Th2 Effector Function Research Articles

Interleukin 4 promotes the development of ex-Foxp3 Th2 cells during immunity to intestinal helminths.

Immunity to intestinal helminth infections requires the rapid activation of T helper 2 cells (Th2 cells). However, simultaneous expansion of CD4+Foxp3+ regulatory T cells (T reg cells) impedes protective responses, resulting in chronic infections. The ratio between T reg and effector T cells can therefore determine the outcome of infection. The redifferentiation of T reg cells into Th cells has been identified in hyperinflammatory diseases. In this study, we asked whether ex-T reg Th2 cells develop and contribute to type-2 immunity. Using multigene reporter and fate-reporter systems, we demonstrate that a significant proportion of Th2 cells derive from Foxp3+ cells after Heligmosomoides polygyrus infection and airway allergy. Ex-Foxp3 Th2 cells exhibit characteristic Th2 effector functions and provide immunity to H. polygyrus Through selective deletion of Il4ra on Foxp3+ cells, we further demonstrate IL-4 is required for the development of ex-Foxp3 Th2 cells. Collectively, our findings indicate that converting T reg cells into Th2 cells could concomitantly enhance Th2 cells and limit T reg cell-mediated suppression.

IL-4 Signal Regulates IL-9-producing Mucosal Mast Cell Differentiation And Function During The Development of Experimental Food Allergy

Abstract The prevalence of food allergy is increasing significantly in developed nations. Accumulating evidences suggest a pivotal role of intestinal mast cells (MCs), TH2 cytokines, and IgE in the pathophysiology of food allergy. We have recently identified the novel IL-9 producing mucosal mast cells (MMC9s), which can produce high levels of IL-9, IL-13, and mast cells mediators. Mice deficient of IL-4 signals fail to develop MMC9s and become resistant to IgE-mediated food allergy. However, it remains unclear whether IL-4 can directly regulate the development and function of MMC9s. In a skin sensitization induced food allergy model, Il4RaF709 mice which have gain-of-function mutations in IL-4Ra were found to produce much higher levels of ovalbumin (OVA) specific IgE and mast cell protease-1 in blood and became more susceptible to experimental food allergy. While the occurrence of MMC9s and type-2 innate lymphoid cells (ILC2s) were comparable between wild type (WT) and Il4RaF709 mice, repeated OVA ingestions induced significant increase of CD4+TH2 cell occurrence and MC numbers in the small intestine of Il4RaF709 mice than those in WT mice. Furthermore, IL-4 treatments induced bone marrow derived mast cells (BMMCs) from Il4RaF709 mice to produce much more IL-13 and TNF-α than those by WT BMMCs. These results suggest that IL-4 signal provided by CD4+TH2 cells may directly potentiate TH2 effector function of MMC9s and promote the differentiation of MMC9s into intestinal mature MCs, resulting in the increased intestinal hypersensitivity to ingested food allergens. Detailed mechanistic studies of IL-4 signaling in MMC9 development and function may provide therapeutic insights into the preventions and treatments of food allergy.

Elevated Galectin-9 Suppresses Th1 Effector Function and Induces Apoptosis of Activated CD4+ T Cells in Osteoarthritis.

T cell immunoglobulin and mucin domain 3 (Tim-3) is a critical regulatory molecule found on activated Th1 cells, exhausted CD8+ T cells, and resting monocytes/macrophages. Galectin-9 (Gal-9) is an identified ligand for Tim-3. Interaction between Tim-3 and Gal-9 is thought to inhibit Th1 responses. The regulation and function of Tim-3 and Gal-9 in osteoarthritis (OA) have not been intensively investigated. We found that in peripheral blood, CD4+ T cells, but not CD8+ T cells or CD14+ monocytes, from OA patients presented significantly elevated Tim-3 and Gal-9 expression compared to those from healthy controls (HC). The CD4+ T cells from OA did not present altered Th1, Th2, and Th17 composition in the peripheral blood, but secreted less Th1 cytokine interleukin 2 (IL-2) and interferon gamma (IFN-γ) after activation. Further investigation demonstrated that Gal-9 induced high levels of apoptosis in activated CD4+ T cells from OA patients. Inhibition of Gal-9 resulted in significantly higher IL-2 and IFN-γ expression that was directly correlated with the number of non-apoptotic cells. In the synovial fluid, both secreted Gal-9 and surface Gal-9 levels were significantly higher in less-severe grade 2 OA patients than in more-severe grade 4 OA patients. Surface Tim-3 was also higher in synovial fluid CD8+ T cells and CD14+ monocytes from grade 2 OA patients and lower in grade 4 OA patients. Together, these results suggested that Tim-3 and Gal-9 could downregulate T cell inflammation in OA, and could be utilized as a novel therapeutic strategy.

