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Objective: T cells and level of the cytokine interferon-γ (IFN-γ) are increased in adipose tissue in obesity. Hedgehog (Hh) signaling has been shown to potently inhibit white adipocyte differentiation. In light of recent findings in neurons that IFN-γ and Hh signaling cross-talk, we examined their potential interaction in the context of adipogenesis.Research Design and Methods: We used Hh reporter cells, cell lines, and primary adipocyte differentiation models to explore costimulation of IFN-γ and Hh signaling. Genetic dissection using Ifngr1(-/-) and Stat1(-/-) mouse embryonic fibroblasts, and ultimately, anti-IFN-γ neutralization and expression profiling in obese mice and humans, respectively, were used to place the findings into the in vivo context.Results: T-cell supernatants directly inhibited hedgehog signaling in reporter and 3T3-L1 cells. Intriguingly, using blocking antibodies, Ifngr1(-/-) and Stat1(-/-) cells, and simultaneous activation of Hh and IFN-γ signaling, we showed that IFN-γ directly suppresses Hh stimulation, thus rescuing adipogenesis. We confirmed our findings using primary mouse and primary human (pre)adipocytes. Importantly, robust opposing signals for Hh and T-cell pathways in obese human adipose expression profiles and IFN-γ depletion in mice identify the system as intact in adipose tissue in vivo.Conclusions: These results identify a novel antagonistic cross-talk between IFN-γ and Hh signaling in white adipose tissue and demonstrate IFN-γ as a potent inhibitor of Hh signaling. [ABSTRACT FROM AUTHOR]

Abstract Background The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. Results The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Conclusion Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.

FOXP3 + regulatory T cells (Treg cells) are key for immune homeostasis. Here, we reveal that nuclear receptor corepressor 1 (NCOR1) controls naïve and effector Treg cell states. Upon NCOR1 deletion in T cells, effector Treg cell frequencies were elevated in mice and in in vitro-generated human Treg cells. NCOR1-deficient Treg cells failed to protect mice from severe weight loss and intestinal inflammation associated with CD4 + T cell transfer colitis, indicating impaired suppressive function. NCOR1 controls the transcriptional integrity of Treg cells, since effector gene signatures were already upregulated in naïve NCOR1-deficient Treg cells while effector NCOR1-deficient Treg cells failed to repress genes associated with naïve Treg cells. Moreover, genes related to cholesterol homeostasis including targets of liver X receptor (LXR) were dysregulated in NCOR1-deficient Treg cells. However, genetic ablation of LXRβ in T cells did not revert the effects of NCOR1 deficiency, indicating that NCOR1 controls naïve and effector Treg cell subset composition independent from its ability to repress LXRβ-induced gene expression. Thus, our study reveals that NCOR1 maintains naïve and effector Treg cell states via regulating their transcriptional integrity. We also reveal a critical role for this epigenetic regulator in supporting the suppressive functions of Treg cells in vivo., Competing Interests: VS, Rd, RR, CZ, TP, PH, DH, MA, LM, MW, DW, AH, BA, MS, TF, TK, SK, CC, KS, MT, AB, CB, NB, WE No competing interests declared, (© 2024, Stolz, de Freitas e Silva et al.)

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103 + dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.

T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation., (© 2023 Sandner et al.)

