Cell Responses In Mouse Model Research Articles

Immunopeptidomics Mapping of Listeria monocytogenes T Cell Epitopes in Mice

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes, we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6000mouse self-peptides presented on MHC class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in the mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, Protected Mice from Inflammatory and Autoimmune Diseases

Pathogenic immune cell responses in inflammatory and autoimmune diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the roles of γc cytokines in driving inflammatory and autoimmune diseases, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic immune cell responses in mouse models of graft-versus-host disease (GVHD), immune aplastic anemia and multiple sclerosis (MS). We first evaluated the efficacy of REGN7257 in xenogeneic and allogeneic mouse models of GVHD. In the xenogeneic model, immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). In the allogeneic GVHD model, irradiated BALB/c mice were engrafted with bone marrow cells and splenocytes from Il2rghu/hu mice. In the xenogeneic GVHD mouse model, we also analyzed the effects of γc cytokine signaling blockade with REGN7257 on inflammation and immune cell infiltration into tissues (e.g. liver, bone marrow), as well as liver fibrosis. Finally, we assessed the effects of IL2RG blockade on pathogenic immune cell responses in an experimental autoimmune encephalomyelitis (EAE) model of MS, looking at immune cell infiltration into the central nervous system (CNS) and associated demyelination. In both xenogeneic and allogeneic models of GVHD, prophylactic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Similarly, in the xenogeneic GVHD model, dosing with REGN7257 starting 3 weeks after huPBMC administration effectively protected mice from weight loss and death. Consistent with the classic pathology of GVHD, tissues of control mice were highly infiltrated by immune cells, including CD4+ and CD8+ T-cells, while γc cytokine signaling blockade strongly reduced immune cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Finally, in an EAE mouse model of MS, inhibition of γc cytokine signaling with REGN7257 alleviated disease progression and significantly prolonged survival. This was associated with reduced inflammation and infiltration by immune cells (CD4+ T-cells, CD8+ T-cells, B-cells and monocytic cells) in the CNS, as well as decreases in myelin oligodendrocyte glycoprotein-specific antibody production. We showed that REGN7257 treatment efficiently protected the spinal cord from demyelination, which was associated with decreased oligodendrocyte injury and neuron protection. Blockade of γc cytokine signaling with REGN7257 protected mice against immune cell-mediated pathology in multiple disease models: an EAE model of MS, an allogeneic GVHD model, and a xenogeneic GVHD model that uniquely presents hallmarks of both acute and chronic GVHD as well as immune aplastic anemia. These data provide evidence of γc cytokines as key drivers of pathogenic immune cell responses, offering a potentially novel strategy for the management of inflammatory and autoimmune diseases, such as GVHD, immune aplastic anemia and MS.

Dual role of the miR-146 family in rhinovirus-induced airway inflammation and allergic asthma exacerbation.

Rhinovirus (RV) infections are associated with asthma exacerbations. MicroRNA‐146a and microRNA‐146b (miR‐146a/b) are anti‐inflammatory miRNAs that suppress signaling through the nuclear factor kappa B (NF‐κB) pathway and inhibit pro‐inflammatory chemokine production in primary human bronchial epithelial cells (HBECs). In the current study, we aimed to explore whether miR‐146a/b could regulate cellular responses to RVs in HBECs and airways during RV‐induced asthma exacerbation. We demonstrated that expression of miR‐146a/b and pro‐inflammatory chemokines was increased in HBECs and mouse airways during RV infection. However, transfection with cell‐penetrating peptide (CPP)‐miR‐146a nanocomplexes before infection with RV significantly reduced the expression of the pro‐inflammatory chemokines CCL5, IL‐8 and CXCL1, increased interferon‐λ production, and attenuated infection with the green fluorescent protein (GFP)‐expressing RV‐A16 in HBECs. Concordantly, compared to wild‐type (wt) mice, Mir146a/b−/− mice exhibited more severe airway neutrophilia and increased T helper (Th)1 and Th17 cell infiltration in response to RV‐A1b infection and a stronger Th17 response with a less prominent Th2 response in house dust mite extract (HDM)‐induced allergic airway inflammation and RV‐induced exacerbation models. Interestingly, intranasal administration of CPP‐miR‐146a nanocomplexes reduced HDM‐induced allergic airway inflammation without a significant effect on the Th2/Th1/Th17 balance in wild‐type mice. In conclusion, the overexpression of miR‐146a has a strong anti‐inflammatory effect on RV infection in HBECs and a mouse model of allergic airway inflammation, while a lack of miR‐146a/b leads to attenuated type 2 cell responses in mouse models of allergic airway inflammation and RV‐induced exacerbation of allergic airway inflammation. Furthermore, our data indicate that the application of CPP‐miR‐146a nanocomplexes has therapeutic potential for targeting airway inflammation.

