Regulation of the effector function of CD8+ T cells by gut microbiota-derived metabolite butyrate.
ABSTRACT: The gut microbiota produces metabolites such as short-chain fatty acids (SCFAs) that regulate the energy homeostasis and impact on immune cell function of the host. Recently, innovative approaches based on the oral administration of SCFAs have been discussed for therapeutic modification of inflammatory immune responses in autoimmune diseases. So far, most studies have investigated the SCFA-mediated effects on CD4+ T cells and antigen presenting cells. Here we show that butyrate and, to a lesser degree, propionate directly modulate the gene expression of CD8+ cytotoxic T lymphocytes (CTLs) and Tc17 cells. Increased IFN-? and granzyme B expression by CTLs as well as the molecular switch of Tc17 cells towards the CTL phenotype was mediated by butyrate independently of its interaction with specific SCFA-receptors GPR41 and GPR43. Our results indicate that butyrate strongly inhibited histone-deacetylases (HDACs) in CD8+ T cells thereby affecting the gene expression of effector molecules. Accordingly, the pan-HDAC inhibitors trichostatin A (TSA) and sodium valproate exerted similar influence on CD8+ T cells. Furthermore, higher acetate concentrations were also able to increase IFN-? production in CD8+ T lymphocytes by modulating cellular metabolism and mTOR activity. These findings might have significant implications in adoptive immunotherapy of cancers and in anti-viral immunity.
Project description:Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs <i>in vitro</i> and <i>in vivo</i> under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-?-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na<sup>+</sup>-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-?1, the expression of T-bet and IFN-? was triggered upon stimulation of CD4<sup>+</sup> T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-? during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.
Project description:Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.
Project description:<h4>Background</h4>IgG<sub>4</sub>-related disease (IgG<sub>4</sub>-RD) is an immune-mediated fibrotic disorder that has been linked to CD4<sup>+</sup> cytotoxic T lymphocytes (CD4<sup>+</sup>CTLs). The effector phenotype of CD4<sup>+</sup>CTLs and the relevance of both CD8<sup>+</sup> cytotoxic T lymphocytes (CD8<sup>+</sup>CTLs) and apoptotic cell death remain undefined in IgG<sub>4</sub>-RD.<h4>Objective</h4>We sought to define CD4<sup>+</sup>CTL heterogeneity, characterize the CD8<sup>+</sup>CTL response in the blood and in lesions, and determine whether enhanced apoptosis may contribute to the pathogenesis of IgG<sub>4</sub>-RD.<h4>Methods</h4>Blood analyses were undertaken using flow cytometry, cell sorting, transcriptomic analyses at the population and single-cell levels, and next-generation sequencing for the TCR repertoire. Tissues were interrogated using multicolor immunofluorescence. Results were correlated with clinical data.<h4>Results</h4>We establish that among circulating CD4<sup>+</sup>CTLs in IgG<sub>4</sub>-RD, CD27<sup>lo</sup>CD28<sup>lo</sup>CD57<sup>hi</sup> cells are the dominant effector subset, exhibit marked clonal expansion, and differentially express genes relevant to cytotoxicity, activation, and enhanced metabolism. We also observed prominent infiltration of granzyme A-expressing CD8<sup>+</sup>CTLs in disease tissues and clonal expansion in the blood of effector/memory CD8<sup>+</sup> T cells with an activated and cytotoxic phenotype. Tissue studies revealed an abundance of cells undergoing apoptotic cell death disproportionately involving nonimmune, nonendothelial cells of mesenchymal origin. Apoptotic cells showed significant upregulation of HLA-DR.<h4>Conclusions</h4>CD4<sup>+</sup>CTLs and CD8<sup>+</sup>CTLs may induce apoptotic cell death in tissues of patients with IgG<sub>4</sub>-RD with preferential targeting of nonendothelial, nonimmune cells of mesenchymal origin.
