samples studied are unsorted PBMCs, which does not allow consideration of differences between similar cell types in the two species. Overall, this study provides an important window into the key assumption made by many immunologists that mice and humans can be considered nearly interchangeable from an immunological perspective. The focus within the work on the differences between human disease and murine models of inflammation will hopefully stimulate a robust discussion around how to chose and validate models, both for basic research and clinical trials.

Of note, a related study came out last month as well, and in Shay et al (PNAS (2013) 110: 2946-2951), there is a demonstration of a highly detailed comparison of two compendia of transcriptional studies of sorted mouse and human cell lineages, during both resting and activated states with evidence of both shared gene transcription programs as well as tantalizing differences between orthologs. Clearly, there will need to be further studies to follow up on this and prior work that will help us continue to improve our understanding of the benefits and limitations of mice as models for understanding the physiology and pathophysiology of the human immune system.

Reviewed by Sarah Henrickson, MD, PhD, Boston Children’s Hospital and Harvard Medical School.


Regulatory B Cells May Facilitate Chronic Joint Inflammation in Rheumatoid Arthritis

A review of Flores-Borja F., Bosma A., Ng D., Reddy V., Ehrenstein M.R., Isenberg D.A., Mauri C. CD19+CD24HICD38HIB Cells Maintain Regulatory T Cells While Limiting TH1 and TH17 Differentiation. Science Translational Medicine. 2013 Feb 20;5 (173):173ra23. PMID: 23427243

Regulatory B cells (Bregs) are a small subset of CD19+CD24HICD38HI B cells that constitute approximately 1% of the peripheral blood leukocyte population. They are thought to regulate T cell function via the production of IL-10 and the engagement of costimulatory molecules, which results in inhibition of interferon-γ (IFNγ) and tumor necrosis factor-γ (TNFγ) by effector T cells. They may also promote the generation of induced regulatory T cells (Tregs). While they have been shown to inhibit autoimmune disease in mouse models, their role in human autoimmune disease is less clearly established. In the manuscript by Flores-Borja, Mauri and colleagues, the authors test the hypothesis that defective Breg function might mediate the defect in Tregs observed in rheumatoid arthritis (RA) patients while permitting the generation of proinflammatory T helper 1 (TH1) and TH17 cells. They compare the frequency and function of Bregs obtained from the peripheral blood of healthy controls and RA patients with active or quiescent disease. There are several interesting findings from this work:

B-regs from healthy individuals limited differentiation of naïve CD4 T cells into TH1 and TH17 cells. They also promoted the generation of CD4+FoxP3+ Tregs from naïve T cells.
B-regs obtained from RA patients with active disease were able to inhibit TH1 differentiation as measured by production of IFNγ and TNFα. However, they failed to prevent TH17 development and were not able to promote differentiation of effector CD4+ T cells into functional Tregs.
Importantly, co-culture of Bregs from healthy controls with RA patient T cells demonstrated that effector T cells from RA patients were not intrinsically refractory to differentiation into TH17 or Tregs with suppressor function. In contrast, RA patient Bregs could not induce the differentiation of Tregs from healthy control naïve CD4+ T cells.
The number of Bregs was significantly lower in the blood of patients with active RA relative to healthy controls and was inversely correlated with C reactive protein (CRP) levels, a marker of disease activity. Interestingly, serial monitoring of RA patients demonstrated that Breg numbers normalized when disease was under control and inactive in RA patients and decreased with disease flares.
Breg number in RA patients did not correlate with the age of the patient or treatment regimen.
Breg numbers in patients with other autoimmune diseases including psoriatic arthritis, Sjogren’s syndrome, and ankylosing spondylitis were similar to healthy controls, suggesting that dysregulation of Bregs is a feature of active RA.

Historically, T cell dysfunction has been thought to play a central role in the pathogenesis of RA. This article is interesting because it highlights a central role for Bregs in RA. The authors postulate that a decrease in Bregs during active disease results in an inability to induce Treg differentiation, thereby enabling the establishment of TH17 cell differentiation and a proinflammatory environment. This raises an important question: What regulates the differentiation and maintenance of Breg numbers in RA patients? It is interesting that the decrease in Breg numbers paralleled disease activity and appeared to not be influenced by the treatment regimen (although the study may not have been appropriately powered to firmly establish this). Addressing this issue will be important as the data to date suggest that enhancing Breg numbers and function could improve the control of RA.

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


Identification of ‘Pseudoexhausted’ T Cells in Chronic Lymphocytic Leukemia (CLL)

A review of Riches J.C., Davies J.K., McClanahan F., Fatah R., Iqbal S., Agrawal S., Ramsay A.G., Gribben J.G. T Cells from CLL Patients Exhibit Features of T Cell Exhaustion but Retain Capacity for Cytokine Production. Blood. 2012 Dec 17. [Epub ahead of print] PMID: 23247726

T cell exhaustion is an acquired state of T cell dysfunction characterized by increased expression of inhibitory receptors such as programmed death-1 (PD1), CD160, and CD244 and poor effector function including decreased proliferative capacity, impaired cytolytic function, and reduced cytokine production. T cell exhaustion is believed to play a critical role in the optimal immune control of chronic viral infections and tumor cells. It has been shown to be a poor prognostic indicator in several malignancies. Consistent with this, inhibiting PD1 in human melanoma patients can reverse T cell exhaustion resulting in significant tumor responses in a subset of patients. While it has long been appreciated that CLL patients often have profound defects in T cell function and increased susceptibility to infections, the mechanisms underlying this dysfunction are poorly understood. In this report, Riches and colleagues explore the role of T cell exhaustion in chronic lymphocytic leukemia patients (CLL). They compare the phenotypic and functional characteristic of peripheral blood T cells in 39 CLL patients with early stage disease and 20 age-matched healthy donors. To control for the potential confounding impact of chronic viral infection, donors were also balanced for CMV seropositivity (56% of CLL patients and 65% of controls). There are several intriguing findings from this work.

