TCR Transgenic Mice Research Articles

The TCR assigns naive T cells to a preferred lymph node.

Naive T cells recirculate between the spleen and lymph nodes where they mount immune responses when meeting dendritic cells presenting foreign antigen. As this may happen anywhere, naive T cells ought to visit all lymph nodes. Here, deep sequencing almost-complete TCR repertoires led to a comparison of different lymph nodes within and between individual mice. We find strong evidence for a deterministic CD4/CD8 lineage choice and a consistent spatial structure. Specifically, some T cells show a preference for one or multiple lymph nodes, suggesting that their TCR interacts with locally presented (self-)peptides. These findings are mirrored in TCR-transgenic mice showing localized CD69 expression, retention, and cell division. Thus, naive T cells intermittently sense antigenically dissimilar niches, which is expected to affect their homeostatic competition.

Regulation of memory CD4+ T cell generation by intrinsic and extrinsic IL-27 signaling during malaria infection.

The generation and maintenance of memory T cells are regulated by various factors, including cytokines. Previous studies have shown that IL-27 is produced during the early acute phase of Plasmodium chabaudi chabaudi AS (Pcc) infection and inhibits the development of Th1-type memory CD4+ T cells. However, whether IL-27 acts directly on its receptor on Plasmodium-specific CD4+ T cells or indirectly via its receptor on other immune cells remains unclear. We aimed to determine the role of IL-27 receptor signaling in different immune cell types in regulating the generation and phenotype of memory CD4+ T cells during Plasmodium infection. We utilized Plasmodium-specific TCR transgenic mice, PbT-II, and Il27rα-/- mice to assess the direct and indirect effects of IL-27 signaling on memory CD4+ T-cell generation. Mice were transferred with PbT-II or Il27rα-/- PbT-II cells and infected with Pcc. Conditional knockout mice lacking the IL-27 receptor in T cells or dendritic cells were employed to discern the specific immune cell types involved in IL-27 receptor signaling. High levels of memory in PbT-II cells with Th1-shift occurred only when both PbT-II and host cells lacked the IL-27 receptor, suggesting the predominant inhibitory role of IL-27 signaling in both cell types. Furthermore, IL-27 receptor signaling in T cells limited the number of memory CD4+ T cells, while signaling in both T and dendritic cells contributed to the Th1 dominance of memory CD4+ T cells. These findings underscore the complex cytokine signaling network regulating memory CD4+ T cells during Plasmodium infection.

Disruption of post-thymic tolerance in skin-reactive TCR transgenic mice through the interaction of lymphopenia and intestinal microbiota.

Autoimmune diseases often arise from conditions where the immune system is compromised. While lymphopenia-induced proliferation (LIP) is crucial for immune system development and maturation, it is also caused by environmental insults, such as infection, and becomes a risk factor for autoimmunity in adults. We used Dsg3H1 TCR transgenic mice, whose T cells are designed to recognize desmogrein-3, a skin antigen, to explore the impact of lymphopenia on post-thymic tolerance. Dsg3H1 mice are known to delete the most highly autoreactive T cells in the thymus, and develop only subtle immune-mediated pathology in the steady state. However, we found that transient lymphopenia induced by total body irradiation (TBI) or cyclophosphamide (CY) results in massive dermatitis in Dsg3H1 mice. The symptoms included expansion and development of self-reactive T cells, their differentiation into CD44high IL-17-producing helper T cells, and severe neutrophilic inflammation. Repopulation of FOXP3+ T regulatory cells after lymphopenia normally occurred, suggesting escape of skin-reactive conventional T cells from control by regulatory T cells. Furthermore, we found that a depletion of the intestinal microbiota by antibiotics prevents CY-induced dermatitis, indicating roles of the commensal intestinal microbiota in LIP and Th17 development in vivo. The current data suggested that post-thymic tolerance of Dsg3H1 mice is established on a fragile balance in the lymphoreplete immune environment and broken by the interplay between lymphopenia and intestinal microbiota. The dynamic phenotypes observed in Dsg3H1 mice prompt a re-evaluation of opportunistic lymphopenia together with the microbiota as pivotal environmental factors, impacting individuals with genetic predispositions for autoimmune diseases.