Serum HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV infection.

Antibody-mediated clearance of hepatitis B surface antigen (HBsAg) from the circulation of chronically infected patients (i.e., seroconversion) is usually associated with increased HBV-specific T cell responsiveness. However, a causative link between serum HBsAg levels and impairment of intrahepatic CD8+ T cells has not been established. Here we addressed this issue by using HBV replication-competent transgenic mice that are depleted of circulating HBsAg, via either spontaneous seroconversion or therapeutic monoclonal antibodies, as recipients of HBV-specific CD8+ T cells. Surprisingly, we found that serum HBsAg clearance has only a minimal effect on the expansion of HBV-specific naive CD8+ T cells undergoing intrahepatic priming. It does not alter their propensity to become dysfunctional, nor does it enhance the capacity of IL-2-based immunotherapeutic strategies to increase their antiviral function. In summary, our results reveal that circulating HBsAg clearance does not improve HBV-specific CD8+ T cell responses in vivo and may have important implications for the treatment of chronic HBV infection.

Extracellular HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV pathogenesis

Extracellular HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV pathogenesis

Evidence for Anti-Viral Effects of Complete Freund’s Adjuvant in the Mouse Model of Enterovirus Infection

Group B coxsackieviruses (CVBs) belonging to the genus, Enterovirus and contain six serotypes that induce various diseases, whose occurrence may involve the mediation of more than one serotype. We recently identified immunogenic epitopes within coxsackieviruses B3 (CVB3) viral protein 1 that induce anti-viral T cell responses in mouse models of CVB infections. In our investigations to determine the protective responses of the viral epitopes, we unexpectedly noted that animals immunized with complete Freund’s adjuvant (CFA) alone and later challenged with CVB3 were completely protected against myocarditis. Similarly, the pancreatitis-inducing ability of CVB3 was remarkably reduced to only 10% in the CFA group as opposed to 73.3% in the control group that received no CFA. Additionally, no mortalities were noted in the CFA group, whereas 40% of control animals died during the course of 21 days post-infection with CVB3. Taken together, our data suggest that the adjuvant effects of CFA may be sufficient for protection against CVB infections. These observations may provide new insights into our understanding of the occurrence of viral infections.

Cell therapy targeting follicular T cells as an innovative approach for treating autoimmune disease

Abstract Systemic lupus erythematosus (SLE) is a debilitating and incurable autoimmune disease characterized by the uncontrolled production of autoantibodies and inflammatory damage to multiple organ systems. Dysregulated follicular helper T cell (TFH) responses are a cardinal feature and putative driver of disease in both mice and humans, where these cells contribute to unrestrained inflammation and autoantibody production by B cells. There is currently no safe and effective way to selectively eliminate TFH as a means to therapeutically alleviate diseases caused by these cells. Although cell surface biomarkers that are unique (lineage-specific targets) to TFH remain undefined, these cells express markedly more programmed cell death protein 1 (PD-1) than other immune cells. We recently engineered a novel programmed death-ligand 1 (PD-L1)-based chimeric antigen receptor (CAR) that when expressed on human natural killer (NK) cells, facilitated discriminate targeting of PD-1high follicular T cells while sparing other PD-1-expressing lymphocytes. The inherent specificity of this approach lies in the lower affinity interactions between PD-L1 and PD-1 compared to that likely achieved with a PD-1-specific single-chain variable fragment-based CAR. Here, we develop a mouse T cell version of the PD-L1 CAR platform to show that selective depletion of PD-1high T cells is safe and effective at undermining harmful autoreactive T and B cell responses in mouse models of SLE-like disease. We believe this affinity-based CAR approach for PD-1high target cell elimination represents a novel clinical tool for treatment of TFH-driven diseases like SLE.