  • CD8+ cells were preferentially expanded in CLL patients irrespective of CMV serostatus.
  • CD4 and CD8 T cells from CLL patients had increased expression of inhibitory markers CD244, CD160, and PD1 relative to healthy controls, suggesting a T cell exhaustion phenotype. Inhibitory receptor expression was highest on CD8+ T cells.
  • CD8+ T cells from CLL patients demonstrated functional defects in proliferation and cytoxicity. The cytotoxic defect was caused by impaired granzyme packaging into vesicles and poor immune synapse formation that resulted in nonpolarized degranulation.
  • A common feature of T cell exhaustion is failure to produce effector cytokines. Surprisingly, CD8 T cells from CLL patients had normal IL-2 production and increased production of INFγ and TNFα. The augmented cytokine production correlated with increased expression of Tbet, a key regulator of TH1 cytokine production.
    Although CMV serostatus modulated the distribution of lymphocyte subsets in patients and controls, it did not impact the augmented expression of inhibitory receptors or the functional defects in T cells from CLL patients.

Taken together, these data indicate that T cells from patients with CLL exhibit some features of T cell exhaustion in regards to poor proliferative responses and defective cytotoxic activity. Intriguingly, unlike classically described T exhaustion, the ability to produce cytokine was retained and even augmented. This suggests T cell dysfunction in the context of malignancy may vary depending on the underlying cancer subtype. These findings also raise the possibility that immunomodulatory approaches such as PD1 blockade may be efficacious in CLL.

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


Opposing Effects of Gemcitabine and 5-Fluorouracil on Antitumor Immunity

A review of Chemotherapy-triggered Cathepsin B Release in Myeloid-derived Suppressor Cells Activates the Nlrp3 Inflammasome and PromotesTtumor Growth. Nature Medicine 2013; 19:57-64. PMID: 23202296

Chemotherapy is a major treatment modality for cancer that is primarily thought to suppress the immune system. However, recent evidence suggests that some chemotherapeutic agents promote antitumor immune responses through various mechanisms including increasing antigen presentation, sensitizing tumor cells to NK and T cell mediated lysis, and selectively killing immunosuppressive cell subsets. Gemcitabine (Gem) and 5-fluorouracil (5FU) are two chemotherapeutic agents used in the clinic that have been shown to promote antitumor immunity through the depletion of myeloid-derived suppressor cells (MDSC), a population of immature myeloid cells known to suppress antitumor T cell responses. In this study, Melanie Bruchard and colleagues demonstrate that the effects of Gem and 5FU on MDSC can also promote tumor growth. This effect occurs prior to the induction of MDSC cell death and is mediated through the activation of NOD-like receptor family, pyrin domain containing-3 protein (NLRP3) inflammasomes in MDSC, leading to IL-1β release and the polarization of pro-tumorigenic TH17 T cell responses.

  • The authors showed that in vitro treatment of MSC-2 cells, a murine MDSC cell line, with 5FU or Gem induced caspase-1 activation prior to, and independently from the induction of MSC-2 cell death. They confirmed their in vitro results in MDSC from EL4 lymphoma-challenged mice treated with 5FU or Gem, and showed that treatment-induced caspase-1 activation was similar in both granulocytic and monocytic MDSC subsets. They also showed that 5FU treatment induced caspase-1 activation in MDSC from metastatic colorectal patients.
  • MDSC from EL4-bearing mice treated with 5FU and Gem were shown to secrete IL-1β, and treatment of EL4-bearing mice with 5FU resulted in elevated serum concentrations of IL-1β. IL-1β serum levels were also found to be increased in 9 of 12 colorectal patients following 5FU treatment. Using caspase-1 and NLRP3 deficient mice, and NLRP3-specific shRNA transduced MDSC, the authors showed that 5FU and Gem induced IL-1β secretion from MDSC through caspase-1 mediated activation of the NLRP3 inflammasome.
  • Chemotherapy-induced MDSC inflammasome activation was shown to be mediated through the permeabilization of lysosomes and the release of mature cathepsin B, and could be inhibited by lysosomal acidification using bafilomycin A, cathepsin B inhibitors, and cathepsin B-specific knockdown using shRNA.
    5FU-induced IL-1β expression by MDSCs markedly decreased the antitumor effect of 5FU in EL4 lymphoma, 4T1 mammary carcinoma, B16F10 melanoma and Lewis lung carcinoma animal models. To confirm the role of IL-1β, the authors demonstrated that the antitumor efficacy of 5FU was enhanced in these cancer models when co-administered with the soluble IL-1 receptor antagonist anak←inra.
  • The pro-tumorigenic effect of 5FU was shown to be mediated through the IL-1β-dependent polarization of TH17 T cell responses. To confirm this, the authors showed that the antitumor effect of 5FU was enhanced in IL17-deficient mice.

The data in this paper demonstrate that 5FU and Gem have opposing effects on antitumor immune responses. On one hand, these chemotherapeutic agents promote antitumor immune responses through the selective depletion of immunosuppressive MDSC. On the other hand, 5FU and Gem abrogate their antitumor immune effect by activating the NLRP3 inflammasome in MDSC, resulting in the secretion of IL-1β and the polarization of pro-tumorigenic TH17 responses. These data help explain why previously reported studies have shown conflicting results for these agents and their effects on antitumor immunity. In addition, these data also suggest that the antitumor efficacy of 5FU and Gem may be enhanced by combining them with IL1 inhibition in the clinic. Importantly, this study provides an excellent example of how chemotherapy and immunotherapy can be appropriately combined to achieve improved antitumor efficacy.