Abstract 5350: Decr2 regulates tumor cell ferroptosis and immunotherapy efficacy

Abstract Checkpoint blockade therapies have transformed the landscape of cancer care. Durable clinical responses have been observed in a subset of patients. However, many patients do not respond, and understanding the mechanisms that determine tumor resistant to checkpoint blockade drugs could potentially benefit more patients. Ferroptosis is a relatively newly described form of regulated cell death distinct from apoptosis and necroptosis. Recently, T cell-promoted tumor ferroptosis was shown to be an anti-tumor mechanism and targeting this pathway could be a potential therapeutic approach. To identify genes critical to immunotherapy resistance, B16.SIY cells were transduced with a genome-scale gRNA lentivirus to generate loss of function mutants. In vitro-primed CD8+ T cells isolated from 2C/Rag2-/- TCR transgenic mice specific for the SIY antigen were co-cultured with transduced B16.SIY tumor cells. Resistant mutants were identified by deep sequencing of survival clones. The gene encoding Decr2, a peroxisomal 2,4-dienoyl-CoA reductase, was identified. Decr2 mutants were relatively resistant to CD8+ T cell killing in vitro. Consistent with this resistance to CD8+ T cell killing, Decr2 knock-down tumors showed minimal response to anti-PD-L1 therapy in vivo. Tumor microenvironment analysis in Decr2 knockdown B16.SIY tumors showed failed antigen specific CD8+ T cells’ expansion and less IFN-γ production after anti-PD-L1 treatment. Knock-down of Decr2 resulted in diminished ferroptosis, triggered either by pharmacologic inducers or antigen-specific CD8+ T cells. Mechanistic studies revealed that Decr2 knockdown resulted in diminished induction of polyunsaturated ether phospholipids (PUFA-ePLs), which is due to reduced polyunsaturated ether phospholipids synthesis rate . Analysis of melanoma tumors from human patients revealed that up-regulation of Decr2 was associated with anti-PD-1 efficacy. Our results proved Decr2 knock down suppressed tumor ferroptosis and confer tumors resistance to anti-PD-L1 therapy by reducing the polyunsaturated ether phospholipids (PUFA-ePLs) synthesis. Citation Format: Shuyin Li, Jason Shapiro, Hardik Shah, Emily F. Higgs, Lishi Xie, Yuanyuan Zha, Jonathan Trujillo, Alexandra Cabanov, Tyler A. Jones, Blake Flood, Ken Hatogai, Justin Kline, Thomas F. Gajewski. Decr2 regulates tumor cell ferroptosis and immunotherapy efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5350.

Antibody-Suppressor CXCR5+CD8+ T Cells Are More Potent Regulators of Humoral Alloimmunity after Kidney Transplant in Mice Compared to CD4+ Regulatory T Cells.

Adoptive cell therapy (ACT), especially with CD4+ regulatory T cells (CD4+ Tregs), is an emerging therapeutic strategy to minimize immunosuppression and promote long-term allograft acceptance, although much research remains to realize its potential. In this study, we investigated the potency of novel Ab-suppressor CXCR5+CD8+ T cells (CD8+ TAb-supp) in comparison with conventional CD25highFoxp3+CD4+ Tregs for suppression of humoral alloimmunity in a murine kidney transplant (KTx) model of Ab-mediated rejection (AMR). We examined quantity of peripheral blood, splenic and graft-infiltrating CD8+ TAb-supp, and CD4+ Tregs in KTx recipients and found that high alloantibody-producing CCR5 knockout KTx recipients have significantly fewer post-transplant peripheral blood and splenic CD8+ TAb-supp, as well as fewer splenic and graft-infiltrating CD4+ Tregs compared with wild-type KTx recipients. ACT with alloprimed CXCR5+CD8+ T cells reduced alloantibody titer, splenic alloprimed germinal center (GC) B cell quantity, and improved AMR histology in CCR5 knockout KTx recipients. ACT with alloprimed CD4+ Treg cells improved AMR histology without significantly inhibiting alloantibody production or the quantity of splenic alloprimed GC B cells. Studies with TCR transgenic mice confirmed Ag specificity of CD8+ TAb-supp-mediated effector function. In wild-type recipients, CD8 depletion significantly increased alloantibody titer, GC B cells, and severity of AMR pathology compared with isotype-treated controls. Anti-CD25 mAb treatment also resulted in increased but less pronounced effect on alloantibody titer, quantity of GC B cells, and AMR pathology than CD8 depletion. To our knowledge, this is the first report that CD8+ TAb-supp cells are more potent regulators of humoral alloimmunity than CD4+ Treg cells.