Gene Expression Profiles of Human Phosphotyrosine Phosphatases Consequent to Th1 Polarisation and Effector Function.

Phosphotyrosine phosphatases (PTPs) constitute a complex family of enzymes that control the balance of intracellular phosphorylation levels to allow cell responses while avoiding the development of diseases. Despite the relevance of CD4 T cell polarisation and effector function in human autoimmune diseases, the expression profile of PTPs during T helper polarisation and restimulation at inflammatory sites has not been assessed. Here, a systematic analysis of the expression profile of PTPs has been carried out during Th1-polarising conditions and upon PKC activation and intracellular raise of Ca2+ in effector cells. Changes in gene expression levels suggest a previously nonnoted regulatory role of several PTPs in Th1 polarisation and effector function. A substantial change in the spatial compartmentalisation of ERK during T cell responses is proposed based on changes in the dose of cytoplasmic and nuclear MAPK phosphatases. Our study also suggests a regulatory role of autoimmune-related PTPs in controlling T helper polarisation in humans. We expect that those PTPs that regulate T helper polarisation will constitute potential targets for intervening CD4 T cell immune responses in order to generate new therapies for the treatment of autoimmune diseases.

Prolonged PD1 Expression on Neonatal Vδ2 Lymphocytes Dampens Proinflammatory Responses: Role of Epigenetic Regulation.

A successful pregnancy depends on the maintenance of tolerance at the fetal-maternal interface; strong inflammation in the placental bed is generally associated with adverse fetal outcomes. Among the mechanisms that foster tolerance and limit inflammation, the fetal immune system favors Th2 or regulatory responses over Th1 responses. The unintended consequence of this functional program is high susceptibility to infections. Human Vδ2 T cells mount innate-like responses to a broad range of microorganisms and are poised for Th1 responses before birth. In infants they likely play a key role in protection against pathogens by exerting early Th1 effector functions, improving function of other innate cells, and promoting Th1 polarization of adaptive responses. However, their propensity to release Th1 mediators may require careful regulation during fetal life to avoid exaggerated proinflammatory responses. We investigated molecules with the potential to act as a rheostat for fetal Vδ2 cells. Programmed death 1 (PD1) is a negative regulator of T cell responses and a determinant of tolerance, particularly at the fetal-maternal interface. Neonatal Vδ2 cells upregulate PD1 shortly after activation and, unlike their adult counterparts, express this molecule for at least 28 d. Engagement of PD1 by one of its ligands, PDL1, effectively dampens TCR-mediated responses (TNF-α production and degranulation) by neonatal Vδ2 cells and may thus help maintain their activity within safe limits. PD1 expression by neonatal Vδ2 cells is inversely associated with promoter DNA methylation. Prolonged PD1 expression may be part of a functional program to control Vδ2 cell inflammatory responses during fetal life.

Enhanced Th2 cell differentiation and function in the absence of Nox2

Patients with chronic granulomatous disease (CGD), whom inherit abnormal function of NADPH oxidase 2 (Nox2), suffer from hyperinflammatory responses in lung as well as bacterial and fungal infection. There have been studies to reveal the function of Nox2 in hyperinflammatory diseases, especially in asthma, but the exact role of Nox2 in asthma is still unclear and controversial. Therefore, we attempted to clarify the exact role of Nox2 in asthma, using various experimental asthma models. Asthma phenotypes were analyzed in response to various allergen-induced experimental asthma using Nox2-deficient mice and recombinase gene-activating-1-deficient mice. To understand the underlying mechanisms of exaggerated Th2 effector functions, we investigated the degree of T-cell activation, levels of activation-induced cell death (AICD), and regulatory T (Treg)-cell differentiation in Nox2-deficient T cells. Asthma phenotypes were increased through enhanced Th2 differentiation and function in Nox2-null mice regardless of dose and route of various allergens. Nox2-deficient T cells also showed hyperactivation, reduced AICD, and diminished Treg-cell differentiation through increased AKT phosphorylation (T308/S473) and enhanced mitochondrial ROS production. Our findings indicate that Nox2 deficiency results in exaggerated experimental asthma, which is caused by enhanced Th2 effector function in a T-cell-intrinsic manner.