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


Immunization Route Matters for Mucosal Tumors

A review of Sandoval F., et al. Mucosal Imprinting of Vaccine-induced CD8+ T Cells is Crucial to Inhibit the Growth of Mucosal Tumors. Science Translational Medicine. 2013; 172,172ra20. PMID: 23100628

Systemic lupus erythematosus (SLE) is associated with the production of autoantibodies reactive against host DNA. Although the role of DNA-specific antibodies in SLE is not clear, anti-DNA antibodies commonly have toxic effects on cells. 3E10 is an anti-DNA monoclonal antibody isolated from a mouse model of SLE that is not toxic to normal cells, but is capable of penetrating the nuclei of cells. Because entry is mediated by a ubiquitous nucleoside transporter, and is independent of the constant domain, nuclei of both normal and malignant cells can be penetrated by full-length 3E10, and its single-chain variable fragment (3E10 scFv). Pre-clinical studies suggest that 3E10 scFv can be used as a vehicle for delivering cargo molecules to cell nuclei. In this study, Peter Glazer and colleagues investigated the potential of using a 3E10 scFv-HSP70 fusion protein to protect cells from ionizing radiation. In the process, they serendipitously discovered that 3E10 by itself can sensitize DNA repair-deficient tumors to DNA-damaging therapy and have provided the rationale for testing 3E10 as a targeted cancer therapy.

  • As expected, the authors showed that in vitro treatment with 3E10 scFv-HSP70 protected the human MCF-7 breast cancer cell line from ionizing radiation. However, unexpectedly they noticed that treatment with 3E10 scFv alone significantly increased MCF-7 sensitivity to radiation. 3E10 scFv treatment also sensitized the U251 human glioma cell line to radiation and the U87 human glioma cell line to the DNA-damaging drug doxorubicin, but not to the microtubule-disrupting drug paclitaxel. Furthermore, in vivo treatment with full 3E10 antibody sensitized U87 glioma xenografts to both radiation and low-dose doxorubicin. These data suggested that both 3E10 and 3E10 scFv specifically sensitizes tumor cells to DNA-damaging therapies.
  • The authors also showed that 3E10 preferentially binds single-strand DNA tails; and sensitizes tumor cells to DNA-damage by inhibiting critical steps involved in both DNA single-strand and double-strand break repair.
  • Given the known sensitivity of BRCA2-deficient tumor cells to DNA-damage, the authors tested 3E10 and 3E10 scFv against BRCA2-deficient and proficient tumor cell lines. As expected, 3E10 and 3E10 scFv exposure was synthetically lethal to BRCA2-deficient human ovarian and pancreatic cancer cell lines, but not BRCA2-proficient ovarian cancer cell lines in vitro.
  • Furthermore, 3E10 treatment dramatically and selectively sensitized BRCA2-deficient, but not BRCA2-proficient, human ovarian cancer cells to doxorubicin and pancreatic cancer cells to radiation.

These data thus demonstrate that the anti-DNA antibody 3E10, and its single chain variable fragment, sensitize DNA repair-deficient tumor cells to DNA-damaging therapies, such as ionizing radiation and doxorubicin, by interfering with DNA single-strand and double-strand break repair. 3E10 has been proven safe in phase I clinical studies testing the antibody as a potential vaccine for SLE. These data support testing 3E10 as a targeted therapy in patients with DNA-repair deficient cancers such as BRCA2-deficient malignancies. The authors also propose that DNA-repair inhibition resulting from anti-DNA autoantibodies could be responsible for lower incidences of breast, prostate and ovarian cancers in SLE patients, since these are cancers that are frequently associated with debilitated DNA repair mechanisms.

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, Official Journal of FOCIS

Epigenetics and Chromatin Remodeling in Henoch-Schönlein Purpura

A review of Luo S., et al. Aberrant Histone Modifications in Peripheral Blood Mononuclear Cells from Patients with Henoch-Schönlein Purpura. Clinical Immunology (2013) 146:165-175. PMID: 23353785

Henoch-Schönlein purpura (HSP) is a systemic vasculitis that classically presents as palpable purpura with arthritis, abdominal pain, or kidney dysfunction in the weeks following an upper respiratory tract infection. The majority of cases of HSP occur in children, although it can affect adults as well, and the pathophysiology is thought to involve IgA-immune complex deposition, which leads to complement activation, neutrophil influx, and microvascular destruction. Despite much interest in HSP, its precise etiology remains unclear and a variety of host and environmental factors have been identified as potential contributors. One area that has not been previously explored in HSP is the role of epigenetics and histone remodeling, which can affect the transcriptional accessibility of specific genetic loci. This avenue of research is of particular interest in light of recent evidence supporting the notion that epigenetic regulation may influence T cell function and susceptibility to autoimmune diseases such as lupus, Sjögren’s syndrome, and rheumatoid arthritis. To test the hypothesis that epigenetic changes might contribute to HSP, Luo et al. analyzed histone modifications and gene expression in PBMCs collected from 24 adult patients with HSP and 22 healthy controls of similar age. The authors found that:

  • Histone modifying genes were differentially expressed in PBMCs of HSP patients relative to healthy controls. HSP patients had increased expression of histone acetyltransferases (e.g. Creb-binding protein) and histone methyltransferases (e.g. SETD1A), as well as reduced expression of histone deacetylases (e.g. HDAC1) and histone demethylases (e.g. LSD1).
  • Acetylation and methylation of particular histones were increased in PBMCs of HSP patients. Specifically, histone H3 acetylation was increased in all HSP patients, while H3K4 methylation was increased only in those HSP patients with concurrent signs of kidney dysfunction.
  • Th2 associated genes (e.g. IL-4, IL-13, GATA3, TIM1, CXCL4) were upregulated in PBMCs of patients with HSP. However, no differences were seen between groups in the expression of Th1 associated genes (e.g. IFN-γ, Tbet).
  • The IL-4 locus was enriched for hyperacetylated histone H3 and hypermethylated histone H3K4 in CD4+ PBMCs of HSP patients, as determined by chromatin immunoprecipitation. However, no differences were seen between groups at the IFN-γ locus with regards to acetylated H3 or methylated H3K4.
    Among patients with HSP, increased H3 acetylation and H3K4 methylation were positively correlated with increasing clinical disease severity and serum levels of inflammatory markers and cytokines (e.g. CRP, ESR, IL-4).

The study authors demonstrate the presence of specific histone modifications, namely hyperacetylated H3 and hypermethylated H3K4, in PBMCs of adult patients with HSP. In addition, the authors correlate these changes with an increase in the expression of Th2-associated genes, such as IL-4 and GATA-3, as well as with worsening clinical disease severity of HSP. These findings represent a novel observation of the role that epigenetics and chromatin remodeling may play in HSP and other vasculitides. In addition, they suggest that Th2-skewing and increased IL-4 production may be important consequences of histone modification in HSP. Further research would be necessary, however, to better demonstrate the extent to which the development of HSP is dependent on epigenetic dysregulation and Th2 activation. Given that HSP predominantly affects children, it would also be important for future work to validate the findings of Luo et al., which were obtained in adult HSP patients, in the pediatric population. By examining the sequelae and outcomes of HSP patients, future research could also provide valuable information as to the prognostic utility of testing for specific histone modifications.

Reviewed by Gabriel Griffin, B.A., Duke University School of Medicine, and Andrew H. Lichtman, MD, PhD, Brigham and Women's Hospital.

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Human Immunophenotyping Update

Miniaturizing Immune Monitoring Assays

Holden T. Maecker, PhD, Stanford University

The most common immune monitoring assays are serological. This stems from the fact that antibody-based assays have a much longer history in immunology than do cellular assays, and because the technical demands for antibody assays tend to be lower. Because of their long history and widespread use, technological advances in antibody-based assays are many. Two of the main areas for advancement have been multiplexing (the ability to measure many analytes at once) and miniaturization (to allow use of smaller samples). Often, the two go hand in hand. Consider the following advances in multiplexing and/or miniaturization of antibody-based assays:

Multiplexed ELISA’s. These include multiplexed assays with readouts other than the traditional colorimetric ELISA (or radiological RIA). A prime example is the electrochemiluminescence platform from MesoScale Discovery (1). By spotting capture reagents for up to 10 analytes per well, the sample volume required per readout is reduced up to 10-fold. Furthermore, the wide linear dynamic range of electrochemiluminescence allows for measurement of samples without multiple serial dilutions.

Bead-based immunoassays. These are available from many vendors, most commonly designed for the Luminex platform (2-4) (see also the previous FOCIS Immunophenotyping Update on this technology). Bead-based arrays allow for a much higher level of multiplexing, theoretically up to several hundred analytes per well with current systems. In practice, most protein-based assays top out at 30-50 analytes, due to considerations such as antibody cross-reactivity and target protein abundance. Still, this considerable level of multiplexing results in a 25 microliter aliquot of serum or plasma being used to measure a large number of targets.

Protein microarrays. Similar to genomic microarrays, these usually have target proteins or peptides spotted on membranes or other substrates. The degree of multiplexing here is enormous. There are commercial arrays containing several thousand human proteins, or thousands of peptides derived from hundreds of microbes. While they are generally considered to be less quantitative than sandwich-type immunoassays, they can quickly read out specificity patterns of potential autoantibodies or exposure to pathogens, for example. A sample of just a few microliters is used to probe all of the targets.

In addition to these direct protein measurement tools, there are multiplexed platforms to perform qPCR for cytokines, and even hybrid methods that use nucleic acid tags as intermediates to detect proteins prior to signal amplification by PCR (Proseek, Olink Technologies).

So, where are the highly multiplexed, miniaturized cellular assays? To be sure, cellular immunology has undergone some technical revolutions, most notably with the development of flow cytometry. No single technology has done as much to further our understanding of the diversity and function of immune cells (5). And, flow cytometry has evolved from one- and two-color assays in the 1980’s to up to 17-color assays that are possible today (6)….with 40+ parameters available by the use of mass labels in the CyTOF (7-9) (See also the FOCIS Immunophenotyping Update, Fall 2011).

In other ways, cellular assays have evolved from messy, bulk radiological tests (3H-thymidine incorporation for proliferation, 51Cr release for cytotoxicity) to dye-labeled single-cell equivalents (including CFSE dilution (10) and CD107-based degranulation assays (11), to name two common ones).