T helper (Th) 17 cells are not only key in controlling infections mediated by extracellular bacteria and fungi but are also triggering autoimmune responses. Th17 cells comprise heterogeneous subsets, some with pathogenic functions. They can cease to secrete their hallmark cytokine IL-17A and even convert to other T helper lineages, a process known as transdifferentiation relying on plasticity. Both pathogenicity and plasticity are tightly linked to IL-23 signaling. Here, we show that the protein tyrosine kinase Tec is highly induced in Th17 cells. Th17 differentiation was enhanced at low interleukin-6 (IL-6) concentrations in absence of Tec, which correlates with increased STAT3 phosphorylation and higher Il23r expression. Therefore, we uncovered a function for Tec in the IL-6 sensing via STAT3 by CD4 + T cells, defining Tec as a fine-tuning negative regulator of Th17 differentiation. Subsequently, by using the IL-17A fate mapping mouse combined with in vivo adoptive transfer models, we demonstrated that Tec not only restrained effector Th17 differentiation but also pathogenicity and plasticity in a T-cell intrinsic manner. Our data further suggest that Tec regulates inflammatory Th17-driven immune responses directly impacting disease severity in a T-cell-driven colitis model. Notably, consistent with the in vitro findings, elevated levels of the IL-23 receptor (IL-23R) were observed on intestinal pre- and postconversion Th17 cells isolated from diseased Tec -/- mice subjected to adoptive transfer colitis, highlighting a fundamental role of Tec in restraining IL-23R expression, likely via the IL-6-STAT3 signaling axis. Taken together, these findings identify Tec as a negative regulator of Th17 differentiation, pathogenicity, and plasticity, contributing to the mechanisms which help T cells to orchestrate optimal immune protection and to restrain immunopathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sandner, Alteneder, Zhu, Hladik, Högler, Rica, Van Greuningen, Sharif, Sakaguchi, Knapp, Kenner, Trauner, Ellmeier and Boucheron.)

Background & Aims: 24-Norursodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid used to treat immune-mediated cholestatic liver diseases, such as primary sclerosing cholangitis (PSC), where dysregulated T cells including CD8 + T cells contribute to hepatobiliary immunopathology. We hypothesized that NorUDCA may directly modulate CD8 + T cell function thus contributing to its therapeutic efficacy., Methods: NorUDCA's immunomodulatory effects were first studied in Mdr2 -/- mice, as a cholestatic model of PSC. To differentiate NorUDCA's immunomodulatory effects on CD8 + T cell function from its anticholestatic actions, we also used a non-cholestatic model of hepatic injury induced by an excessive CD8 + T cell immune response upon acute non-cytolytic lymphocytic choriomeningitis virus (LCMV) infection. Studies included molecular and biochemical approaches, flow cytometry and metabolic assays in murine CD8 + T cells in vitro. Mass spectrometry was used to identify potential CD8 + T cell targets modulated by NorUDCA. The signaling effects of NorUDCA observed in murine cells were validated in circulating T cells from patients with PSC., Results: NorUDCA demonstrated immunomodulatory effects by reducing hepatic innate and adaptive immune cells, including CD8 + T cells in the Mdr2 -/- model. In the non-cholestatic model of CD8 + T cell-driven immunopathology induced by acute LCMV infection, NorUDCA ameliorated hepatic injury and systemic inflammation. Mechanistically, NorUDCA demonstrated strong immunomodulatory efficacy in CD8 + T cells affecting lymphoblastogenesis, expansion, glycolysis and mTORC1 signaling. Mass spectrometry identified that NorUDCA regulates CD8 + T cells by targeting mTORC1. NorUDCA's impact on mTORC1 signaling was further confirmed in circulating PSC CD8 + T cells., Conclusions: NorUDCA has a direct modulatory impact on CD8 + T cells and attenuates excessive CD8 + T cell-driven hepatic immunopathology. These findings are relevant for treatment of immune-mediated liver diseases such as PSC., Lay Summary: Elucidating the mechanisms by which 24-norursodeoxycholic acid (NorUDCA) works for the treatment of immune-mediated liver diseases, such as primary sclerosing cholangitis, is of considerable clinical interest. Herein, we uncovered an unrecognized property of NorUDCA in the immunometabolic regulation of CD8 + T cells, which has therapeutic relevance for immune-mediated liver diseases, including PSC., Competing Interests: Conflict of interest M. Trauner has served as speaker for Falk Foundation, Gilead, Intercept and MSD; he has advised for Albireo, BiomX, Boehringer Ingelheim, Falk Pharma GmbH, Genfit, Gilead, Intercept, Jannsen, MSD, Novartis, Phenex, Regulus and Shire. He further received travel grants from Abbvie, Falk, Gilead and Intercept and research grants from Albireo, Alnylam, Cymabay, Falk, Gilead, Intercept, MSD Takeda and UltraGenyx. He is also co-inventor of patents on the medical use of NorUDCA filed by the Medical Universities of Graz and Vienna. All the other authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