Abstract 576: A novel intestinal microbiome-derived peptide modulates host T cell activation

Abstract The microbiome shapes the metabolic and immunological landscape of individuals in health and disease and represents a new robust source of bioactive molecules with therapeutic potential. Second Genome’s large and curated microbiome database coupled with its proprietary bioinformatics and machine learning pipeline enables the discovery of novel microbiome-derived drug candidates across multiple disease areas, including immuno-oncology (IO). Microbial genus Bifidobacterium previously showed a positive association with antitumor T cell responses in mouse models and was overrepresented in melanoma patients responding to immunotherapy with anti-PD-1 antibody. Our hypothesis is that bioactive molecules derived from these Bifidobacteria help drive these antitumor responses and could function as cancer therapeutics or adjuvants for cancer immunotherapy. Thus, we nominated protein and peptide candidates with high scores of secretability, uniqueness, stability, and expressability from Bifidobacterium breve and Bifidobacterium longum genomes for screening in cell-based assays. Initial evaluation of more than 50 Bifidobacterium peptides revealed candidates capable of inducing immune activation. Here, we describe a B. breve-derived peptide (termed SG-B) that induced up-regulation of co-stimulatory (OX40 and ICOS) and inhibitory (PD-1) molecules on CD4+ and CD8+ T cells in both: i) a purified human pan-T cell system; and ii) PBMC cultures stimulated with low-dose anti-CD3 antibody. Furthermore, SG-B stimulated secretion of effector cytokines by in vitro-cultured purified T cells (TNF-a, IL-2, IFN-g, and IL-10) and PBMCs (IL-1b, IL-6, and IL-8). These effects were dose-dependent and evident across multiple human blood donors. In the CT26 syngeneic tumor model, peri-tumoral administration of SG-B induced an increase in the proportion of NK cells and CD4+ T cells within the tumor. The peptide also induced up-regulation of activation marker on CD8+ T cells (CD25, Ki67, and OX40). Collectively, these data suggest that SG-B can modulate adaptive immunity via T cell potentiation and may enhance tumor inflammation in vivo. The peptide’s ability to up-regulate key co-stimulatory and checkpoint molecules on T cells provides a strong rationale for its potential future use in combination with approved or IO agents in current development. These results validate the capability of the Second Genome drug discovery platform to identify novel microbial agents of potential therapeutic relevance in IO and demonstrate a unique approach that can identify microbial factors involved in modulating immune cell effector functions and/or immune cell differentiation. Citation Format: Yuliya V. Katlinskaya, Dhwani Haria, Lily McLaughlin, Sunit Jain, Shoko Iwai, Todd DeSantis, Thomas Weinmaier, Toshi Takeuchi, Anu Hoey, Karim Dabbagh, Kareem Graham, Helena Kiefel. A novel intestinal microbiome-derived peptide modulates host T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 576.

B Cell-Mediated Antigen Presentation through MHC Class II Is Dispensable for Atherosclerosis Progression.