But none of these assays are truly miniaturized. When performed on cryopreserved PBMC, the requirement in terms of starting blood volume is particularly high, owing to the loss that comes with cryopreservation. Often, a 10 cc tube of blood gets processed to a single cryovial of PBMC and devoted to one flow cytometry assay. The high cell requirement could be substantially reduced if whole blood were assayed directly. One way to facilitate this is through point-of-collection devices that can stimulate and fix blood for later functional assays (Smart Tube, Inc.). Although this commercial system is currently designed for 1 mL blood per stimulation condition, it could certainly be further miniaturized without loss of reproducibility. And presumably, plate-based versions could be developed that would allow for further multiplexing of samples and stimulators. If combined with a CyTOF readout, many hundreds of cell subsets could be enumerated and their functional capacities interrogated for each stimulation condition. Such an assay, currently in development in our laboratory, would read out more information from 1-2 mL of blood than current state-of-the art assays can read out from ten times that volume.

One of the ways that cells are lost in current assays is through washing steps. However, using acoustic waves to move and align cells in microfluidic chambers, it is possible to achieve efficient buffer exchange without loss of any cells (12, 13). Devices that perform such operations, even automating the staining and washing of cells in a microchip device, could revolutionize the front-end processing currently required for flow cytometry. In fact, these devices might be coupled to flow cytometers, or else placed in scanning cytometers for readout. The latter has the advantage that, in theory, the same cells could be repetitively analyzed in situ, to follow dynamic processes in real time.

At least one group has used microdevices to trap immune cells in nano-scale wells and perform multiple assays on them over time (14-16). Cell supernatants are printed onto slides for assay of secreted cytokines, while the cells themselves are phenotyped using fluorescent-labeled antibodies and detection by fluorescent microscopy. This system, while currently a bit labor-intensive, allows for the maximum information content to be derived from a relatively small starting population of cells.

Taking this idea a step further, the immunoassay of the future could possibly be something like the following. A few hundred microliters of blood are pipetted into a microchip, where plasma and cellular components are separated. The plasma is run over a series of biosensors to measure soluble cytokines and to probe antibody types and specificities. The cellular fraction is separated by acoustic or electrical means into erythrocytes and leukocytes. Since this is the future, there will probably be important assays to be performed on the erythrocytes as well as the leukcocytes, but these will likely not be done in a mixture. At any rate, the leukocytes might be directed into microchambers where they are stimulated with a battery of cytokines, antigens, etc. They might be stained in situ with the addition of antibodies, whose binding is read by laser-scanning cytometry. Meanwhile, their secreted products can be analyzed in real time using biosensors. Alternately, the cells could be sent after staining into a flow (or mass) cytometer.

A bioinformatics module would take the many readouts from such a chip and summarize them in ways such as:

  • Absolute numbers and percentages of hundreds of phenotypic cell subsets
  • Functional profiles of each of these cell types with regards to major signaling pathways
  • Antigen reactivity of B, T, and NK cells to major antigens of interest
  • Circulating levels of a number of cytokines
  • Levels of immunoglobulin isotypes and reactivity profile of antibodies

One could envision that such a super-assay could be customized for particular purposes, by varying the stimuli, antibody probes, etc. But a somewhat standardized version of it would be a tremendously valuable (and easy to implement) biomarker discovery tool. With enough data from healthy and diseased individuals, normal ranges could be established and metrics of diseases identified. This could become the cellular immunologist’s version of a whole-genome gene expression array, but in many ways more powerful, because its information would be generated on a single-cell level. Because it would presumably be automated, largely self-contained, and performed with a small amount of blood, it could become a powerful clinical diagnostic tool as well. Of course, it would require a major engineering effort to create. But we cellular immunologists can dream.

References:

  1. S. V. Sennikov et al., Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method. J Immunol Methods 275, 81 (Apr 1, 2003).
  2. S. Pang et al., A comparability study of the emerging protein array platforms with established ELISA procedures. J Immunol Methods 302, 1 (Jul, 2005).
  3. J. F. Djoba Siawaya et al., An evaluation of commercial fluorescent bead-based luminex cytokine assays. PloS one 3, e2535 (2008).
  4. M. F. Elshal, J. P. McCoy, Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods 38, 317 (Apr, 2006).
  5. L. A. Herzenberg, D. Parks, B. Sahaf, O. Perez, M. Roederer, The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48, 1819 (Oct, 2002).
  6. S. P. Perfetto, P. K. Chattopadhyay, M. Roederer, Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4, 648 (Aug, 2004).
  7. D. R. Bandura et al., Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry. Analytical chemistry, (Jul 14, 2009).
  8. O. Ornatsky et al., Highly multiparametric analysis by mass cytometry. J Immunol Methods, (Jul 21, 2010).
  9. S. C. Bendall et al., Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687 (May 6, 2011).
  10. P. K. Wallace et al., Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution. Cytometry. Part A : the journal of the International Society for Analytical Cytology 73, 1019 (Nov, 2008).
  11. M. R. Betts et al., Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281, 65 (Oct 1, 2003).
  12. J. Shi, X. Mao, D. Ahmed, A. Colletti, T. J. Huang, Focusing microparticles in a microfluidic channel with standing surface acoustic waves (SSAW). Lab on a chip 8, 221 (Feb, 2008).
  13. M. E. Piyasena et al., Multinode acoustic focusing for parallel flow cytometry. Analytical chemistry 84, 1831 (Feb 21, 2012).
  14. N. Varadarajan et al., A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis. J. Clin. Invest. 121, 4322 (Nov 01, 2011).
  15. Q. Han et al., Polyfunctional responses by human T cells result from sequential release of cytokines. Proceedings of the National Academy of Sciences 109, 1607 (Feb 31, 2012).
  16. N. Varadarajan et al., Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving. Proceedings of the National Academy of Sciences 109, 3885 (Apr 06, 2012).