CD4 + T cell trafficking is a fundamental property of adaptive immunity. In this study, we uncover a novel role for histone deacetylase 1 (HDAC1) in controlling effector CD4 + T cell migration, thereby providing mechanistic insight into why a T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis (EAE). HDAC1-deficient CD4 + T cells downregulated genes associated with leukocyte extravasation. In vitro, HDAC1-deficient CD4 + T cells displayed aberrant morphology and migration on surfaces coated with integrin LFA-1 ligand ICAM-1 and showed an impaired ability to arrest on and to migrate across a monolayer of primary mouse brain microvascular endothelial cells under physiological flow. Moreover, HDAC1 deficiency reduced homing of CD4 + T cells into the intestinal epithelium and lamina propria preventing weight-loss, crypt damage and intestinal inflammation in adoptive CD4 + T cell transfer colitis. This correlated with reduced expression levels of LFA-1 integrin chains CD11a and CD18 as well as of selectin ligands CD43, CD44 and CD162 on transferred circulating HDAC1-deficient CD4 + T cells. Our data reveal that HDAC1 controls T cell-mediated autoimmunity via the regulation of CD4 + T cell trafficking into the CNS and intestinal tissues., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo., (© 2020 Federation of American Societies for Experimental Biology.)

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4 + T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4 + T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4 + CCR6 + cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA., Competing Interests: Declaration of competing interest None. This research has received support from the Innovative Medicines Initiative (IMI) Joint Undertaking under grant agreement n°115142 (BTCure), and grant agreement n(o)777357 (RTCure) resources of which are composed of financial contribution from the European Union's Framework Programmes and EFPIA companies' in kind contribution. The work has been supported by the Austrian Science Fund (FWF) project F26193 (to WE) and the FWF special research program SFB F70 (subproject F7003 to MB, F7004 to NB and F7005 to WE)., (Copyright © 2019. Published by Elsevier Ltd.)

Some effector CD4+ T cell subsets display cytotoxic activity, thus breaking the functional dichotomy of CD4+ helper and CD8+ cytotoxic T lymphocytes. However, molecular mechanisms regulating CD4+ cytotoxic T lymphocyte (CD4+ CTL) differentiation are poorly understood. Here we show that levels of histone deacetylases 1 and 2 (HDAC1-HDAC2) are key determinants of CD4+ CTL differentiation. Deletions of both Hdac1 and 1 Hdac2 alleles (HDAC1cKO-HDAC2HET) in CD4+ T cells induced a T helper cytotoxic program that was controlled by IFN-γ-JAK1/2-STAT1 signaling. In vitro, activated HDAC1cKO-HDAC2HET CD4+ T cells acquired cytolytic activity and displayed enrichment of gene signatures characteristic of effector CD8+ T cells and human CD4+ CTLs. In vivo, murine cytomegalovirus-infected HDAC1cKO-HDAC2HET mice displayed a stronger induction of CD4+ CTL features compared with infected WT mice. Finally, murine and human CD4+ T cells treated with short-chain fatty acids, which are commensal-produced metabolites acting as HDAC inhibitors, upregulated CTL genes. Our data demonstrate that HDAC1-HDAC2 restrain CD4+ CTL differentiation. Thus, HDAC1-HDAC2 might be targets for the therapeutic induction of CD4+ CTLs.

