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T cells need to adapt their cellular metabolism for effector cell differentiation. This relies on alterations in mitochondrial physiology. Which signals and molecules regulate those alterations remains unclear. We recently reported, that the mitochondrial protein TCAIM inhibits activation-induced changes in mitochondrial morphology and function and thus, CD4+effector T cell formation. Using conditional TCAIM knock-in (KI) and knockout (KO) mice, we now show that it also applies to CD8+T cells and more importantly, delineate the molecular processes in mitochondria by which TCAIM controls effector cell differentiation. TCAIM KI resulted in reduced activation-induced HIF1α protein expression. Metabolomics and transcriptional data in combination with mathematical flux modeling revealed an impaired induction of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis in TCAIM KI CD8+T cells. Addition of cholesterol completely rescued HIF1α protein expression, activation and proliferation of TCAIM KI CD8+T cells. At the molecular level, TCAIM delayed activation-induced mitochondria-ER contact (MERC) formation by binding to MERC promoting proteins such as RMD3 and VDAC2. In summary, we demonstrate that TCAIM suppresses effector cell differentiation by inhibiting MERC formation, which induce HIF1α-mediated increase in cellular metabolism and cholesterol biosynthesis.

Significance T helper type (Th) 9 cells demonstrate both pro- and antiinflammatory properties, pointing to a functional heterogeneity not examined so far. Applying single cell gene expression analysis of alloreactive Th9 cells, we revealed the existence of two major subsets, CD96high and CD96low Th9 cells, with strongly opposing inflammatory and, especially, colitis-inducing potential. Mechanistically, we found that CD96 controls cytokine and colitis-inducing potential of Th9 cells, providing strong evidence for an inhibitory role of CD96 in controlling CD4+ T-cell effector functions. Thus, interfering with CD96-mediated immune inhibition would be a promising approach in preventing Th9-mediated diseases, such as ulcerative colitis, or reinforcement of Th9-mediated immune control of tumors and infections., Recent findings demonstrated proinflammatory functions of interleukin (IL)-9–producing T helper type (Th) 9 cells in the pathogenesis of intestinal bowel diseases (IBDs). However, also antiinflammatory properties have been ascribed to Th9 cells, pointing to a functional heterogeneity. To dissect the specific expression pattern and, especially, diversity of murine antigen-specific Th9 cells, we applied single cell transcription profiling. Th9 cells displayed reduced expression of typical activation markers, such as Cd40 ligand and Cd96, whereas expression of Cd25 and Cd83 was increased compared with other Th subsets. Importantly, we identified two subsets of Th9 cells differing above all in their CD96 expression. The heterogeneous CD96 expression was specific for Th9 cells and not observed for other Th subtypes, such as Th1 cells. Lower CD96 expression was also observed in human IL-9+ compared with IFN-γ+ T cells. Although Il9 was highly transcribed by all Th9 cells, IL-9 mRNA and protein expression was increased in CD96low cells. Transfer of CD96low Th9 cells into recombination activating gene 1-deficient (Rag1−/−) mice caused severe weight loss, intestinal and colonic inflammation, and destruction of allogeneic skin grafts and thus showed high inflammatory potential. This was associated with their expansion and tissue accumulation. Contrastingly, CD96high Th9 cells did not cause colitis and showed reduced expansion and migratory potential. Blockade of CD96 completely restored the expansion and inflammatory properties of CD96high Th9 cells. Collectively, our data suggest an inhibitory role for the cosignaling receptor CD96 in Th9 cells, raising new opportunities in the treatment of IL-9–associated inflammations such as IBD.

Type 1 regulatory T (Tr1) cells are induced by interleukin-27 (IL-27) and have critical roles in the control of autoimmunity and resolution of inflammation. Here, we show that the transcription factors IRF1 and BATF are induced early during treatment with IL-27 and are required for the differentiation and function of Tr1 cells in vitro and in vivo. Epigenetic and transcriptional analyses reveal that both transcription factors influence chromatin accessibility and expression of genes required for Tr1 cell function. IRF1 and BATF deficiencies uniquely alter the chromatin landscape, suggesting that these factors serve a pioneering function during Tr1 cell differentiation.

All memory T cells mount an accelerated response on antigen reencounter, but significant functional heterogeneity is present within the respective memory T-cell subsets as defined by CCR7 and CD45RA expression, thereby warranting further stratification. Here we show that several surface markers, including KLRB1, KLRG1, GPR56, and KLRF1, help define low, high, or exhausted cytokine producers within human peripheral and intrahepatic CD4+ memory T-cell populations. Highest simultaneous production of TNF and IFN-γ is observed in KLRB1+KLRG1+GPR56+ CD4 T cells. By contrast, KLRF1 expression is associated with T-cell exhaustion and reduced TNF/IFN-γ production. Lastly, TCRβ repertoire analysis and in vitro differentiation support a regulated, progressive expression for these markers during CD4+ memory T-cell differentiation. Our results thus help refine the classification of human memory T cells to provide insights on inflammatory disease progression and immunotherapy development., Despite the current human CD4 memory T cell stratification by CD45RA/CCR7, functional heterogeneities still exist within the respective subsets. Here the authors show that several surface markers, including KLRB1, KLRG1, GPR56 and KLRF1, help to further refine the subsetting of human CD4 memory T cells and provide insights for their differentiation.

