TCR Tg Research Articles

DOK1 and DOK2 regulate CD8 T cell signaling and memory formation without affecting tumor cell killing

Targeting intracellular inhibiting proteins has been revealed to be a promising strategy to improve CD8+ T cell anti-tumor efficacy. Here, we are focusing on intracellular inhibiting proteins specific to TCR signaling: DOK1 and DOK2 expressed in T cells. We hypothesized that depletion of intracellular inhibition checkpoint DOK1 and DOK2 could improve CD8+ T-cell based cancer therapies. To evaluate the role of DOK1 and DOK2 depletion in physiology and effector function of CD8+ T lymphocytes and in cancer progression, we established a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) in WT and Dok1/Dok2 DKO (double KO) mice. We showed that both DOK1 and DOK2 depletion in CD8+ T cells after an in vitro pre-stimulation induced a higher percentage of effector memory T cells as well as an up regulation of TCR signaling cascade- induced by CD3 mAbs, including the increased levels of pAKT and pERK, two major phosphoproteins involved in T cell functions. Interestingly, this improved TCR signaling was not observed in naïve CD8+ T cells. Despite this enhanced TCR signaling essentially shown upon stimulation via CD3 mAbs, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxic capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. Indeed, our results allow us to conclude that DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.

Colitis in a transgenic mouse model of autoimmune uveitis may be induced by neoantigen presentation in the bowel

Undifferentiated uveitis (intraocular inflammation, IOI) is an idiopathic sight-threatening, presumed autoimmune disease, accountable for ~ 10% of all blindness in the developed world. We have investigated the association of uveitis with inflammatory bowel disease (IBD) using a mouse model of spontaneous experimental autoimmune uveoretinitis (EAU). Mice expressing the transgene (Tg) hen egg lysozyme (HEL) in the retina crossed with 3A9 mice expressing a transgenic HEL-specific TCR spontaneously develop uveoretinitis at post-partum day (P)20/21. Double transgenic (dTg TCR/HEL) mice also spontaneously develop clinical signs of colitis at ~ P30 with diarrhoea, bowel shortening, oedema and lamina propria (LP) inflammatory cell infiltration. Single (s)Tg TCR (3A9) mice also show increased histological LP cell infiltration but no bowel shortening and diarrhoea. dTg TCR/HEL mice are profoundly lymphopenic at weaning. In addition, dTg TCR/HEL mice contain myeloid cells which express MHC Class II-HEL peptide complexes (MHCII-HEL), not only in the inflamed retina but also in the colon and have the potential for antigen presentation. In this model the lymphopenia and reduction in the absolute Treg numbers in dTg TCR/HEL mice is sufficient to initiate eye disease. We suggest that cell-associated antigen released from the inflamed eye can activate colonic HEL-specific T cells which, in a microbial micro-environment, not only cause colitis but feedback to amplify IOI.

Influence of the Th1 Cytokine Environment on CCL5 Production from Langerhans Cells.

In the chronic skin lesions of atopic dermatitis (AD), T helper type 1 (Th1) cells appear in addition to Th2 cells, but the role played by Th1 cells in skin inflammation during the chronic phase remains unknown. Here we examined CCL5 production from Langerhans cells (LCs) in the Th1 cytokine environment. LCs were generated from mouse bone marrow cells, then stimulated with anti-CD40 antibody and the Th1 cytokine, interferon (IFN)-γ. Their CCL5 production was then measured. In addition, the LCs were incubated with naïve CD4+ T cells from a DO 11.10 TCR Tg mouse in the presence of ovalbumin peptide for 5 d, and their IFN-γ, interleukin-4 and CCL5 production was then measured. When LCs were stimulated with the anti-CD40 antibody in the presence of IFN-γ, significant levels of CCL5 production were confirmed. Furthermore, when LCs presented antigen to Th cells in the Th1 cytokine environment, significant levels of CCL5 production were induced. This CCL5 production was associated with IFN-γ activity and CD40L expression by Th cells in the culture. Our present data suggest that LCs augment CCL5 production by responding to IFN-γ while presenting antigen to Th cells, and that this augmentation of CCL5 production would likely contribute to infiltration of eosinophils and other Th1 cells into skin lesions, followed by expansion of chronic inflammation in the skin.