Multiple sclerosis (MS) is a human neurodegenerative disease characterized by the invasion of autoreactive T cells from the periphery into the CNS. Application of pan-histone deacetylase inhibitors (HDACi) ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model for MS, suggesting that HDACi might be a potential therapeutic strategy for MS. However, the function of individual HDAC members in the pathogenesis of EAE is not known. In this study we report that mice with a T cell-specific deletion of HDAC1 (using the Cd4-Cre deleter strain; HDAC1-cKO) were completely resistant to EAE despite the ability of HDAC1cKO CD4 + T cells to differentiate into Th17 cells. RNA sequencing revealed STAT1 as a prominent upstream regulator of differentially expressed genes in activated HDAC1-cKO CD4 + T cells and this was accompanied by a strong increase in phosphorylated STAT1 (pSTAT1). This suggests that HDAC1 controls STAT1 activity in activated CD4 + T cells. Increased pSTAT1 levels correlated with a reduced expression of the chemokine receptors Ccr4 and Ccr6, which are important for the migration of T cells into the CNS. Finally, EAE susceptibility was restored in WT:HDAC1-cKO mixed BM chimeric mice, indicating a cell-autonomous defect. Our data demonstrate a novel pathophysiological role for HDAC1 in EAE and provide evidence that selective inhibition of HDAC1 might be a promising strategy for the treatment of MS., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nuclear receptor corepressor 1 (NCOR1) is a transcriptional regulator bridging repressive chromatin modifying enzymes with transcription factors. NCOR1 regulates many biological processes, however its role in T cells is not known. Here we show that Cd4-Cre-mediated deletion of NCOR1 (NCOR1 cKO Cd4 ) resulted in a reduction of peripheral T cell numbers due to a decrease in single-positive (SP) thymocytes. In contrast, double-positive (DP) thymocyte numbers were not affected in the absence of NCOR1. The reduction in SP cells was due to diminished survival of NCOR1-null postselection TCRβ hi CD69 + and mature TCRβ hi CD69 - thymocytes. NCOR1-null thymocytes expressed elevated levels of the pro-apoptotic factor BIM and showed a higher fraction of cleaved caspase 3-positive cells upon TCR stimulation ex vivo. However, staphylococcal enterotoxin B (SEB)-mediated deletion of Vβ8 + CD4SP thymocytes was normal, suggesting that negative selection is not altered in the absence of NCOR1. Finally, transgenic expression of the pro-survival protein BCL2 restored the population of CD69 + thymocytes in NCOR1 cKO Cd4 mice to a similar percentage as observed in WT mice. Together, these data identify NCOR1 as a crucial regulator of the survival of SP thymocytes and revealed that NCOR1 is essential for the proper generation of the peripheral T cell pool.

T cells must tightly regulate their metabolic processes to cope with varying bioenergetic demands depending on their state of differentiation. The metabolic sensor AMPK is activated in states of low energy supply and modulates cellular metabolism toward a catabolic state. Although this enzyme is known to be particularly active in regulatory T (T reg ) cells, its impact on T helper (T h )-cell differentiation is poorly understood. We investigated the impact of several AMPK activators on T reg -cell differentiation and found that the direct activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), but not the indirect activators metformin and 2-deoxyglucose, strongly enhanced T reg -cell induction by specifically enhancing T reg -cell expansion. Conversely, T h 17 generation was impaired by the agent. Further investigation of the metabolic background of our observations revealed that AICAR enhanced both cellular mitochondrogenesis and fatty acid uptake. Consistently, increased T reg induction was entirely reversible on inhibition of fatty acid oxidation, thus confirming the dependence of AICAR's effects on metabolic pathways alterations. Translating our findings to an in vivo model, we found that the substance enhanced T reg cell generation on IL-2 complex-induced immune stimulation. We provide a previously unrecognized insight into the delicate interplay between immune cell function and metabolism and delineate a potential novel strategy for metabolism-targeting immunotherapy.-Gualdoni, G. A., Mayer, K. A., Göschl, L., Boucheron, N., Ellmeier, W., Zlabinger, G. J. The AMP analog AICAR modulates the T reg /T h 17 axis through enhancement of fatty acid oxidation., (© FASEB.)

Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8+ T cell function remains elusive. By using conditional gene targeting approaches, we show that LckCre-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8+CD4- cells within the CD3/TCRβlo population, indicating that HDAC1 is essential for the efficient progression of immature CD8+CD4- cells to the DP stage. Moreover, CD44hi effector CD8+ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44hi CD8+ T cells produced more IFNγ upon ex vivo PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44l°CD62L+) HDAC1-null CD8+ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their in vivo cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8+ T cell response upon LCMV infection and impaired expansion of virus-specific CD8+ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8+ T cell homeostasis and for an efficient in vivo expansion and activation of CD8+ T cells in response to LCMV infection.

Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFβ complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFβ complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.

Mast cells are key players in type I hypersensitivity reactions in humans and mice and their activity has to be tightly controlled. Previous studies implicated the transcription factor MAZR in the regulation of mast cell function. To study the role of MAZR in mast cells, we generated a conditional Mazr allele and crossed Mazr (F/F) mice with the Vav-iCre deleter strain, which is active in all hematopoietic cells. MAZR-null BM-derived mast cells (BMMC) were phenotypically indistinguishable from wild-type BMMCs, although the numbers of IL-3 generated Mazr (F/F) Vav-iCre BMMCs were reduced in comparison to Mazr (F/F) BMMCs, showing that MAZR is required for the efficient generation of BMMC in vitro. A gene expression analysis revealed that MAZR-deficiency resulted in the dysregulation of 128 genes, with more genes up- than down-regulated in the absence of MAZR, indicating that MAZR acts as a transcriptional repressor in mast cells. Among the up-regulated genes were the chemokines Ccl5, Cxcl10, Cxcl12, the chemokine receptor Ccr5 and the cytokine IL18, suggesting an immunoregulatory role for MAZR in mast cells. Enforced expression of MAZR in mature Mazr-deficient BMMCs rescued the altered expression pattern of some genes tested, suggesting direct regulation of these genes by MAZR. Upon FcεRI stimulation, Mazr expression was transiently down-regulated in BMMCs. However, early and late effector functions in response to FcεRI-mediated stimulation were not impaired in the absence of MAZR, with the exception of IL-6, which was slightly decreased. Taken together, out data indicate that MAZR preferentially acts as a transcriptional repressor in mast cells, however MAZR plays only a minor role in the transcriptional networks that regulate early and late effector functions in mast cells in response to FcεRI stimulation.

ABSTRACT Members of the Tec kinase family (Tec, Btk, Itk, Rlk, and Bmx) play an important role during innate and adaptive immune responses, and mutations in Tec family kinases are linked with immunodeficiencies in humans and mice. Three members of the Tec kinase family are expressed in T cells (Tec, Itk, and Rlk), and biochemical and genetic studies have revealed important roles for Tec family kinases during T-cell development and in the control of T-cell function. Here the authors review the role of Tec family kinases in the regulation of T-helper-cell differentiation.

Cd8a and Cd8b1 coreceptor gene (Cd8) expression is tightly controlled during T-cell development by the activity of five Cd8 enhancers (E8(I)-E8(V)). Here we demonstrate a unique transcriptional program regulating CD8 expression during CD8(+) effector T-cell differentiation. The Cd8 enhancer E8(I) and Runx/core-binding factor-β (CBFβ) complexes were required for the establishment of this regulatory circuit, because E8(I)-, Runx3-, or CBFβ-deficient CD8(+) T cells down-regulated CD8α expression during activation. This finding correlated with enhanced repressive histone marks at the Cd8a promoter in the absence of E8(I), and the down-regulation of CD8α expression could be blocked by treating E8(I)-, Runx3-, or CBFβ-deficient CD8(+) T cells with the histone deacetylase inhibitor trichostatin A. Moreover, Runx/CBFβ complexes bound the Cd8ab gene cluster in activated CD8(+) T cells, suggesting direct control of the Cd8a locus. However, CD8(+) effector T cells maintained high levels of CD8α when CBFβ was conditionally deleted after activation. Thus, our data suggest an E8(I)- and Runx3/CBFβ-dependent epigenetic programming of the Cd8a locus during T-cell activation, leading to Runx/CBFβ complex-independent maintenance of CD8α expression in effector T cells.