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Concepts in Basic Immunology: Principles and Therapeutic Applications

A New Wave of Rational Therapeutics for Immunological Disorders: Small Molecule Signaling Inhibitors

Shiv Pillai, MD, PhD, Massachusetts General Hospital

History
For over two millenia, compounds with therapeutic efficacy have largely been discovered by chance - at best by a process of trial and error. In ancient Greece, India and China and later in the Islamic world and pre-Columbian America, arsenals of efficacious medicinal preparations were discovered centuries ago by physicians and herbalists and some of these preparations are still in use today. A little over a hundred years ago, Paul Ehrlich searched for drugs derived from dyes and related organic compounds and early in the 20th century gave us our first chemotherapeutic, Salvarsan. This opened the doors to more discoveries that helped bring synthetic chemists into the brave new world of pharmacology. Alexander Fleming's discovery in the 1940s of the first antibiotic derived from a fungal spore led to Ernst Boris Chain's chemical synthesis of penicillin. Organic chemists have since created large compendiums of compound libraries - these libraries as well as collections of molecules derived from natural products have long formed the core of large screens aimed at identifying random molecules that happen to possess some biological efficacy of relevance to the treatment of disease.

The first baby steps towards rational therapy coincided with the birth of modern immunology. Behring and Kitasato injected large animals with bacterial toxins that had been chemically modified using iodoform in order to generate anti-toxins. Passive serum therapy with anti-toxins was a huge success. The discovery of monoclonal antibody technology by Kohler and Milstein in the 1970s paved the way for the first true rational therapeutic. This emanated from the discovery by Maini and Schulman that the synovium in rheumatoid arthritis subjects contains large amounts of TNF. Their pioneering experiments with monoclonal antibodies against TNF in the treatment of rheumatoid arthritis ushered in the era of biologics, largely monoclonal antibodies and some immunoglobulin-receptor ectodomain fusion proteins that typically block receptor function. Some of the most successful biologics used in immunological disorders have included TNF inhibitors, anti-CD20 antibodies for B cell depletion, antibodies that block integrins and antibodies that negate inhibitory signaling by PD-1 - among many, many others.

Targeting Signaling Pathways
Since the mid 1980s there has been an explosion in our understanding of signaling pathways in vertebrate cells - including immune cells and cancer cells. A highly touted example of rational therapy in cancer in the past decade was also something of an accident. In chronic myelogenous leukemia, a fusion protein containing a cytosolic protein called Bcr (not the antigen receptor in B cells!) and the c-Abl tyrosine kinase is generated from the vicinity of the translocation breakpoint in the Philadelphia chromosome. The Bcr-Abl tyrosine kinase is therefore cancer specific - it is not found in normal cells. Attempts to generate a kinase inhibitor that inhibited protein kinase C led to the identification of a molecule that was actually far more capable of inhibiting the Bcr-Abl kinase. This inhibitor, Gleevec or Imatinib, has emerged as a remarkable therapeutic and remains one of the poster children for rational therapy. It remains the first small molecule rational therapeutic.

A number of small molecules have of course been used as therapeutics in transplantation and autoimmunity for decades. Although the mechanism of action of drugs like cyclosporin A, FK506 and rapamycin have been worked out in detail they were identified from fungal extracts and their mechanisms of actions were discovered after they had already proven to be biologically efficacious. It is not an accident that these compounds, and indeed the majority of small molecule therapeutics, are enzyme inhibitors. Small molecules typically function by binding tightly to hydrophobic clefts in proteins. It has been estimated that only about 15% of all proteins possess a hydrophobic cleft. Enzymes by nature of their ability to interact closely with and "re-organize" the structure of substrates invariably contain hydrophobic clefts amenable to small molecule attack while the overwhelming majority of non-catalytic proteins lack such a feature. The growing knowledge about the enzymes that are activated downstream of antigen receptors, cytokine receptors and other receptors in the immune system has generated a series of targets for rational therapy aimed at disorders involving the immune system.

There are a number of advantages to small molecule inhibitors. The first is “dosability “- achieving the right therapeutic dose is considerably easier with a molecule given a couple of times a day rather than once every few months. Side effects can be more easily reversed by altering doses of a small molecule compared with a biologic. In addition, there are large swaths of the world in which biologics will remain a pipe dream because of issues of delivery and cost. Small molecules therefore hold great promise for a very wide variety of reasons.

Small Molecule Inhibitors Under Development
A number of therapeutic trials are currently in progress involving small molecules targeting enzymes that are crucial in immune cells. We will briefly discuss here inhibitors of three particular categories of protein tyrosine kinases that have already exhibited great promise in disorders affecting the immune system and in certain tumors. Not all the inhibitors currently in use are unique in their target specificity. One interesting example involves inhibitors for Btk (Bruton's tyrosine kinase). Btk is an enzyme activated downstream of the B cell receptor in B cells; it is also activated by immune receptors in myeloid cells. B cell receptor activation involves the sequential activation of members of three different families of tyrosine kinases. Src family kinases such as Lyn are first activated, followed by the recruitment and activation of Syk, a tyrosine kinase with tandem phosphotyrosine specific SH2 domains, and the consequent activation of Btk. Btk is a tyrosine kinase of the Tec family and Btk inhibitors currently in trials are fairly specific molecules which covalently bind to a cysteine residue in this kinase and thus irreversibly inactivate it. This particular residue of Btk, cysteine -481, lacks a counterpart in the vast majority of protein kinases, as a result of which these inhibitors are very specific. These covalent inhibitors are quite potent and have already shown to be of value in B lymphoid malignancies, particularly those in which B cell receptor signaling plays a presumed trophic role. These malignancies include chronic lymphocytic leukemia, small cell lymphoma and diffuse large B cell lymphoma. Irreversible inhibitors might be more suited to malignancies that to chronic inflammatory diseases. Attempts are ongoing to generate non-covalent, reversible Btk inhibitors that may prove useful in autoimmune disorders such as systemic lupus erythematosus.