Repeated α-GalCer Administration Induces a Type 2 Cytokine-Biased iNKT Cell Response and Exacerbates Atopic Skin Inflammation in Vα14Tg NC/Nga Mice.

We have previously shown that Vα14 TCR Tg (Vα14Tg) NC/Nga (NC) mice contain increased numbers of double-negative (DN) invariant natural killer T (iNKT) cells that protect against spontaneous development of atopic dermatitis (AD). iNKT cells can regulate immune responses by producing various cytokines such as IFNγ and IL4 rapidly upon stimulation with α-galactosylceramide (α-GalCer), a prototypical iNKT cell agonist. However, the precise role of α-GalCer-activated iNKT cells in AD development remains unclear. Therefore, we examined whether repeated activation of iNKT cells with α-GalCer can regulate the pathogenesis of AD in Vα14Tg NC mice. We found that Vα14Tg NC mice injected repeatedly with α-GalCer display exacerbated AD symptoms (e.g., a higher clinical score, IgE hyperproduction, and increased numbers of splenic mast cells and neutrophils) compared with vehicle-injected Vα14Tg NC mice. Moreover, the severity of AD pathogenesis in α-GalCer-injected Vα14Tg NC mice correlated with increased Th2 cells but reduced Th1 and Foxp3+ Treg cells. Furthermore, the resulting alterations in the Th1/Th2 and Treg/Th2 balance were strongly associated with a biased expansion of type 2 cytokine-deviated iNKT cells in α-GalCer-treated Vα14Tg NC mice. Collectively, our results have demonstrated the adverse effect of repeated α-GalCer treatment on skin inflammation mediated by type 2 immunity.

Modular approach to generating long-lived effector/memory (Teff/Tem) T cells to prolong malaria immunity

Abstract Immunity to Plasmodium infection or vaccination is known to decay. In mouse models, both B and T cells are required for immunity; however, decay of protection correlates with loss of malaria-specific T cells, not antibody. Whole parasite vaccines, such as the infection and drug cure protocol, protect. However, sterile protection from P. chabaudi or vaccination last less than 200 days. Therefore, we have reverse-engineered a vaccine strategy that first induces a long-lived protective antibody response using live parasite vaccination, and then boosts Teff and Tem response to prolong protection. Cytomegalovirus (CMV), is a known chronic vaccine vector that induces T cell mediated immunity and shows promise against SIV, tuberculosis and liver-stage malaria. Therefore, we used chronic vaccination with MCMV to test our hypothesis that promoting longer generation of Teff in mice would prolong protection by a live vaccine. MCMV encoding P. chabaudi MSP-1 epitope B5 (MCMV-B5) was used to follow the malaria- specific T cell response. Upon infection of mice with MCMV-B5, both adoptively transferred B5 TCR Tg T cells and MCMV-specific CD4 T cells proliferated and were maintained over 2.5 months. Coinciding with this protection, the MCMV vector worked to prolong the protection generated by live malaria vaccine. In investigating the mechanisms of protection, we found that IFN-γ induced by MCMV prolongs the protection, potentially through promotion of IL-12 producing and antigen presenting cell function. Our findings suggest the methods and the mechanisms to effectively prolong immunity to Plasmodium infection based on CMV mechanism to redress the problem of short-lived protection to malaria infection and vaccines.