OT-I TCR Transgenic Mice to Study the Role of PTPN22 in Anti-cancer Immunity.

Phosphotyrosine phosphatase non-receptor type 22 (PTPN22) is a key regulator of immune cell activation and responses. Genetic polymorphisms of PTPN22 have been strongly linked with an increased risk of developing autoimmune diseases, while analysis of PTPN22-deficient mouse strains has determined that PTPN22 serves as a negative regulator of T cell antigen receptor signaling. As well as these key roles in maintaining immune tolerance, PTPN22 acts as an intracellular checkpoint for T cell responses to cancer, suggesting that PTPN22 might be a useful target to improve T cell immunotherapies. To assess the potential for targeting PTPN22, we have crossed Ptpn22-deficient mice to an OT-I TCR transgenic background and used adoptive T cell transfer approaches in mouse cancer models. We provide basic methods for the in vitro expansion of effector OT-I cytotoxic T lymphocytes, in vitro phenotypic analysis, and in vivo adoptive T cell transfer models to assess the role of PTPN22 in anti-cancer immunity.

Antigen recognition reinforces regulatory T cell mediated Leishmania major persistence

Cutaneous Leishmania major infection elicits a rapid T cell response that is insufficient to clear residually infected cells, possibly due to the accumulation of regulatory T cells in healed skin. Here, we used Leishmania-specific TCR transgenic mice as a sensitive tool to characterize parasite-specific effector and immunosuppressive responses in vivo using two-photon microscopy. We show that Leishmania-specific Tregs displayed higher suppressive activity compared to polyclonal Tregs, that was mediated through IL-10 and not through disrupting cell-cell contacts or antigen presentation. In vivo expansion of endogenous Leishmania-specific Tregs resulted in disease reactivation that was also IL-10 dependent. Interestingly, lack of Treg expansion that recognized the immunodominant Leishmania peptide PEPCK was sufficient to restore robust effector Th1 responses and resulted in parasite control exclusively in male hosts. Our data suggest a stochastic model of Leishmania major persistence in skin, where cellular factors that control parasite numbers are counterbalanced by Leishmania-specific Tregs that facilitate parasite persistence.

Pparβ/δ Has an Indispensable Role in T Cell Function and Anti-Tumor Responses

The peroxisome proliferator-activated receptors (PPARs) are a group of transcriptional factor of the nuclear hormone receptor superfamily, which promote ligand-dependent transcription of target genes that regulate substrate delivery, oxidative phosphorylation (OXPHOS), and energy balance. The PPAR superfamily consists of three subtypes, PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ), with distinct tissue allocations. In addition to their roles in energy homeostasis and carbohydrate and lipid metabolism in metabolic organs, an emerging role of PPARs involves the differentiation and fate of immune cells. PPARβ/δ is ubiquitously expressed and activated by fatty acid ligands. While the effects of PPARα and PPARγ on M2 macrophage polarization and Th1 inhibition are well established, only few studies have investigated the effects of PPARβ/δ on the immune responses. It has been proposed that PPARβ/δ might inhibit T cell differentiation towards T helper 1 cells (Th1) and T helper 17 cells (Th17), and synthetic ligands of PPARβ/δ might alleviate inflammation in autoimmune diseases by downregulating pro-inflammatory mediators. Conversely, other studies have observed that PPARβ/δ agonists might enhance anti-tumor T cell responses by reprogramming immunometabolism toward fatty acid oxidation and increasing T cell longevity. In the present study, we sought to determine the role of PPARβ/δ in T cell function by combining genetic and pharmacologic approaches. We generated mice with conditional targeting of Pparβ/δ gene and crossed them with CD4Cre ( Pparβ/δ f/fCD4Cre) to delete Pparβ/δ in T cells. In vitro stimulation of T cells from Pparβ/δ f/fCD4Cre mice with anti-CD3/TCR-and-CD28 mediated signaling resulted in diminished expansion of CD4 + and CD8 + T cells. Consistent with these findings, treatment of antigen-specific CD4 + and CD8 + T cells from OTII and OTI TCR-transgenic mice with the Pparβ/δ-specific antagonist GSK3787 diminished responses to cognate antigen as determined by DNA synthesis, expression of activation markers and differentiation to T effector cells. Furthermore, under these conditions, GSK 3787 induced a dose-dependent decrease in the production of IL-2, TNF-α, and IFN-γ by OTII antigen-specific CD4 + T cells. To investigate the T cell-specific role of Pparβ/δ in anti-tumor responses, we used Pparβ/δ f/fCD4Cre and control Pparβ/δ f/f mice and implanted them with MC38 colon adenocarcinoma tumor cells. We found that Pparβ/δ f/fCD4Cre mice had enhanced tumor growth compared to control. No quantitative differences were detected in tumor infiltrating CD4 +, CD8 + and Treg cells. However, Treg cells from tumor-bearing Pparβ/δ f/fCD4Cre mice had a diminished activation state as determined by lower expression of CD44 and PD-1. CD4 + T cells in tumors and tumor draining lymph nodes also had decreased expression of CD44 and ICOS but no differences in other activation or checkpoint markers, including GITR, TIGIT, CD69 and CD44 compared to their counterparts from tumor-bearing control mice. In contrast, CD8 + T cells from tumor-bearing Pparβ/δ f/fCD4Cre mice had increased expression of Tox and multiple checkpoint inhibitors including CTLA4, PD-1, and Lag3 and decreased expression of IFN-g compared to CD8 + cells from control tumor-bearing mice. These results reveal a previously unappreciated indispensable role of PPARβ/δ in regulating anti-tumor immune responses and protecting CD8 + tumor infiltrating T cells from developing an exhaustion immunological and functional phenotype. Our findings suggest that pharmacologic PPARβ/δ-specific agonists might prevent tumor-mediated exhaustion and support anti-tumor function of CD8 + T effector cells.