Depletion of B cells attenuates plaque development and modulates T cell responses in mouse models of atherosclerosis, suggesting that Ag presentation by B cells may promote disease progression. Thus, we set out to determine the role of B cell-mediated MHC class II (MHC II) Ag presentation during atherosclerotic plaque development. We developed murine conditional MHC II deletion and expression systems under control of the B cell-restricted CD19 promoter in an experimental model of atherosclerosis. Mice lacking MHC II expression only on B cells exhibited systemic shifts in germinal center and marginal zone B cell populations, leading to a reduced Ab response compared with littermate control animals. However, all populations were present and normal cholesterol uptake was detected in the plasma following high-fat diet treatment. In a second model, in which conditional expression of MHC II is limited only to B cells, showed similar overall cellularity characteristics compared with mice with complete MHC II deficiency. High-fat diet feeding showed no major changes in atherosclerotic plaque size or plaque cellular content in either conditional deletion or conditional expression approaches, compared with control animals. By testing the necessity and sufficiency of MHC II on B cells in the progression of atherosclerosis, we determine that MHC II on B cells does not directly regulate lesion development in murine models.

Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis

Integrin αvβ6 is rapidly up-regulated on cells of epithelial lineage during tissue injury, where one of its primary functions is activation of latent transforming growth factor beta 1 (TGFβ1). In human liver cirrhosis, αvβ6 is overexpressed by cells comprising the ductular reaction, and its inhibition suppresses experimental biliary fibrosis in rodents. Here, we show that αvβ6 is expressed on the actively proliferating subset of hepatic progenitor cells and is required for their progenitor function in vivo and in vitro through integrin αvβ6-dependent TGFβ1 activation. Freshly isolated αvβ6(+) liver cells demonstrate clonogenic potential and differentiate into cholangiocytes and functional hepatocytes in vitro, whereas colony formation by epithelial cell adhesion molecule-positive progenitor cells is blocked by αvβ6-neutralizing antibody and in integrin beta 6-deficient cells. Inhibition of progenitors by anti-αvβ6 antibody is recapitulated by TGFβ1 neutralization and rescued by addition of bioactive TGFβ1. Genetic disruption or selective targeting of αvβ6 with 3G9 antibody potently inhibits progenitor cell responses in mouse models of chronic biliary injury and protects from liver fibrosis and tumorigenesis, two conditions clinically associated with exacerbated ductular reaction. These results suggest that αvβ6 is a promising target for chronic fibrotic liver diseases and associated cancers.

Vaccine-induced Aβ-specific CD8+ T cells do not trigger autoimmune neuroinflammation in a murine model of Alzheimer's disease.

BackgroundActive immunization against Aβ was reported to have a therapeutic effect in murine models of Alzheimer’s disease. Clinical Aβ vaccination trial AN1792 was interrupted due to the development in 6 % of the patients of meningoencephalitis likely involving pro-inflammatory CD4+ T cells. However, the potential implication of auto-aggressive anti-Aβ CD8+ T cells has been poorly investigated.MethodsPotential MHC-I-restricted Aβ-derived epitopes were first analyzed for their capacity to recruit functional CD8+ T cell responses in mouse models. Their impact on migration of CD8+ T cells into the brain parenchyma and potential induction of meningoencephalitis and/or neuronal damage was investigated upon vaccination in the APPPS1 mouse model of AD.ResultsWe identified one nonamer peptide, Aβ33-41, which was naturally processed and presented in association with H-2-Db molecule on neurons and CD11b+ microglia. Upon optimization of anchor residues for enhanced binding to H-2-Db, immunization with the modified Aβ33-41NP peptide elicited Aβ-specific IFNγ-secreting CD8+ T cells, which are cytotoxic towards Aβ-expressing targets. Whereas T cell infiltration in the brain of APPPS1 mice is dominated by CD3+CD8− T cells and increases with disease evolution between 4 and 7 months of age, a predominance of CD3+CD8+ over CD3+CD8− cells was observed in 6- to 7-month-old APPPS1 but not in WT animals, only after vaccination with Aβ33-41NP. The number of CD11b+ mononuclear phagocytes, which significantly increases with age in the brain of APPPS1 mice, was reduced following immunization with Aβ33-41NP. Despite peripheral activation of Aβ-specific CD8+ cytotoxic effectors and enhanced infiltration of CD8+ T cells in the brain of Aβ33-41NP-immunized APPPS1 mice, no clinical signs of severe autoimmune neuroinflammation were observed.ConclusionsAltogether, these results suggest that Aβ-specific CD8+ T cells are not major contributors to meningoencephalitis in response to Aβ vaccination.