Helios expression in CD4+Foxp3− T cells

Abstract The transcription factor Helios is expressed in the majority of Foxp3+ Treg cells, where it is necessary for the maintenance of self-tolerance. Mice with a Foxp3-specific deficiency in Helios develop systemic immune activation with increased T follicular helper (Tfh) and T helper 1 (Th1) effector responses. Notably, mice with a CD4-specific deletion of Helios do not exhibit autoimmunity, pointing to an additional role for Helios in CD4+ T cells. In fact, Helios is expressed in a minor population of conventional CD4+ T cells and its expression is upregulated upon activation of CD4+ T cells, yet the role of Helios in these cells remains unknown. We generated TCR Tg mice with a CD4-specific Helios deletion and have shown that Helios deficient naive CD4+ T cells are fully able to differentiate into Th1, Th2 and Tfh effector cells in vivo. Helios deficient CD4+T cells show no defect in expansion or the production of cytokines. However, Helios deficiency increased the propensity of naive T cells to differentiate into antigen-specific pTregs under inflammatory as well as tolerizing conditions and attenuated the induction of a memory response. To further characterize CD4+Foxp3−Helios+ cells, Next-generation sequencing (NGS) analysis was performed. This analysis has yielded evidence for a heterogenous population that is comprised of Tfh, Tmem, iNKT and a population of cells that have Treg-like features. In vitro activation assays and qPCR have validated differentially expressed cytokines between Helios+ and Helios− CD4+ T cell populations, in agreement with the NGS data. Together, these findings suggest that Helios may play a role in stabilizing effector function.

Continuous generation of effector (Teff) and effector memory (Tem) T cells with mouse cytomegalovirus (MCMV) vaccination to prolong malaria immunity

Abstract Immunity to Plasmodium infection or vaccination decays without continuous exposure. This decay correlates with loss of malaria-specific T cells, not antibody. We recently demonstrated that Teff protect better than memory T cells, and that Ifng+ Teff are responsible for the protection provided by persistent P. chabaudi infection. Based on this data, we have reverse-engineered a vaccine strategy that first induces a long-lived antibody response, and then boosts the Teff and Tem response to prolong protection. We chose live vaccination with “infection and drug cure”, which generates protection that decays over 200 days. The boost uses MCMV as it is an excellent T cell-inducing vector with promise against SIV, tuberculosis and liver-stage malaria. First, we observed that MCMV infection strongly reduced parasite growth, as previously shown for Leishmania, suggesting a useful adjuvant effect. To promote continuous induction of specific T cells, we generated MCMV expressing the P. chabaudi T cell epitope B5. Vaccination with MCMV-B5 activated both adoptively transferred B5 TCR Tg T cells and MCMV-specific CD4 T cells, generating protective Teff and Tem phenotypes for over 2.5 months. Importantly, we show that boosting with the MCMV vector prolongs protection generated by the live vaccine, as shown by better protection from re-infection at day 200. We are currently investigating the contribution of activated Plasmodium-specific T cells in prolonging protective immunity by MCMV-B5, and mechanisms of protection. Our findings suggest that chronic vaccination can redress the problem of short-lived protection to malaria infection and vaccines, which would be beneficial for any efficacious but short-term vaccine strategy.

GVHD Is Sustained By T Cell Maintenance within Target Tissues: Insights from Clonal Tracking and Parabiosis