Antigen-driven T cell-macrophage interactions mediate the interface between innate and adaptive immunity in histidyl-tRNA synthetase-induced myositis.

Previous work in humans has demonstrated that both innate and adaptive immune signaling pathways contribute to the pathogenesis of idiopathic inflammatory myopathy (IIM), a systemic autoimmune disease targeting muscle as well as extra-muscular organs. To better define interactive signaling networks in IIM, we characterized the cellular phenotype and transcriptomic profiles of muscle-infiltrating cells in our established murine model of histidyl-tRNA synthetase (HRS)-induced myositis. Myositis was induced in wild type (WT) and various congenic/mutant strains of C57BL/6 mice through intramuscular immunization with recombinant HRS. Histopathological, immunohistochemical, flow cytometric, and transcriptomic assessments were used to characterize the functional relationship between muscle-infiltrating cell populations in these strains lacking different components of innate and/or adaptive immune signaling. RAG1 KO mice developed markedly reduced muscle inflammation relative to WT mice, demonstrating a key requirement for T cells in driving HRS-induced myositis. While the reduction of mononuclear cell infiltrates in CD4-Cre.MyD88fl/fl conditional knockout mice and OT-II TCR transgenic mice highlighted roles for both innate and TCR-mediated/adaptive immune signaling in T cells, diminished inflammation in Lyz2-Cre.MyD88fl/fl conditional knockout mice underscored the importance of macrophage/myeloid cell populations in supporting T cell infiltration. Single cell RNA sequencing-based clustering of muscle-infiltrating subpopulations and associated pathway analyses showed that perturbations of T cell signaling/function alter the distribution and phenotype of macrophages, fibroblasts, and other non-lymphoid cell populations contributing to HRS-induced myositis. Overall, HRS-induced myositis reflects the complex interplay between multiple cell types that collectively drive a TH1-predominant, pro-inflammatory tissue phenotype requiring antigen-mediated activation of both MyD88- and TCR-dependent T cell signaling pathways.

Repetitive Antigen Responses of LDL-Reactive CD4+ T Cells Induce Tr1 Cell-Mediated Immune Tolerance.