Abstract 2815: Induction of both OVA specific CD4+ and CD8+ T cells by using PepTivator® OVA-pulsed DCs in mouse model

Abstract Antigen specific immune response by both MHC class I-restricted CD8+ T cells and MHC class II-restricted CD4+ T cells is a key event in cancer immunotherapy. In DC vaccine therapy, the short epitope peptides (9 or 10-mer) are mainly used to load, but these short peptides are presented on MHC class I molecule and can stimulate only CD8+ T cells. Using whole protein as antigen might be better to induce both antigen specific CD8+ and CD4+ T cells. The PepTivator® is a peptide pool: consisting mainly of 15-mer sequences with 11 amino acids overlap and covering the complete sequence of target protein. In this study, we used PepTivator® derived from ovalbumin protein (PepTivator® OVA) instead of whole protein as antigen, and evaluated the inducing activity of antigen specific CD8+ and CD4+ T cell responses in mouse model. In order to examine the antigen presenting ability of PepTivator® OVA-pulsed DCs, CD8+ T cells (106 cells/mL) isolated from OT-I transgenic mouse (OT-I CD8T) or CD4+ T cells (106 cells/mL) isolated from OT-II transgenic mouse (OT-II CD4T) were cultured with PepTivator® OVA-pulsed DCs (5×104 cells/mL) for 4 days. As positive control, OT-I epitope peptide (OVA257-264aa) and OT-II epitope peptide (OVA323-339aa) were used. DCs were prepared from bone marrow cells of C57BL/6 mice after cultivation with medium containing GM-CSF, IL-4 and LPS for 10 days. In preparation of peptide-pulsed DCs, DCs were treated with each peptide at 2 µg/mL for 4 hours and washed with medium to deplete free peptide before use. During the cultivation of OT-I CD8T or OT-II CD4T cells with each peptide-pulsed DCs, the responsive T cell proliferation was measured daily by flow cytometric analysis with absolute count beads and CFSE dye dilution. In addition, cytokines (IL-2 and IFN-gamma) in culture supernatant were measured by ELISA. As a result, the proliferation of OT-I CD8T cells cocultured with PepTivator® OVA-pulsed DCs seemed to be similar to that in coculture with OT-I epitope peptide-pulsed DCs. In the case of proliferation assay using OT-II CD4T cells, PepTivator® OVA-pulsed DCs showed similar T cell expansion in comparison with OT-II epitope peptide-pulsed DCs. As for cytokines secretion, no difference was observed in concentration of IL-2 and IFN-gamma in both cases using OT-I CD8T and OT-II CD4T cells when PepTivator® OVA was compared with OT-I or OT-II epitope peptide, respectively. These results showed that PepTivator® OVA pulsed-DCs could induce both antigen specific CD8+ and CD4+ T cell responses in mouse model. It suggests that PepTivator® OVA is at least superior to short epitope peptide in inducing antigen specific T cell response. In addition, it might be big advantage that PepTivator® derived from tumor antigen could be useful in many candidate patients without their HLA restriction. It is expected that therapeutic benefit of PepTivator® pulsed-DCs will be shown in clinical. Citation Format: Kenji Miki, Koji Nagaoka, Hermann Bohnenkamp, Takayuki Yoshimoto, Takashi Kamigaki, Ryuji Maekawa. Induction of both OVA specific CD4+ and CD8+ T cells by using PepTivator® OVA-pulsed DCs in mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2815. doi:10.1158/1538-7445.AM2014-2815

Inflammatory T Cell Responses Rely on Amino Acid Transporter ASCT2 Facilitation of Glutamine Uptake and mTORC1 Kinase Activation