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloSCT). In GVHD, donor T cells recognize recipient tissues as non-self and mount a broad attack that results in multi-organ damage. While there has been some success in diminishing the incidence of GVHD, less progress has been made in treating established or steroid-refractory disease without severe global immunosuppression. This is in part due to a lack of understanding of the underlining mechanisms that sustain GVHD despite chronic T cell antigen exposure.To address this, we developed a mouse GVHD model that allows us to track the progeny of single alloreactive T cell clones. We used this model to test hypotheses on GVHD maintenance: 1) GVHD is driven by the continuous output and trafficking of alloreactive T cells from secondary lymphoid tissues (SLT) into GVHD target organs; and 2) once tissues are seeded with alloreactive T cells from SLT, GVHD is maintained locally within affected tissues. We reasoned that if GVHD is maintained by the continuous SLT output of T cells, the progeny of single alloreactive clones in a target tissue would come into equilibrium with those in SLT and other target tissues. Alternatively, if there is a degree of local tissue GVHD maintenance, our model predicts that clonal progeny should be unequally distributed and not in equilibrium with SLT.We first tested these possibilities using a GVHD model wherein BALB/c RAG2-/- TCR transgenic (Tg) CD4 T cells (TS1) that recognize the S1 peptide derived from influenza hemagglutinin (HA) induce GVHD in BALB/c RAG2-/- mice that ubiquitously express HA (HA104 mice). We generated TS1 TCR Tg mice on 9 congenic backgrounds based on the expression of CD45.1/2, Thy1.1/2 and GFP. We transferred 500 naïve TS1 cells of 1 clonotype (to induce GVHD) along with single naïve TS1 cells from the remaining 8 clonotypes and BALB/c RAG2-/- bone marrow (BM) into lethally irradiated HA104 mice. We recovered a total of 432 single-cell derived TS1 clones (72% of input clones) from tissues of 79 mice, analyzed 7-35 days after transfer. We enumerated the TS1 clonal composition of each tissue (expressed as the % of all TS1 of that tissue) and found disparate clonal distribution across tissues within individual mice. For example, in a representative mouse analyzed at day 33 (Figure 1), the fractions of a single TS1 clone were relatively high in the colon (1.4%) and small intestine intraepithelial lymphocyte (IEL) (4.6%) when compared to the spleen (0.07%), BM (0.02%) and liver (0.03%). These data support that TS1 clones are not equally distributed among tissues and are not in equilibrium with SLT, suggesting that GVHD is at least in part maintained locally. We also analyzed TS1 clonal frequency distribution in a second model. BALB/c RAG2-/- BM and polyclonal T cells were transplanted into F1 (BALB/c HA104xB10.D2) recipients along with 8 single distinct TS1 cells. In this system, GVHD is induced by polyclonal BALB/c cells and TS1 cells are trackers of reactivity to HA. Preliminary experiments also indicate unequal distributions of TS1 clones across tissues in individual mice.In a second approach to test whether GVHD is maintained locally, irradiated HA104 mice were reconstituted with either 500 Thy1.1 or 500 Thy1.2 TS1 cells and CD45.1 or CD45.2 BALB/c RAG2-/- BM. One partner also received congenic TS1 single cells. We performed parabiosis of mice from one group to the other 21-28 days later. We analyzed 4 pairs 4 weeks post-joining, looking first at the blood to establish a baseline for TS1 crossover from the parabiotic partner. In blood, 18.9% ±1.9 of all TS1 were derived from the partner. Importantly, relative to equilibration in blood, there were far fewer partner-derived TS1 cells in all other tissues (Figure 2). Only a few single cell-derived TS1 clones were detected in the partner mouse at very low frequencies, even when they were dominant in tissues of the corresponding partner. Together, these data indicate that once GVHD is established, local maintenance dominates over new TS1 entry. Consistent with this, TS1 cells incorporate BrdU in vivo even at late time points. We are combining proliferation and clonality data at multiple timepoints to develop a mathematical model of GVHD establishment and maintenance. We are also extending our observations in a polyclonal GVHD model wherein the progeny from single alloreactive CD8 cells can be enumerated. DisclosuresShlomchik:NapaJen: Consultancy.

Donor T Cells Require Notch Signals but Not Alloantigen Presentation from Specialized Secondary Lymphoid Organ Fibroblasts to Drive Graft-Versus-Host Disease