Objectives: Stable long-term functioning of liver cells after transplantation in humans is still not achieved successfully. A new approach for successful engraftment of liver cells may be the transplantation of syngeneic cells into an allogeneic liver graft. We therefore developed a new rat model for combined liver and liver cell transplantation (cLCTx) under stable immunosuppression. Materials and Methods: After inducing a mitotic block, liver grafts from female donor rats (Dark Agouti) were transplanted into female recipients (Lewis). In male Lewis rats, liver cell proliferation was induced with subsequent cell isolation and transplantation into female recipients after organ transplantation. Y-chromosome detection of the transplanted male cells was performed by quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FisH) with localization of transplanted cells by immunohistochemistry. Results: Immunohistochemistry demonstrated the engraftment of transplanted cells, as confirmed by FisH, showing repopulation of the liver graft with 15.6% male cells (± 1.8 SEM) at day 90. qPCR revealed 14.15% (± 5.09 SEM) male DNA at day 90. Conclusion: Engraftment of transplanted syngeneic cells after cLCTx was achieved for up to 90 days under immunosuppression. Immunohistochemistry indicated cell proliferation, and the FisH results were partly confirmed by qPCR. This new protocol in rats appears feasible for addressing long-term functioning and eventually the induction of operational tolerance in the future.

Recent data suggest that donor-specific memory T cells (T(mem)) are an independent risk factor for rejection and poor graft function in patients and a major challenge for immunosuppression minimizing strategies. Many tolerance induction protocols successfully proven in small animal models e.g. costimulatory blockade, T cell depletion failed in patients. Consequently, there is a need for more predictive transplant models to evaluate novel promising strategies, such as adoptive transfer of regulatory T cells (Treg). We established a clinically more relevant, life-supporting rat kidney transplant model using a high responder (DA to LEW) recipients that received donor-specific CD4(+)/ 8(+) GFP(+) T(mem) before transplantation to achieve similar pre-transplant frequencies of donor-specific T(mem) as seen in many patients. T cell depletion alone induced long-term graft survival in naïve recipients but could not prevent acute rejection in T(mem)(+) rats, like in patients. Only if T cell depletion was combined with permanent CNI-treatment, the intragraft inflammation, and acute/chronic allograft rejection could be controlled long-term. Remarkably, combining 10 days CNI treatment and adoptive transfer of Tregs (day 3) but not Treg alone also induced long-term graft survival and an intragraft tolerance profile (e.g. high TOAG-1) in T(mem)(+) rats. Our model allows evaluation of novel therapies under clinically relevant conditions.

CD44 is a prominent activation marker which distinguishes memory and effector T cells from their naïve counterparts. It also plays a role in early T cell signaling events as it is bound to the lymphocyte-specific protein kinase and thereby enhances T cell receptor signalling. Here, we investigated whether IFN-γ and IL-17 producing T helper cells differ in their CD44 expression and their dependence of CD44 for differentiation. Stimulation of CD4+ T cells with allogeneic dendritic cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo- reactive IL-17 producing T helper cells were mainly CD44+++ as compared to IFN-γ+ T helper cells, which were CD44++. This effect was enhanced under polarizing conditions. T helper 17 polarization led to a shift towards the CD44+++ population, whereas T helper 1 polarization diminished this population. Furthermore, blocking CD44 decreased IL-17 secretion, while IFN-γ was barely affected. Titration experiments revealed that low T cell receptor and CD28 stimulation supported T helper 17 rather than T helper 1 development. Under these conditions CD44 could act as a co-stimulatory molecule and replace CD28. Indeed, rested CD44+++CD4+ T cells contained already more total and especially phosphorylated zeta-chain-associated protein kinase 70 as compared to CD44++ cells. Our results support the notion, that CD44 enhances T cell receptor signaling strength by delivering lymphocyte-specific protein kinase, which is required for induction of IL-17 producing T helper cells.

Primary T cell activation and effector cell differentiation is required for rejection of allogeneic grafts in naive recipients. It has become evident, that mitochondria play an important role for T cell activation. Expression of several mitochondrial proteins such as TCAIM (T cell activation inhibitor, mitochondrial) is down-regulated upon T cell receptor triggering. Here we report that TCAIM inhibited spontaneous development of memory and effector T cells. CD4(+) T cells from Tcaim knock-in (KI) mice showed reduced activation, cytokine secretion and proliferation in vitro. Tcaim KI T cells tolerated allogeneic skin grafts upon transfer into Rag-1 KO mice. CD4(+) and CD8(+) T cells from these mice did not infiltrate skin grafts and kept a naive or central memory phenotype, respectively. They were unable to acquire effector phenotype and functions. TCAIM altered T cell activation-induced mitochondrial distribution and reduced mitochondrial reactive oxygen species (mROS) production. Thus, TCAIM controls T cell activation and promotes tolerance induction probably by regulating TCR-mediated mitochondrial distribution and mROS production.