Glutamine has been implicated as an immunomodulatory nutrient, but how glutamine uptake is mediated during T cell activation is poorly understood. We have shown that naive T cell activation is coupled with rapid glutamine uptake, which depended on the amino acid transporter ASCT2. ASCT2 deficiency impaired the induction of T helper 1 (Th1) and Th17 cells and attenuated inflammatory T cell responses in mouse models of immunity and autoimmunity. Mechanistically, ASCT2 was required for T cell receptor (TCR)-stimulated activation of the metabolic kinase mTORC1. We have further shown that TCR-stimulated glutamine uptake and mTORC1 activation also required a TCR signaling complex composed of the scaffold protein CARMA1, the adaptor molecule BCL10, and the paracaspase MALT1. This function was independent of IKK kinase, a major downstream target of the CARMA1 complex. These findings highlight a mechanism of T cell activation involving ASCT2-dependent integration of the TCR signal and a metabolic signaling pathway.

Evaluating antigen-specific cytotoxic T lymphocyte responses by a novel mouse granzyme B ELISPOT assay

We have established novel ELISA- and ELISPOT-based assays specific for the detection of a potent cytotoxic mediator, granzyme B (GrB), for the assessment of antigen-specific cytotoxic T lymphocyte responses in mice. The sensitivity and specificity of our assays was demonstrated by ELISA using purified mouse GrB and supernatants and cell lysates of cytotoxic lymphocytes derived from GrB-deficient mice. No reactivity was observed by the GrB ELISA in GrB-deficient cells. The mouse GrB ELISPOT was successfully employed to detect antigen-specific effector cell responses of two CTL clones, producing GrB ELISPOT results that correlated strongly with target cell lysis, as assessed by 51Cr-release. Furthermore, we were able to demonstrate direct correlations between GrB ELISPOT and killing by LCMV gp33-specific effector and memory T cells generated in vivo. Thus, the mouse GrB ELISPOT may be used to detect cytotoxic responses, at the single-cell level, for the functional assessment of antigen-specific CD8 + T cell responses in mouse models of infection.

181 POSTER Parathyroid Hormone-related Protein (PTHrP) expression in human bone and liver metastases

Glutamine has been implicated as an immunomodulatory nutrient, but how glutamine uptake is mediated during T cell activation is poorly understood. We have shown that naive T cell activation is coupled with rapid glutamine uptake, which depended on the amino acid transporter ASCT2. ASCT2 deficiency impaired the induction of T helper 1 (Th1) and Th17 cells and attenuated inflammatory T cell responses in mouse models of immunity and autoimmunity. Mechanistically, ASCT2 was required for T cell receptor (TCR)-stimulated activation of the metabolic kinase mTORC1. We have further shown that TCR-stimulated glutamine uptake and mTORC1 activation also required a TCR signaling complex composed of the scaffold protein CARMA1, the adaptor molecule BCL10, and the paracaspase MALT1. This function was independent of IKK kinase, a major downstream target of the CARMA1 complex. These findings highlight a mechanism of T cell activation involving ASCT2-dependent integration of the TCR signal and a metabolic signaling pathway.

183 POSTER TGF-beta-induced Stat3 phosphorylation mediated growth regulation in lung cancer

Glutamine has been implicated as an immunomodulatory nutrient, but how glutamine uptake is mediated during T cell activation is poorly understood. We have shown that naive T cell activation is coupled with rapid glutamine uptake, which depended on the amino acid transporter ASCT2. ASCT2 deficiency impaired the induction of T helper 1 (Th1) and Th17 cells and attenuated inflammatory T cell responses in mouse models of immunity and autoimmunity. Mechanistically, ASCT2 was required for T cell receptor (TCR)-stimulated activation of the metabolic kinase mTORC1. We have further shown that TCR-stimulated glutamine uptake and mTORC1 activation also required a TCR signaling complex composed of the scaffold protein CARMA1, the adaptor molecule BCL10, and the paracaspase MALT1. This function was independent of IKK kinase, a major downstream target of the CARMA1 complex. These findings highlight a mechanism of T cell activation involving ASCT2-dependent integration of the TCR signal and a metabolic signaling pathway.