Inflammation triggered by the deposition of LDL (low-density lipoprotein) in the arterial wall leads to the development of atherosclerosis. Regulatory T (Treg) cells inhibit vascular inflammation through the induction of immune tolerance toward LDL-related antigens. However, tolerogenic mechanisms that promote the generation of LDL-specific Treg cells in vivo remain unclear. We identified LDL-specific T cells by activation-induced marker expression and analyzed expression profiles and suppressive functions of TCR (T-cell antigen receptor)-transgenic T cells upon repetitive transfer into antigen-transgenic mice via flow cytometry. We investigated the naturally occurring Treg-cell response against human LDL in standard chow diet-fed mice that are transgenic for human ApoB100 (apolipoprotein B100). We found that IL (interleukin)-10 expression in LDL-specific T cells from spleen increases with age, albeit LDL-specific populations do not enlarge in older mice. To investigate the generation of IL-10-producing LDL-specific T cells, we transferred naive CD4+ T cells recognizing human ApoB100 from TCR-transgenic mice into human ApoB100-transgenic mice. Adoptive transfer of human ApoB100-specific T cells induced immune tolerance in recipient mice and effectively inhibited activation of subsequently transferred naive T cells of the same specificity in vivo. Moreover, repetitive transfers increased the population of Treg type 1 cells that suppress ApoB100-specific responses via IL-10. In a translational approach, LDL-specific Treg type 1 cells from blood of healthy donors suppressed the activation of monocytic THP-1 cells in an IL-10-dependent manner. We show that repetitive transfer of naive ApoB100-specific T cells and recurrent LDL-specific T-cell stimulation induces Treg type 1 cell-mediated immune tolerance against LDL in vivo. Our results provide insight into the generation of autoantigen-specific anti-inflammatory T cells under tolerogenic conditions.

MHV-68 induces central nervous system autoimmunity in an MBP-specific TCR transgenic mouse model of multiple sclerosis

Abstract Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. Latent infection with Epstein-Barr virus (EBV) has been identified as a necessary yet insufficient factor in the development of the disease. Several groups have demonstrated that the murine EBV analogue, MHV-68, exacerbates disease severity in experimental autoimmune encephalomyelitis (EAE). However, the induction of EAE requires immunization and does not fully replicate the clinical features of MS. We sought to develop a virus-inducible model that accurately represents MS etiology and does not rely on classical EAE immunization. Using a transgenic mouse line with a myelin basic protein-specific T cell receptor, we found that MHV-68 induces demyelinating disease in 72% of infected mice. These mice exhibit clinical features such as optic neuritis, incontinence, and head tilt, which are commonly observed in MS patients but not in mice with EAE. This phenomenon was specific to MHV-68 and was not seen with other viral pathogens including PR8 influenza. Our work demonstrates that latent gammaherpesvirus infection can drive CNS autoimmunity in genetically predisposed mice without prior EAE induction, thus providing a more natural murine model of MS. National Institute of Health R01AI140741-04

Regulatory T cells aid Leishmania majorimmune evasion by suppressing effector responses against the parasite in an antigen-specific manner

Abstract Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete and leaves a pool of chronically infected cells. Understanding of the persistence is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells at the site of infection has been proposed to prevent parasite clearance in vivo. Here, we used a novel TCR transgenic mouse model, where CD4 +T cells recognize an immunodominant peptide derived from Leishmania-glycosomal phosphoenolpyruvate carboxykinase (PEPCK), to visualize the dynamics of anti-L. major CD4 +T cell responses and characterize mechanisms which restrain their effector function. We show that PEPCK-specific Tregs are able to suppress nearly 80% of IFNg produced by PEPCK-specific Th1 cells in vitro though the production of IL-10, while decreasing IL-12, TNF, and IL-2. Intravital microscopy studies characterizing PEPCK-specific CD4 +T cell migration dynamics in live mice show a significant recruitment of adoptively transferred cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition, yet augmented at the chronic stage, indicating a fundamentally altered environment. Injection of heat-killed L. major into mice which have already healed a L. major infection, distal to the original lesion, induced a rapid expansion of PEPCK-specific Tregs at the original site of infection, which suppressed endogenous anti-Leishmania response through IL-10 and led to parasite expansion and lesion reactivation. Our results show that antigen recognition by Tregs plays an important role in suppressing effector responses against L. major and thus maintaining concomitant immunity. Research funded by a grant from the GSK-CIHR Partnered Chair Program

Exogenous catalase treatment enhances CD8+ T cell functionality and hinders T cell exhaustion