Dual Blockade of OX40 and CD30 Signaling Reduces Memory Th2-driven Lung Inflammatory Responses to House Dust Mite Allergen

Abstract Allergic asthma is an inflammatory disease that is induced by immune responses to airborne allergens in the lungs. Several studies have identified T cells as being important players in asthma, with Type 2 helper T cells (Th2) being a critical subset. The selective removal of pre-existing pathogenic memory Th2 cells could be a key process leading to enhanced tolerance. We have attempted to identify signaling molecules in the TNF superfamily that regulate memory Th2 cells in a house dust mite (HDM) model of allergic lung inflammation. C57BL/6J mice up-regulated OX40L to higher levels than other TNF family members measured in the lungs in response to HDM exposure. We then tested whether OX40-OX40L interactions were required for memory Th2 cell responses driven by HDM. While OX40-deficient mice displayed strongly reduced overall lung inflammation and Th2 development when challenged with HDM, therapeutic blocking of OX40L at the time of memory T cell reactivation did not result in similar inhibition of allergic responses, implying OX40L may have been important for initiation but not the memory response. Similar negative results were gained by targeting another TNF superfamily protein, CD30L, that was also up-regulated by HDM exposure. However, we observed significant reduction in airway inflammation when OX40L was simultaneously neutralized with CD30L. Dual blockade was found to target memory Th2 cell expansion and effector function but did not prevent accumulation of pTreg cells. This suggests that persisting memory responses to complex allergens are controlled by several co-stimulatory interactions, and only abrogation of signaling through multiple TNF superfamily members might promote airway tolerance.

Src-Like Adaptor Protein 2 regulates NKT lineage diversification

Abstract Natural Killer T (NKT) cells are a class of αβ T cells that express a semi-invariant T Cell Receptor (TCR) and recognize glycolipids presented by the non-polymorphic MHC molecule, CD1d. NKT cells are considered to be innate-like lymphocytes because they produce a robust cytokine response within hours of stimulation. This ‘poised effector’ state is acquired during development in the thymus, where NKT precursors differentiate into one of three distinct subsets and acquire TH1, TH2, or TH17 effector functions. It is not known which signals control the divergence of NKT1, NKT2 and NKT17 subsets. We have identified a novel role for Src-like adaptor protein 2 (SLAP2) in regulation of NKT cell development. We have found that SLAP2-deficiency causes accumulation of thymic NKT cells and alters lineage diversification. In the absence of SLAP2, the frequency and number of thymic NKT2 and NKT17 cells are increased with a concomitant decrease in NKT1 cells. SLAP2−/− NKT cells express elevated levels of PLZF and higher levels of CD5, both of which are downstream of TCR signals. Although SLAP2 has been shown to negatively regulate TCR signaling in vitro, analysis of SLAP2−/− thymocytes revealed that TCR levels and signaling in CD4+CD8+ double positive thymocytes are normal. These data suggest that SLAP2 does not directly regulate TCR during development, in vivo. The numbers and proportions of conventional αβ T cells are largely unaffected by SLAP2-deficiency. In light of these data, we predict that SLAP2 regulates signals that are important for NKT lineage diversification but are dispensable for conventional T cell development.