Notch signals drive pathogenic T cell alloreactivity in mouse models of allogeneic bone marrow transplantation (allo-BMT) leading to lethal graft-versus-host disease (GVHD). In a mouse model of MHC-mismatched allo-BMT with high intensity whole body conditioning (11 Gy), residual recipient or donor hematopoietic cells were dispensable sources of Notch signals to drive T cell alloreactivity. Instead, abrogating the expression of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands in Ccl19-Cre+ secondary lymphoid organ (SLO) fibroblastic stromal cells protected mice from GVHD (Chung et al., JCI 2017). In this study we sought to 1) understand the expression and regulation of Notch ligands in fibroblastic stromal cells during allo-BMT; 2) test whether fibroblastic stromal cells are still critical sources of Notch signals in models of allotransplantation with reduced or no conditioning (when other radiation-sensitive sources of Notch ligands are better preserved); and 3) assess whether SLO fibroblastic stromal also present alloantigens to T cells during GVHD.To characterize stromal sources of Notch ligands early after allo-BMT, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele in Dll4-mCherry BAC reporter mice. We observed combined Ccl19-Cre activity and Dll4-mCherry reporter expression in CD157hi fibroblastic reticular cells, MAdCAM1+ marginal reticular cells, CD21+ follicular dendritic cells, and a subset of MAdCAM1+ lymphatic endothelial cells. 12 hours after high intensity conditioning and allo-BMT, Dll4 mRNA and cell surface expression increased in fibroblastic but not in hematopoietic or endothelial cellular compartments. Dll4 upregulation required transfer of alloreactive T cells and was potentiated by the intensity of myeloablative conditioning, suggesting that fibroblastic stromal cells have increased availability of Notch ligands to drive GVHD after high-intensity conditioning.To assess the functional impact of conditioning on the cellular sources of Notch ligands, we studied a parent-to-F1 allo-BMT model in which recipients are tolerant to donor-derived T cells, and the conditioning intensity can be titrated (C57BL/6 [H-2b] or BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b]). Cell-specific genetic loss of function systems combining floxed alleles for Dll1 and Dll4 with Ccl19-Cre+ and Mx-Cre+ transgenes allowed conditional deletion in SLO fibroblastic stromal cells or hematopoietic cells, respectively. Across all conditioning intensities tested, upregulation of core-2 O-glycosylation machinery in alloreactive T cells was critically regulated by Notch signals. Thus, induction of the core-2 glycoform of CD43 could be used as a sensitive surrogate readout of Notch signals received by alloreactive T cells. In models of allotransplantation with high-intensity (11 Gy), low-intensity (3 Gy), and no conditioning, fibroblastic stromal cells remained essential and hematopoietic cells were dispensable as sources of Notch signals driving both T cell core-2 O-glycosylation and lethal alloimmunity.Because previous studies indicated that non-hematopoietic cells can serve as antigen-presenting cells in CD4+ T cell mediated GVHD, we tested if fibroblastic stromal cells could present MHC class II alloantigens in addition to Notch ligands. We used Ccl19-Cre+ crossed to mice with a floxed I-A allele to delete the cell-intrinsic ability of fibroblastic stromal cells to present MHC class II alloantigens. In multiple models of high-intensity conditioned CD4+ T cell mediated GVHD (BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b] and TEa TCR Tg into BALB/c x C57BL/6 F1 [H-2d/b]), fibroblastic stromal cells were dispensable as sources of alloantigen to drive T cell alloreactivity and lethal GVHD. Together, these results indicate that across varying conditioning intensities, specialized subsets of SLO fibroblastic stromal cells critically regulate T cell alloreactivity through presentation of Notch ligands independently of antigen presentation. Selective targeting of Delta-like Notch ligand expression on SLO fibroblastic cells may prevent T cell immunopathology while preserving other beneficial antigen-dependent T cell responses after both high-intensity conditioned allo-BMT and allotransplantation with reduced intensity conditioning. DisclosuresBlazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Siebel:Genentech: Employment.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Thymic Development of Beta-Cell Specific Regulatory T Cells in Neonatal NOD Mice