Abstract ROS are small short-lived oxygen-containing molecules which are highly chemically reactive and involved in the regulation of biological processes like T cell activation and proliferation. The tumor microenvironment (TME) is a metabolically hostile environment with a deficiency in nutrients like glucose and glutamine, accumulation of tumor-driven metabolic waste, and extreme hypoxia. Hypoxia results in impaired mitochondrial function and generation of excess levels of ROS, which have been known to promote T cell exhaustion. Here, we investigated how exogenous treatment of antioxidant catalase altered CD8+ T cell effector responses and T cell exhaustion using OT-I TCR transgenic mice. During CD8+ T cell effector differentiation in vitro by stimulation with cognate antigen followed by culture with IL-2, addition of catalase significantly enhanced T cell activation and effector differentiation. Catalase-treated effector cells had higher expression of critical activation markers including CD69 and CD25, increased expression of the stemness marker TCF-1, and enhanced secretion of effector cytokines IFNγ and TNFα. During induction of T cell exhaustion in vitro by co-culture with B16 melanoma expressing ovalbumin (B16-Ova), catalase-treated OT-I T cells had significantly increased cytotoxic activity, enhanced expression of CD25 and lower co-expression of PD-1 and TIM-3 relative to non-treated OT-I T cells. Our findings uncover a new role for catalase in altering the fate of CD8+ effector T cells and potentially a new therapeutic tool for managing T cell exhaustion. NIH (R01 CA212605)

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity invivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

Lack of Herpes Virus Entry Mediator Signals in Thymocytes Impairs Conventional CD8 T Cell Selection and Promotes Memory-like CD8 T Cell Development.

Thymocytes having diverse Ag specificities are selected in response to self-MHC-peptide expressed in thymic epithelial cells, which contributes to the formation of a T cell repertoire. However, it is not well understood whether additional signals from epithelial cells are required to drive positive selection. In this study, we found that one of the TNFR superfamily members, herpes virus entry mediator (HVEM), when expressed on thymocytes provides signals for positive selection. HVEM deficiency in double-positive (DP) thymocytes impaired positive selection of CD8 thymocytes. HVEM-deficient thymocytes in OT-1 TCR transgenic mice exhibited significant defects in positive selection and impaired CD69 upregulation of selected thymocytes. HVEM ligands (lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, and B and T lymphocyte attenuator) were expressed in cortical thymic epithelial cells. Weak TCR ligation combined with HVEM signals enhanced ERK activation in DP thymocytes developed invitro. Insufficient signals for positive selection in HVEM-deficient DP thymocytes led to the development of innate memory-like CD8 T cells expressing high levels of CD122, along with the increased development of PLZF+ NKT cells. These results suggest that thymocytes receive activation signals through HVEM during positive selection. Thus, our findings provide evidence that the threshold of thymocyte positive selection is set by signals from TCR in association with HVEM.

Heterozygous OT-I mice reveal that antigen-specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly.

Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I+/+ and OT-I+/- mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I+/- mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I+/- mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I+/- model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzyme-mediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation.

Low TCR Binding Strength Results in Increased Progenitor-like CD8+ Tumor-Infiltrating Lymphocytes.

T-cell receptor (TCR) binding strength to peptide-MHC antigen complex influences numerous T-cell functions. However, the vast diversity of a polyclonal T-cell repertoire for even a single antigen greatly increases the complexity of studying the impact of TCR affinity on T-cell function. Here, we determined how TCR binding strength affected the protein and transcriptional profile of an endogenous, polyclonal T-cell response to a known tumor-associated antigen (TAA) within the tumor microenvironment (TME). We confirmed that the staining intensity by flow cytometry and the counts by sequencing from MHC-tetramer labeling were reliable surrogates for the TCR-peptide-MHC steady-state binding affinity. We further demonstrated by single-cell RNA sequencing that tumor-infiltrating lymphocytes (TIL) with high and low binding affinity for a TAA can differentiate into cells with many antigen-specific transcriptional profiles within an established TME. However, more progenitor-like phenotypes were significantly biased towards lower affinity T cells, and proliferating phenotypes showed significant bias towards high-affinity TILs. In addition, we found that higher affinity T cells advanced more rapidly to terminal phases of T-cell exhaustion and exhibited better tumor control. We confirmed the polyclonal TIL results using a TCR transgenic mouse possessing a single low-affinity TCR targeting the same TAA. These T cells maintained a progenitor-exhausted phenotype and exhibited impaired tumor control. We propose that high-affinity TCR interactions drive T-cell fate decisions more rapidly than low-affinity interactions and that these cells differentiate faster. These findings illustrate divergent forms of T-cell dysfunction based on TCR affinity which may impact TIL therapies and antitumor responses.