Enhanced innate CD161+ T cell responses in mucosal tissues of nonhuman primates

Abstract Innate T cells recognize non-peptide antigens presented by non-classical MHC molecules, such as CD1 and MR1. The three major populations within the innate T cell group, namely, γδ T cells, MAIT cells, and NKT cells express high levels of the C-type lectin molecule CD161. To understand the significance of CD161 expressing innate T cells in mucosal immunity, we investigated the phenotype and functions of these cells in mucosal tissues, lymph nodes and blood of rhesus macaques. The frequency of CD161+ T cells was higher in the lungs and colonic mucosa (9.4%±3.8 and 6.2%±1.9) in comparison to peripheral blood (2.9%±1) and mesenteric lymph nodes (0.5%±0.3). Regardless of the tissue compartment, 30 to 80% of the CD161+ T cells comprised of γδ T cells and TCR Vα 7.2+ MAIT cells in approximately equal proportions. However, only 1 to 6% consisted of CD1d tetramer+ NKT cells. Moreover, the mucosal tissue CD161+ T cells displayed a highly activated phenotype based on CD69 and HLA-DR expression in comparison to blood and lymph node cells. HMBPP stimulated γδ T cells from lungs and colonic mucosa produced significantly higher amounts of IFN-γ, Perforin and IL-17 than those in blood. Similarly, E.coli stimulated MAIT cells from mucosal tissues produced significantly higher amounts of IFN-γ and IL-17 than those in blood. The enhanced Th1 and Th17 type effector functions of mucosal innate T cells along with their cytotoxic potential suggests that they can mount early immune responses to mucosal pathogens like HIV and Mtb prior to the development of adaptive immune responses and thereby play a role in the initial control of pathogen burden/dissemination.

Hierarchy Low CD4+/CD8+ T-Cell Counts and IFN-γ Responses in HIV-1+ Individuals Correlate with Active TB and/or M.tb Co-Infection.

ObjectiveDetailed studies of correlation between HIV-M.tb co-infection and hierarchy declines of CD8+/CD4+ T-cell counts and IFN-γ responses have not been done. We conducted case-control studies to address this issue.Methods164 HIV-1-infected individuals comprised of HIV-1+ATB, HIV-1+LTB and HIV-1+TB- groups were evaluated. Immune phenotyping and complete blood count (CBC) were employed to measure CD4+ and CD8+ T-cell counts; T.SPOT.TB and intracellular cytokine staining (ICS) were utilized to detect ESAT6, CFP10 or PPD-specific IFN-γ responses.ResultsThere were significant differences in median CD4+ T-cell counts between HIV-1+ATB (164/μL), HIV-1+LTB (447/μL) and HIV-1+TB- (329/μL) groups. Hierarchy low CD4+ T-cell counts (<200/μL, 200-500/μL, >500/μL) were correlated significantly with active TB but not M.tb co-infection. Interestingly, hierarchy low CD8+ T-cell counts were not only associated significantly with active TB but also with M.tb co-infection (P<0.001). Immunologically, HIV-1+ATB group showed significantly lower numbers of ESAT-6-/CFP-10-specific IFN-γ+ T cells than HIV-1+LTB group. Consistently, PPD-specific IFN-γ+CD4+/CD8+ T effector cells in HIV-1+ATB group were significantly lower than those in HIV-1+LTB group (P<0.001).ConclusionsHierarchy low CD8+ T-cell counts and effector function in HIV-1-infected individuals are correlated with both M.tb co-infection and active TB. Hierarchy low CD4+ T-cell counts and Th1 effector function in HIV-1+ individuals are associated with increased frequencies of active TB, but not M.tb co-infection.

Disparate Effects of Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis and Cuprizone-Induced Demyelination.

Mesenchymal stem cells (MSCs) are pleiotropic cells with potential therapeutic benefits for a wide range of diseases. Because of their immunomodulatory properties they have been utilized to treat autoimmune diseases such as multiple sclerosis (MS), which is characterized by demyelination. The microenvironment surrounding MSCs is thought to affect their differentiation and phenotype, which could in turn affect the efficacy. We thus sought to dissect the potential for differential impact of MSCs on central nervous system (CNS) disease in T cell mediated and non-T cell mediated settings using the MOG35–55 experimental autoimmune encephalomyelitis (EAE) and cuprizone-mediated demyelination models, respectively. As the pathogeneses of MS and EAE are thought to be mediated by IFNγ-producing (TH1) and IL-17A-producing (TH17) effector CD4+ T cells, we investigated the effect of MSCs on the development of these two key pathogenic cell groups. Although MSCs suppressed the activation and effector function of TH17 cells, they did not affect TH1 activation, but enhanced TH1 effector function and ultimately produced no effect on EAE. In the non- T cell mediated cuprizone model of demyelination, MSC administration had a positive effect, with an overall increase in myelin abundance in the brain of MSC-treated mice compared to controls. These results highlight the potential variability of MSCs as a biologic therapeutic tool in the treatment of autoimmune disease and the need for further investigation into the multifaceted functions of MSCs in diverse microenvironments and the mechanisms behind the diversity.