The generation of Th17 cells has to be tightly controlled during an immune response. In this study, we report an increase in a CD44(high)CD62L(-) Th17 subset in mice deficient for the protein tyrosine kinase Tec. CD44(high)CD62L(-) Tec(-/-) CD4(+) T cells produced enhanced IL-17 upon activation, showed increased expression levels of IL-23R and RORγt, and IL-23-mediated expansion of Tec(-/-) CD4(+) T cells led to an increased production of IL-17. Tec(-/-) mice immunized with heat-killed Streptococcus pneumoniae displayed increased IL-17 expression levels in the lung postinfection with S. pneumoniae, and this correlated with enhanced pneumococcal clearance and reduced lung inflammation compared with Tec(+/+) mice. Moreover, naive Tec(-/-) OT-II CD4(+) T cells produced higher levels of IL-17 when cultured with OVA peptide-loaded bone marrow-derived dendritic cells that have been previously activated with heat-killed S. pneumoniae. Taken together, our data indicated a critical role for Tec in T cell-intrinsic signaling pathways that regulate the in vivo generation of CD44(high)CD62L(-) effector/memory Th17 populations.

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells.

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon Fc epsilon RI stimulation of BM-derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C(4) levels were reduced in Tec(-) (/) (-) BMMC. Furthermore, the production of IL-4 was severely impaired, and GM-CSF, TNF-alpha and IL-13 levels were also diminished. Finally, a comparison of WT, Tec(-) (/) (-), Btk(-) (/) (-) and Tec(-) (/) (-)Btk(-) (/) (-) BMMC revealed a negative role for Btk in the regulation of IL-4 production, while for the efficient production of TNF-alpha, IL-13 and GM-CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well-characterized family member Btk.

Vav1 and the Tec family kinase Itk act in similar T-cell activation pathways. Both molecules interact with members of the Cbl family of E3 ubiquitin ligases, and signaling defects in Vav1(-/-) T cells are rescued upon deletion of Cbl-b. In this study we investigate the relation between Itk and Cbl-b or Vav1 by generating Itk/Cbl-b and Itk/Vav1 double-deficient mice. Deletion of Cbl-b in Itk(-/-) CD4(+) T cells restored proliferation and partially IL-2 production, and also led to a variable rescue of IL-4 production. Thus, Itk and Vav1 act mechanistically similarly in peripheral T cells, since the defects in Itk(-/-) T cells, as in Vav1(-/-) T cells, are rescued if cells are released from the negative regulation mediated by Cbl-b. In addition, only few peripheral CD4(+) and CD8(+) T cells were present in Vav1(-/-)Itk(-/-) mice due to severely impaired thymocyte differentiation. Vav1(-/-)Itk(-/-) thymocyte numbers were strongly reduced compared with WT, Itk(-/-) or Vav1(-/-) mice, and double-positive thymocytes displayed increased cell death and impaired positive selection. Therefore, our data also reveal that the combined activity of Vav1 and Itk is required for proper T-cell development and the generation of the peripheral T-cell pool.

Transcriptional pathways controlling the development of CD44(hi) memory phenotype (MP) T cells with "innate-like" functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44(hi), but not in CD44(lo), CD4(+) T cells. Transgenic expression of PLZF during T cell development and in CD4(+) and CD8(+) T cells induced a T cell intrinsic program leading to an increase in peripheral CD44(hi) MP CD4(+) and CD8(+) T cells and a corresponding decrease of naïve CD44(lo) T cells. The MP CD4(+) and CD8(+) T cells produced IFNgamma upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4(+) transgenic T cells showed reduced IL-2 and IFNgamma production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44(hi)CD62L(+) subset. Our data indicate that PLZF is a novel regulator of the development of CD44(hi) MP T cells with a characteristic partial innate-like phenotype.

CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.

Members of the Tec kinase family (Bmx, Btk, Itk, Rlk and Tec) are primarily expressed in the hematopoietic system and form, after the Src kinase family, the second largest class of non-receptor protein tyrosine kinases. During lymphocyte development and activation Tec kinases have important functions in signaling pathways downstream of the antigen receptors. Tec family kinases are also expressed in cells of the myeloid lineage. However, with the exception of mast cells and platelets, their biological role in the myeloid system is only poorly understood. This review summarizes the current knowledge about the function of Tec family kinases in hematopoietic cells of the myeloid lineage., (Copyright 2004 S. Karger AG, Basel)

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.

Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.