Syk functions downstream of the B cell receptor in B cells and plays a similar role in myeloid cells downstream typically of Fc receptors for IgG in many phagocytic cells and distal to the high affinity Fc receptor in eosinophils and mast cells. Inhibitors of the Syk tyrosine kinase have been tried successfully in B cell malignancies, rheumatoid arthritis and in allergic lung disease and the spectrum of diseases in which this category of small molecules may prove effective is likely to widen.

There are four Jak tyrosine kinases (sometimes called Janus kinases) that are activated downstream of a large number of cytokine receptors. They are known as Jak1, Jak2, Jak3, and Tyk2. Ligation of Type I and Type II cytokine receptors activates specific Jak kinases which in turn cause the phosphorylation, dimerization and nuclear transport of specific STAT transcription factors. Inhibitors that preferentially target Jak3 (specificity is not absolute) compromise signaling via common γ chain cytokines such as IL-2, IL-4, IL-7, IL-15, and IL-21. These inhibitors can therefore compromise allergic inflammation (Th2) and also attenuate Th17 type responses. They have shown promise in allergic diseases, psoriasis and rheumatoid arthritis. Jak1/Jak2 inhibitors can block signaling via IFN-γ and IL-6. They can therefore compromise Th1 and Th17 responses and have been shown to be effective in psoriasis, rheumatoid arthritis, myelofibrosis, acute myelogenous leukemias and refractory lymphomas.

Numerous other small molecule inhibitors continue to be developed at an astonishing rate even as signaling pathways continue to be parsed and three-dimensional structures of more enzymes of interest determined. The small molecule therapeutic wave has hit the shore and the ripples will flow for a long time.

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Selected Recent Clinical Trial Results

Edited by Carla J. Greenbaum, MD, Benaroya Research Institute

Dimethyl Fumarate in Relapsing Multiple Sclerosis: The DEFINE and CONFIRM Studies

Clinical Trials:

Fox J. et al., Placebo-Controlled Phase 3 Study of Oral BG-12 or Glatiramer in Multiple Sclerosis. New England Journal of Medicine 2012; 367:1087-97.) PMID: 22992072

Gold R et al., Placebo-Controlled Phase 3 Study of Oral BG-12 for Relapsing Multiple Sclerosis. New England Journal of Medicine 2012; 367:10107. PMID: 22992073

Disease: Multiple Sclerosis

Study Design:

  • Two large Phase 3 studies of dimethyl fumarate (BG-12) in relapsing Multiple Sclerosis (MS) were recently reported in the same issue of the NEJM.
  • The DEFINE study was a double-blind, randomized, three-arm trial over 96 weeks evaluating BG-12 in two regimens (240mg twice daily and 240mg thrice daily) versus placebo. The primary endpoint was the proportion of patients who experienced a relapse of MS during the study. Secondary endpoints included the annualized relapse rate, the time to disability progression (defined as sustained, 1.0 point increase in the Expanded
  • Disability Status Scale [EDSS]), and several MRI measures of MS lesions. Eligible patients (n = 1,237) were adults with relapsing-remitting MS (RRMS) and 1 or more relapses within the last 12 months.
  • The CONFIRM study had a similar design, except it included an open-label arm of glatiramer acetate 20 mg SQ DAILY. The primary endpoint was the annual relapse rate at the end of study (96 weeks). Secondary endpoints included the proportion of patients with relapse within the study period, disability progression, and MRI measures. Eligible patients (n = 1430) had identical eligibility criteria.

Drug:

  • Dimethyl Fumarate (BG-12) is an orally-administered fumaric acid ester that has been used in the EU as a treatment for severe systemic psoriasis since the 1990s. Development in MS resulted from observation by German clinicians that oral fumarate stabilized MS in psoriatic patients with MS. Mechanism of action is not fully understood. It may have neuroprotective effects via activation of the E2-related transcription factor 2 (Nrf2) – which upregulates NAD(P)H;quinone reductase, increasing glutathione levels (an important antioxidant). Among other potentially beneficial effects, BG-12 may cause a shift from a TH1 to TH2 cytokine profile, downregulate ICAM-1 and VCAM, and downregulate differentiation antigens on (and induce apoptosis of) dendritic cells.

Results:

  • In DEFINE, the proportion of patients with relapse was 27%, 26%, and 46% for BG-12 twice daily, BG-12 thrice daily and placebo, respectively (P
  • Interestingly, the reduction of disease progression was not significant for BG-12 or glatiramer in CONFIRM, while it was significant for both BG-12 doses versus placebo in DEFINE (38% and 34%, P
  • The main adverse events with use of BG-12 were flushing, GI events such as diarrhea or upper abdominal pain, decreased WBC counts and elevated liver enzymes. There was no association with risk of serious infection, opportunistic infection, or neoplasm.

Why is the trial is of interest to the broader FOCIS community?

BG-12 has features that make it a model for the kind of therapies that will be readily approved and widely adopted for use in chronic inflammatory diseases in the future. First, because it has been used to treat psoriasis in the EU since the 1990s, BG-12 has a two-decade safety record that will address any long-term safety concerns raised by regulatory agencies. Second, it is an orally-administered small molecule, which is appealing to both patients who will likely be very adherent on the drug. Of course, equally important to the future success of BG-12 is the unexpectedly robust efficacy data in these two Phase 3 studies as measured by MRI and relapse rates. The key question as to whether this therapy does or does not affect disability progression will require additional studies.