The MAPK-Activated Kinase MK2 Attenuates Dendritic Cell–Mediated Th1 Differentiation and Autoimmune Encephalomyelitis

Dendritic cell (DC)-mediated inflammation induced via TLRs is promoted by MAPK-activated protein kinase (MK)-2, a substrate of p38 MAPK. In this study we show an opposing role of MK2, by which it consolidates immune regulatory functions in DCs through modulation of p38, ERK1/2-MAPK, and STAT3 signaling. During primary TLR/p38 signaling, MK2 mediates the inhibition of p38 activation and positively cross-regulates ERK1/2 activity, leading to a reduction of IL-12 and IL-1α/β secretion. Consequently, MK2 impairs secondary autocrine IL-1α signaling in DCs, which further decreases the IL-1α/p38 but increases the anti-inflammatory IL-10/STAT3 signaling route. Therefore, the blockade of MK2 activity enables human and murine DCs to strengthen proinflammatory effector mechanisms by promoting IL-1α-mediated Th1 effector functions in vitro. Furthermore, MK2-deficient DCs trigger Th1 differentiation and Ag-specific cytotoxicity in vivo. Finally, wild-type mice immunized with LPS in the presence of an MK2 inhibitor strongly accumulate Th1 cells in their lymph nodes. These observations correlate with a severe clinical course in DC-specific MK2 knockout mice compared with wild-type littermates upon induction of experimental autoimmune encephalitis. Our data suggest that MK2 exerts a profound anti-inflammatory effect that prevents DCs from prolonging excessive Th1 effector T cell functions and autoimmunity.

Analysis of Tpl2-deficient mice reveals functional redundancies in the regulation of IFNγ production by NK cells during MCMV infection (INM8P.401)

Abstract The serine-threonine kinase, Tpl2, promotes innate inflammatory responses and is considered as a therapeutic target for autoimmunity. We have demonstrated that Tpl2 is required for efficient IFNγ secretion by CD4+ T cells and Th1 effector functions, but little is known about how Tpl2 regulates IFNγ production by other cells, including natural killer (NK) cells. Herein, we addressed the contribution of Tpl2 to the development and function of NK cells. NK cell development and cytotoxic activity occurred independently of Tpl2. In contrast, IL-12-induced IFNγ secretion was nearly abrogated in Tpl2-/- NK cells but could be restored by concomitant IL-18 stimulation. Impaired IFNγ secretion was not attributed to differences in IL-12R expression or Stat4-dependent signals. Furthermore, only a modest reduction in IL-12-induced IFNγ mRNA expression was observed, suggesting that Tpl2 regulates IFNγ production primarily via translational control. Consistent with in vitro findings, Tpl2-/- mice mounted a diminished NK-derived systemic IFNγ response to murine cytomegalovirus (MCMV) infection, which could be compensated for in ex vivo restimulation assays by the presence of MCMV infection-induced IL-18 secretion. These data demonstrate the importance of Tpl2 in IL-12-induced NK cell IFNγ secretion. Furthermore, the synergism and functional redundancy of the IL-18 pathway with the IL-12-Tpl2 pathway underscores the criticality of ensuring appropriate NK-derived IFNγ to anti-viral immunity.

CD4 signaling and Pneumocystis clearance: a critical role of IL-21R and STAT3 for effector function (MPF2P.745)

Abstract Pneumocystis jirovecii is an opportunist infection that is a significant cause of disease in immunocompromised patients. Patients whose blood CD4 counts drop below 200 cell/μl due to HIV infection or immunosuppressive therapies are at high risk of developing Pneumocystis jirovecii pneumonia (PJP). However, the exact role of CD4 cells and the specific effector subtype that is required for clearance has yet to be determined. We investigated this question with several cytokine and STAT knock out mice using both in vivo infection as well as the assessment of effector CD4+ T-cell function by adoptive transfer into Pneumocystis infected Rag1-/- mice. We observe that classical Th1, Th2, Th17, and Tfh cell effector functions are dispensable for organism clearance. However we found an essential role for both IL21 receptor signaling on CD4 cells as well as CD4+ T-cell intrinsic STAT3 activation are required for clearance. These data are consistent with the recent identification of human IL21R deficiency and susceptibility to PJP. We are currently examining potential novel downstream mechanisms that may mediate immunity against Pneumocystis pneumonia in this context.