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that involves the destruction of insulin producing β cells found within the pancreatic islets of Langerhans. Recent attention to neonatal thymocyte development has revealed a window during which central tolerance is established in an Autoimmune Regulator (Aire) dependent manner. However, expression of Chromogranin A (ChgA) within the thymus has not been detected; therefore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) presenting ChgA to developing thymocytes. The relative contribution of thymically and peripherally derived antigens in the generation of neonatal β cell antigen specific CD4+ regulatory T cells (Tregs) is not well understood. For example, post-translational modification (PTM) of ChgA by fusion with an insulin peptide increases epitope immunogenicity by forming neo-antigens. Pathogenic T cells that are specific for neo-antigens uniquely expressed in the pancreas may escape thymic selection. Therefore, we utilized two experimental designs to test the role of peripheral antigen exposure on neonatal development of β cell specific Tregs. Firstly, we adoptively transferred dendritic cells pulsed with ChgA peptides into BDC2.5 TCR Tg mice and WT NOD mice to study the role of migratory DCs on neonatal development of ChgA Tregs. Secondly, we targeted thymic Langerin+ dendritic cells with anti-Langerin linked to ChgA peptides. In both experimental approaches, we found a robust increase in the ratio and number of ChgA specific Tregs in the presence of the hybrid insulin/chromogranin A peptide (2.5HIP) while also observing an increase in negative selection. The expansion of thymic Tregs was observed in both adult and neonatal mice. Together our data suggests neonatal exposure to PTM peptides enhances thymic development of β cell specific Tregs and may alter T1D pathogenesis. Disclosure Y. Lin: None. T. Lee: None. M. Bettini: None. M. Bettini: None.

Thymic development of regulatory T cells by dendritic cells presenting modified peripheral beta cell antigens.

Abstract Type I Diabetes (T1D) is a T cell-mediated autoimmune disease that destroys insulin producing β cells found within the pancreatic islets of Langerhans. Insulin and Chromogranin A (ChgA) are the only currently known β cell antigens (βAg) necessary to initiate autoimmune diabetes in the non-obese diabetic (NOD) mice; however, expression of ChgA within medullary thymic epithelial cells (mTECs) has not yet been detected. Therefore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) presenting ChgA to developing thymocytes and naïve T cells. For example, post-translational modification (PTM) of ChgA by fusion with an insulin peptide increases epitope immunogenicity by forming the neo-antigen, insulin-ChgA fusion peptide (2.5HIP). Pathogenic T cells that are specific for 2.5HIP, which is uniquely expressed in the pancreas, may escape thymic selection due to immunologic ignorance. The relative contribution of thymic and peripheral derived Ag in the selection of βAg specific CD4+ T cells and regulatory T cells (Tregs) remains unclear. To test the role of peripheral Ag exposure on thymic development of βAg specific Tregs, we generated a 50:50 mixed bone marrow chimera using NOD WT (CD45.2 × CD45.1) and BDC2.5 TCR Tg (CD45.1) bone marrow to study the role of peripheral DCs (CD45.2) on developing ChgA T cells. Next, we targeted thymic Langerin+ DCs with anti-Langerin linked to ChgA peptides in BDC2.5 TCR Tg mice. In both experiments, we found robust increase in the ratio and number of ChgA specific Tregs in the presence of the 2.5HIP, in adult and neonatal mice. The results suggest thymic exposure to PTM peptides by DCs enhances β cell specific Treg development and may alter T1D pathogenesis.

An unadjuvanted model for oral immunization revealing complex regulatory control of gut IgA responses to soluble protein

Abstract We have identified a unique mouse model in which oral immunization with soluble protein, without a potent mucosal adjuvant, is highly effective at stimulating strong gut IgA responses. In fact, cholera toxin (CT), which is the gold standard for oral adjuvants, did not enhance the gut IgA response to ovalbumin (OVA) in this model. As we dissected the mechanism allowing for an unadjuvanted strong gut IgA response we found that adoptively transferred OVA-peptide-specific TCR Tg DO11.10 CD4 T cells into nude Balb/c mice were sufficient to promote the gut IgA response. However, only CD4 T cells with a rearranged TCR a-chain, reactive to unknown, but possibly microbial, antigens in the gut, were effective, because SCID DO11.10 CD4 T cells failed to support a gut IgA response. Co-transfer of wild-type CD4 T cells with DO11.10 CD4 T cells into nude Balb/c hosts completely obliterated the response, but not if the CD4 T cells were isolated from IL-10−/− mice. Hence, the gut IgA response was under strict regulatory CD4 T cell control. By contrast, if CT adjuvant was admixed with OVA we completely rescued the gut IgA response following oral immunization. Thus, oral CT adjuvant primarily exerted a modulating effect on the regulatory CD4 T cell control in Peyer’s patches (PP), impacting on the balance between follicular helper and regulatory T cells. While both Th17 and CD25+ Tregs, have been claimed sufficient for supporting gut IgA responses, our model clearly suggests that the regulatory environment in PP is more complex, as will be discussed.