Transfer of mesenchymal stem cell mitochondria to CD4+ T cells contributes to repress Th1 differentiation by downregulating T-bet expression

BackgroundMesenchymal stem/stromal cells (MSCs) are multipotent cells with strong tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses, and in particular T cell responses, they show therapeutic potential for the treatment of autoimmune diseases, organ rejection and graft versus host disease. MSCs have the remarkable ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 responses is unknown.Methods and resultsIn this report we have utilized CD4+ T cells from HNT TCR transgenic mice that develop Th1-like responses upon antigenic stimulation in vitro and in vivo. Allogeneic bone marrow-derived MSCs reduced the diabetogenic potential of HNT CD4+ T cells in vivo in a transgenic mouse model of disease. In co-culture experiments, we have shown that MSCs were able to reduce HNT CD4+ T cell expansion, expression of key effector markers and production of the effector cytokine IFNγ after activation. This was associated with the ability of CD4+ T cells to acquire mitochondria from MSCs as evidenced by FACS and confocal microscopy. Remarkably, transfer of isolated MSC mitochondria to CD4+ T cells resulted in decreased T cell proliferation and IFNγ production. These effects were additive with those of prostaglandin E2 secreted by MSCs. Finally, we demonstrated that both co-culture with MSCs and transfer of isolated MSC mitochondria prevent the upregulation of T-bet, the master Th1 transcription factor, on activated CD4+ T cells.ConclusionThe present study demonstrates that transfer of MSC mitochondria to activated CD4+ T cells results in the suppression of Th1 responses in part by downregulating T-bet expression. Furthermore, our studies suggest that MSC mitochondrial transfer might represent a general mechanism of MSC-dependent immunosuppression.

The TCR Cα Domain Regulates Responses to Self-pMHC Class II.

T cells play a central role in adaptive immunity by recognizing peptide Ags presented by MHC molecules (pMHC) via their clonotypic TCRs. αβTCRs are heterodimers, consisting of TCRα and TCRβ subunits that are composed of variable (Vα, Vβ) and constant (Cα, Cβ) domains. Whereas the Vα, Vβ, and Cβ domains adopt typical Ig folds in the extracellular space, the Cα domain lacks a top β sheet and instead has two loosely associated top strands (C- and F-strands) on its surface. Previous results suggest that this unique Ig-like fold mediates homotypic TCR interactions and influences signaling in vitro. To better understand why evolution has selected this unique structure, we asked, what is the fitness cost for development and function of mouse CD4+ T cells bearing a mutation in the Cα C-strand? In both TCR retrogenic and transgenic mice we observed increased single-positive thymocytes bearing mutant TCRs compared with those expressing wild-type TCRs. Furthermore, our analysis of mutant TCR transgenic mice revealed an increase in naive CD4+ T cells experiencing strong tonic TCR signals, increased homeostatic survival, and increased recruitment of responders to cognate pMHC class II upon immunization compared with the wild-type. The mutation did not, however, overtly impact CD4+ T cell proliferation or differentiation after immunization. We interpret these data as evidence that the unique Cα domain has evolved to fine-tune TCR signaling, particularly in response to weak interactions with self-pMHC class II.

B Cells Drive MHC Class I-Restricted CD4 T Cells to Induce Spontaneous Central Nervous System Autoimmunity.

Multiple sclerosis (MS) is an inflammatory, demyelinating CNS disease believed to be mediated by CD4 T cells specific for CNS self-antigens. CD8 T cells are also implicated in MS but their function is not well understood. MS lesions are heterogeneous and may reflect variation in the contribution of different types of lymphocytes. Understanding how lymphocytes with different effector functions contribute to MS is essential to develop effective therapies. We investigated how T cells expressing an MHC class I-restricted transgenic TCR specific for myelin basic protein (MBP) contribute to CNS autoimmunity using the mouse model of MS, experimental autoimmune encephalomyelitis. Virus infection triggered cytotoxic TCR-transgenic CD8 T cells to initiate acute experimental autoimmune encephalomyelitis in an IFN-γ- and perforin-dependent manner. Unexpectedly, spontaneous CNS autoimmunity developed in the TCR-transgenic mice that was accelerated by IFN-γ-deficiency. Spontaneous disease was associated with CD4 T cells that develop via endogenous TCR rearrangements but retain specificity for the MHC class I-restricted MBP epitope. The CD4 T cells produced TNF-α without other inflammatory cytokines and caused lesions with striking similarity to active MS lesions. Surprisingly, B cells were the predominant cell type that cross-presented MBP, and their depletion halted disease progression. This work provides a new model of spontaneous CNS autoimmunity with unique similarities to MS that is mediated by T cells with a distinct effector phenotype.