Nedd4 family interacting protein 1 limits effector CD4 T cell abundance, cytokine production and inflammation. (HUM3P.248)

Abstract When effector T cells fail to appropriately terminate signaling responses, autoimmune diseases such as multiple sclerosis and inflammatory bowel disease may occur. Signaling in a cell may be terminated by the ubiquitylation and degradation of key signaling transducers. The ubiquitylation cascade is a well studied sequence of orchestrated reactions by which ubiquitin is added on to a substrate protein to, among other things, target that protein for degradation. Ndfip1, or Nedd4 family interacting protein 1, activates the function of E3 ligases, which play important roles in the ubiquitylation cascade. Ndfip1 works with Itch and Nedd4-2 E3 ligases to degrade the IL-4 transcription factor, JunB. Thus in mice that lack Ndfip1, there is unrestricted IL-4 secretion, which leads to an inflammatory phenotype. However, this fails to explain why human SNPs in Ndfip1 have been linked to two autoimmune conditions where TH1 and/or TH17 effector function is unrestrained, namely, multiple sclerosis and inflammatory bowel disease. Mice lacking Ndfip1 show increased overall frequencies of activated T cells and of IFNγ+ and IL-17A+ CD4 T cells, which suggests a general defect in limiting effector T cell abundance. We present data indicating that Ndfip1 limits inflammation in two models of inflammatory bowel disease. Understanding how Ndfip1 limits effector T cell function could clarify how human diseases such as inflammatory bowel disease and multiple sclerosis develop.

Interleukin-21 suppresses the differentiation and functions of T helper 2 cells.

T helper type 2 (Th2) cells, which produce interleukin-4 (IL-4), IL-5 and IL-13, control immunity to all forms of allergic inflammatory responses. Interleukin-21 (IL-21) reduces allergic symptoms in murine models and inhibits IL-4-induced IgE secretion by B cells. However, whether or not IL-21 directly affects Th2 cells, which leads to reduced allergic symptoms, is unclear. In this study, we investigated the effects of IL-21 on the differentiation and effector functions of Th2 cells. We found that IL-21 reduced the number of differentiated Th2 cells and these Th2 cells showed a diminished Th2 cytokine production. Interleukin-21 suppressed Th2 cytokine production of already polarized Th2 cells by down-regulation of transcription factor GATA-3. It also induced apoptosis of Th2 cells with decreased anti-apoptotic factor Bcl-2. Intranasal administration of IL-21 at the beginning of ovalbumin (OVA) sensitization or before OVA challenge decreased Th2 cytokines in the bronchoalveolar lavage fluid of OVA/alum-immunized allergic mice. In addition, the inhibitory effects of IL-21 on Th2 effector functions can also be found in allergic patients. Our results demonstrate that IL-21 suppresses the development of Th2 cells and functions of polarized Th2 cells. Hence, the administration of IL-21 may be considered for use as a preventive and therapeutic approach when dealing with Th2-mediated allergic diseases.

Immune-based antitumor effects of BRAF inhibitors rely on signaling by CD40L and IFNγ.

B-Raf(V600E) inhibitors have been suggested to promote tumor regression with the help of host immunity, but this hypothesis has not been examined directly in detail. In this study, we profiled immunologic changes in the tumor microenvironment and tumor-infiltrating lymphocytes (TIL) in a B-RafV600E/Pten-driven murine model of melanoma after administration of the B-Raf(V600E) small molecule inhibitor PLX4720. In this model, we found that as tumors developed, they gradually acquired immunosuppressive features, including accumulation of regulatory T cells (Treg) and CD11b(+)/Gr-1(+) myeloid cells and loss of Th1 effector functions on CD4(+) TILs, such as CD40L and IFNγ expression. PLX4720 administration promoted development of a more immune stimulatory microenvironment associated with a relative increase in CD40L and IFNγ expression on intratumoral CD4(+) TILs and a reduced accumulation of Tregs and CD11b(+)/Gr-1(+) myeloid cells. Strikingly, CD40L or IFNγ blockade compromised the ability of PLX4720 to inhibit melanoma growth. Supporting this result, agonistic CD40 antibody was sufficient to evoke antitumor immunity and suppress tumor growth in tumor-bearing mice. Taken together, our results establish the critical role of immune-related changes, with key contributions for CD40L and IFNγ signaling in the antitumor responses triggered in vivo by B-Raf(V600E) inhibitors.