Potentiation of T Cell Stimulatory Activity by Chemical Fixation of a Weak Peptide-MHC Complex.

The stability of peptide-MHC complex (pMHC) is an important factor to shape the fate of peptide-specific T cell immune response, but how it influences on T cell activation process is poorly understood. To better understand that, we investigated various T cell activation events driven by Ld MHCI loaded with graded concentrations of P2Ca and QL9 peptides, respectively, with 2C TCR Tg T cells; the binding strength of P2Ca for Ld is measurably weaker than that of QL9, but either peptides in the context of Ld interact with 2C TCR with a similar strength. When their concentrations required for early T cell activation events, which occur within several minutes to an hour, were concerned, EC50s of QL9 were about 100 folds lower than those of P2Ca, which was expected from their association constants for Ld. When EC50s for late activation events, which takes over several hours to occur, were concerned, the differences grew even larger (> 300 folds), suggesting that, due to weak binding, Ld/P2Ca dissociate from each other more easily to lose its antigenicity in a short time. Accordingly, fixation of Ld/P2Ca with paraformaldehyde resulted in a significant improvement in its immunogenicity. These results imply that binding strength of a peptide for a MHC is a critical factor to determine the duration of pMHC-mediated T cell activation and thus the attainment of productive T cell activation. It is also suggested that paraformaldehyde fixation should be an effective tool to ameliorate the immunogenicity of pMHC with a poor stability.

Next Generation Veto Cells for Non-Myeloablative Haploidentical HSCT: Combining Anti-Viral and Graft Facilitating Activity

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm).When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance.Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2).The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3).In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection.*E.B.L and N.O.G contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBachar Lustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Lack of TNFR2 expression by CD4+ T cells limits IL-2 production and promotes Th17 differentiation (BA11P.138)

Abstract Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.

Commensal flagellated A4 bacteria inhibit intestinal Th2 responses through induction of innate cell TGFβ production (MUC9P.742)

Abstract Interaction between host and microbiota maintains intestinal immune homeostasis. While commensal bacteria promotes Th1, Th17, and Tregs cells in lamina propria (LP) in the steady condition, it suppresses mucosal Th2 cells. Some specific commensal organisms have defined effects on mucosal immune function, e.g., SFB preferably promotes intestinal Th17 while certain Clostridium spp induce Tregs. However, it is still unclear whether there are specific commensal organisms down-regulating Th2 responses, and the mechanism involved. In this report, we showed that there were more LP Th2 cells in germ-free mice compared to the mice under SPF conditions. A4 bacteria, a commensal isolated from mouse cecum and produces immunodominant microbiota antigen, CBir1 flagellin, presented in intestinal lumen. In CBir1 TCR Tg mice, Th1, Th17 and Treg but not Th2 cells were present in the intestinal LP. When stimulated with CBir1 flagellin, naïve CBir1 Tg T cells differentiated into Th1, Th17 and Tregs but resisted Th2 differentiation. Aaddition of A4 bacteria, as well as pretreatment of APC with A4 bacteria, inhibited Th2 differentiation of OTII T cells stimulated with OVA and of wild-type T cells stimulated with anti-CD3 under Th2 polarizing conditions. A4 bacteria stimulated DC production of TGFβ, blockade of TGFβ abrogated A4 inhibition of Th2 cell development. Collectively, our data reveal that A4 bacteria inhibit intestinal Th2 responses through induction of innate cell production of TGFβ.

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells.

BackgroundWe previously reported that the peripheral regulatory T cells (pTregs) generated ‘on-demand’ in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo.MethodsTransgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (βgal) as retinal neo-self antigen and βgal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination.ResultsLocal depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in βgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal βgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local βgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after βgal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation.ConclusionsOur results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.