Reviewed by Carla Greenbaum, MD and Steven Lamola, MD Benaroya Research Institute


Phase 1/2a Open-Label, Dose-Escalation Study of Ovalbumin-Specific Treg Cells in Refractory Crohn’s Disease (Crohn’s and Treg Cells Study [CATS1])

Clinical Trial:
Desreumaux P et al., Gastroenterology 2012; 143:1207-1217. PMID: 22885333

Disease: Crohn's Disease

Study Design:

  • 12-week, open-label, single-injection of escalating-dose of Treg cells. Phase 1/2a study in 20 patients with refractory Crohn’s Disease.
  • Key inclusion criteria were age 18-65, diagnosis of Crohn’s Disease > 12 months, with active disease and prior inadequate response or intolerance to conventional treatment. Key exclusion criteria were active infection or malignancy, and immunosuppressant or anti-TNF- therapy.
  • Safety and efficacy were assessed at follow-up visits at weeks 1, 2, 3, 5, 8 and 12. Efficacy was primarily assessed by measure of Crohn’s Disease Activity Score (CDAI). Proliferation of PBMCs and serum cytokines were also evaluated, as were C-reactive protein (CRP).

Drug:

  • Treg cells isolated from patients’ PBMCs were exposed to ovalbumin. At day 0, patients received a single IV injection of 106 (n = 8), 107 (n = 3), 108 (n = 3) or 109 (n = 6) of these ovalbumin-specific type-1 Treg cells. Patients also ingested a meringue cake to enable activation of ovalbumin-specific Tregs in the inflamed gut.

Results:

  • Both pro-inflammatory CD14+ CD16+ monocytes and FoxP3-expressing Tregs cells decreased in responding patients – however serum cytokine levels did not. Ovalbumin-specific proliferative responses of PBMCs were significantly lower in responders vs. nonresponders (especially in the lowest dose group) at week 8. Mean change in CRP at week 1 was significantly lower in CDAI responders at week 5 (n = 8) vs. nonresponders.
    Overall the treatment was well-tolerated, with only 2 related AEs and 3 related SAEs (no deaths, and all recovered). Response to treatment, per standard definition of 100 point reduction in CDAI, was observed in 40% of patients at weeks 5 and 8, and in 75% of the lowest dose group (106 Treg cells). It was maximal at 5 weeks post-treatment, and subsequently declined.

Why the trial is of interest to the broader FOCIS community?

This is the first-in-man study of antigen specific Treg therapy in human inflammatory bowel disease (Tregs previously been used in Type 1 diabetes and graft-versus-host disease). As in any small, open-label trial, results should be interpreted with caution.

Reviewed by Carla Greenbaum, MD and Steven Lamola, MD Benaroya Research Institute


Phase 2a Study of Secukinumab in Crohn’s Disease

Clinical Trial:
Hueber, W., et al. Gut. 2012; 61:1693-1700. PMID: 22595313

Diseases: Crohn's Disease

Study Design:

  • Double-blind, randomized, placebo-controlled Phase 2a trial of 59 adult patients with moderate-to-severe Crohn’s Disease assigned in a 2:1 ratio to either secukinumab ( IL-17A monoclonal antibody) or placebo.
  • Safety and efficacy evaluated at weeks 1, 2, 3, 4, 5, 6, 10, 14 and 18.
  • The primary endpoint was the probability of the study drug achieving a reduction in the Crohn’s Disease activity index (CDAI) at week 6

Drug:

  • Secukinumab, an IL-17A monoclonal antibody, given at a dose of 10 mg/kg and administered as 2 hour IV infusions on days 1 and 22.

Results:

  • The trial was prematurely terminated when it was determined at a scheduled interim analysis of data from the first 41 patients that pre-specified futility criteria had been met. Secondary analysis showed statistically significant worsening of disease with secukinumab treatment during weeks 4-10.
    Worsening of disease in the active treatment arm was further reflected in a high rate of Crohn’s Disease related serious adverse effects compared with placebo. Additionally, 44% (17/39) of patients on secukinumab developed an infection (compared to none on placebo), including 4 fungal infections.

Why the trial is of interest to the broader FOCIS community?

Although larger studies of secukinumab have demonstrated clinical efficacy and safety in psoriasis and rheumatoid arthritis, the drug has been shown in this Phase 2a study to lack efficacy in Crohn’s Disease -- and furthermore be harmful in at least a subset of patients. Brodalumab, a monoclonal antibody directed against IL-17RA, with efficacy in psoriasis has also failed in Crohn’s Disease. As pointed out in an accompanying editorial (1) this divergence has come to pass despite substantial shared genetic risk between psoriasis and Crohn’s Disease (including having the same risk haplotypes at IL23R) – highlighting the hazards of predicting pathophysiology from genetic risk alone. These divergent results highlight the complexity of TH17 biology and emphasize the need for cross-autoimmune disease evaluation of pathway similarity and differences.

References:

  1. Raine, T and Kaser, A. Gut. 2012; 61:1653-4

Reviewed by Carla Greenbaum, MD and Steven Lamola, MD Benaroya Research Institute


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Permissions: Copyright © 2013 Federation of Clinical Immunology Societies
Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the Copyright, Designs and Patent Act 1988, this publication may be reproduced, stored or transmitted, in any form or by any means, only with the prior permission in writing of the publishers, or in the case of reprographic reproduction, in accordance with the terms and licenses issued by the Copyright Licensing Agency.
Translational Immunology Update is published bimonthly by the Federation of Clinical Immunology Societies. You may opt-out of receiving the publication at any time by clicking the unsubscribe link in the email.

 

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