IL-35-Producing T Cells (iTR35) Inhibit Th2 Effector Function, Induce Infectious Tolerance and Are Elevated Following Grass Pollen Sublingual Immunotherapy

IL-35-Producing T Cells (iTR35) Inhibit Th2 Effector Function, Induce Infectious Tolerance and Are Elevated Following Grass Pollen Sublingual Immunotherapy

Escalating regulation of 5T4-specific IFN-γ+ CD4+ T cells distinguishes colorectal cancer patients from healthy controls and provides a target for in vivo therapy.

The relationship between the adaptive CD4+ T cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed on many human carcinomas, including colorectal cancer (CRC) cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4+ T cells in a proportion of CRC patients, and we extended this study to examine whether the quality or quantity of the T cell response reflects tumor stage. An overlapping peptide library spanning 5T4 was used as a target to enumerate cognate IFN-γ+CD4+ T-cells (measured as spot forming cells [SFC]/105 cultured T cells) in peripheral blood-derived lymphocytes following a 12-day in vitro culture period comparing patients pre-operatively (n = 27) to healthy controls (n = 17). Robust 5T4-specific T cell responses were present in 100% of healthy donors. There was a steady loss of T cell responses with advancing tumors with a significant negative correlation from stage I to III (P = 0.008). The predictability of the decline meant < 200 SFC/105 was only found in subjects with stage III CRC. The mechanism of loss of T cell response is independent of HLA-DR type or patient age, but does correspond to increases in Foxp3+ regulatory T cells (Tregs). Using low-dose cyclophosphamide to reduce the proportion of Tregs in vivo resulted in increased anti-5T4 T cell responses in CRC patients. The selective loss of 5T4-specific IFN-γ+CD4+ T cell responses implies a link between tumor stage and antitumor Th1 effector function; depleting Tregs can enhance such responses.

95 : Type I interferon regulates acute IL-5 and IL-13 expression in human memory CD4+ T cells

Type I Interferon (IFN- α / β ) is a critical anti-viral cytokine and is currently being used to treat Hepatitis C viral infections and multiple sclerosis, among other diseases. Although the positive regulatory effects of this cytokine have been studied, less is known about its role as a negative regulator of gene expression. Previously, we demonstrated that IFN- α / β suppresses Th2 development in human naive CD4+ T cells by negatively regulating GATA3 expression, leading to the suppression of IL-4, IL-5 and IL-13. Based on this data, we were interested in how IFN- α / β might affect other T cell types. In the current study, we interrogated the effects of IFN- α / β on human memory CD4+ T cells. We hypothesized that IFN- α / β has similar negative regulatory effects on memory CD4+ T cells, leading to decreased Th2 cytokine expression. Human memory CD4+ T cells were acutely stimulated in the presence of IFN- α . Interestingly, IL-5 and IL-13 gene expression was suppressed, however, IL-4 expression was unaffected. Further, IFN- α / β signaling, but not IFN- γ or IFN- λ , suppressed IL-5 and IL-13 expression. Nuclear run-on studies demonstrated that IFN- α / β signaling suppressed de novo transcription of IL-5 and IL-13, and this suppression did not require the translation of interferon-sensitive genes. Our data indicate that this negative regulatory pathway acted upstream of gene expression, and suggested that IFN- α / β was able to acutely suppress specific gene expression. Currently, we are assessing direct STAT2 binding by ChIP-Seq, which will identify potential cis-regulatory regions necessary for IL-5 and L-13 gene modulation. Together, these results suggest that IFN- α / β could have many benefits in suppressing multiple aspects of Th2 development and effector function in atopic asthma.