Evidence implicating the commensal microbiota in shaping anti-tumor immunity in melanoma

T cell infiltration of solid tumors is found in a subset of cancer patients and is associated with favorable patient outcomes. This demonstrates the capacity of the immune system to mount protective anti-tumor responses in some cancer patients, yet the underlying mechanisms mediating the presence or absence of a T cell infiltrate are not well understood. Individuals exhibit remarkable diversity in their commensal microbiota, which in turn influences systemic immune responses, yet the role of microbiota in shaping spontaneous immunity against tumors is not known. Here we sought to examine the effect of microbial composition on the immune response to melanoma by comparing subcutaneous B16 melanoma growth in genetically identical C57BL/6 mice derived from two different facilities, Jackson laboratories and Taconic farms, which have been shown to differ in their commensal microbes. We found that B16.SIY tumors exhibited an increased growth rate in mice derived from Taconic (p < 0.02), which was associated with reduced induction of tumor-specific IFN-γ-producing CD8+ T cells (p < 0.009). A markedly increased number of SIY antigen-specific CD8+ T cells was found to accumulate within the tumor microenvironment in Jackson versus Taconic mice (p < 0.03). Strikingly, the differences in tumor growth, endogenous T cell priming, and SIY antigen-specific T cell infiltration were ablated in animals cohoused for at least two weeks prior to injection of tumor cells, consistent with a microbiota-derived effect. We thus asked whether transfer of Jackson fecal material into Taconic mice by oral gavage prior to tumor inoculation was sufficient to confer protective anti-tumor responses. Indeed, Taconic mice that received Jackson feces exhibit dramatically slower tumor growth (p < 0.015) and increased numbers of SIY antigen-specific CD8+ T cells at the tumor site, compared to Taconic mice that received Taconic feces. To pursue mechanisms that might explain these differences, we transferred CFSE-labeled SIY-specific 2C TCR Tg T cells into tumor-bearing Jackson and Taconic mice and tested their proliferation and acquisition of IFN-γ production ex vivo. 2C T cells responding to tumors in Taconic mice produced significantly less IFN-γ than 2C T cells responding to tumors in Jackson mice, likely pointing to a difference at the level of antigen-presenting cells. No difference in Th17 induction was observed. Our data provide evidence implicating the microbiota in shaping anti-tumor immunity in a pre-clinical model of melanoma. Gaining further understanding of the mechanisms underlying this effect may provide the foundation for rational manipulation of commensal microbes as a cancer therapeutic.

CD8+ TCR transgenic strains expressing public versus private TCR targeting the respiratory syncytial virus K(d)M2(82-90) epitope demonstrate similar functional profiles.

Our previous work has characterized the functional and clonotypic features of two respiratory syncytial virus (RSV) epitope-specific T cell responses in mice. Following single-cell sequencing, we selected T cell receptor sequences to represent both a public and a private clone specific for the dominant KdM282–90 epitope for the generation of T cell receptor transgenic (TCR Tg) mice. We evaluated cells from these TCR Tg strains for three major functions of CD8+ T cells: proliferation, cytokine production and cytolytic activity. In vitro comparisons of the functional characteristics of T cells from the newly-generated mice demonstrated many similarities in their responsiveness to cognate antigen stimulation. Cells from both TRBV13-1 (private) and TRBV13-2 (public) TCR Tg mice had similar affinity, and proliferated similarly in vitro in response to cognate antigen stimulation. When transferred to BALB/c mice, cells from both strains demonstrated extensive proliferation in mediastinal lymph nodes following RSV infection, with TRBV13-2 demonstrating better in vivo proliferation. Both strains similarly expressed cytokines and chemokines following stimulation, and had similar Granzyme B and perforin expression, however cells expressing TRBV13-1 demonstrated better cytolytic activity than TRBV13-2 cells. These new, well-characterized mouse strains provide new opportunities to study molecular mechanisms that control the phenotype and function of CD8+ T cell responses.