Project description:Cancer immunity is mediated through the effective priming and activation of tumour-specific class I MHC molecule-restricted CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs). DEC-205<sup>+</sup> dendritic cells (DCs) can cross-present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumour-specific CD8<sup>+</sup> CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of impaired CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8<sup>+</sup> CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8<sup>+</sup> CTLs with tumour-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide (α-GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC-205<sup>+</sup> DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α-GalCer every 48 hr appeared to convert tolerogenic DEC-205<sup>+</sup> DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a stronger cross-presentation capacity, which primed CTL precursors and induced tumour-specific CD8<sup>+</sup> CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205<sup>+</sup> DCs within tumours into immunogenic DCs through the sequential administration of an immuno-potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co-stimulatory molecules prime and activate tumour-specific CD8<sup>+</sup> CTLs within the tumour to control tumour growth.
Project description:CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met<sup>+</sup> CTLs). Phenotypic and functional analysis of c-Met<sup>+</sup> CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met<sup>-</sup> CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met<sup>+</sup> CTLs in cell-mediated cytotoxicity reactions <i>in vitro</i> and <i>in vivo</i> and abrogates T-cell responses against metastatic melanoma <i>in vivo</i> Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met<sup>+</sup> CTLs are a naturally occurring CD8<sup>+</sup> T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8<sup>+</sup> T-cell-mediated anti-tumor immunity.
Project description:<h4>Objectives</h4>To better understand how immune responses may be harnessed against breast cancer, we investigated which immune cell types and signalling pathways are required for spontaneous control of a mouse model of mammary adenocarcinoma.<h4>Methods</h4>The NOP23 mammary adenocarcinoma cell line expressing epitopes derived from the ovalbumin model antigen is spontaneously controlled when orthotopically engrafted in syngeneic C57BL/6 mice. We combined this breast cancer model with antibody-mediated depletion of lymphocytes and with mutant mice affected in interferon (IFN) or type 1 conventional dendritic cell (cDC1) responses. We monitored tumor growth and immune infiltration including the activation of cognate ovalbumin-specific T cells.<h4>Results</h4>Breast cancer immunosurveillance required cDC1, NK/NK T cells, conventional CD4<sup>+</sup> T cells and CD8<sup>+</sup> cytotoxic T lymphocytes (CTLs). cDC1 were required constitutively, but especially during T-cell priming. In tumors, cDC1 were interacting simultaneously with CD4<sup>+</sup> T cells and tumor-specific CTLs. cDC1 expression of the XCR1 chemokine receptor and of the T-cell-attracting or T-cell-activating cytokines CXCL9, IL-12 and IL-15 was dispensable for tumor rejection, whereas IFN responses were necessary, including cDC1-intrinsic signalling by STAT1 and IFN-γ but not type I IFN (IFN-I). cDC1 and IFNs promoted CD4<sup>+</sup> and CD8<sup>+</sup> T-cell infiltration, terminal differentiation and effector functions. In breast cancer patients, high intratumor expression of genes specific to cDC1, CTLs, CD4<sup>+</sup> T cells or IFN responses is associated with a better prognosis.<h4>Conclusion</h4>Interferons and cDC1 are critical for breast cancer immunosurveillance. IFN-γ plays a prominent role over IFN-I in licensing cDC1 for efficient T-cell activation.
Project description:Memory CD8<sup>+</sup> T cells are ideal candidates for cancer immunotherapy because they can mediate long-term protection against tumors. However, the therapeutic potential of different <i>in vitro</i>-generated CD8<sup>+</sup> T cell effector subsets to persist and become memory cells has not been fully characterized. Type 1 CD8<sup>+</sup> T (Tc1) cells produce interferon-? and are endowed with high cytotoxic capacity, whereas IL-17-producing CD8<sup>+</sup> T (Tc17) cells are less cytotoxic but display enhanced self-renewal capacity. We sought to evaluate the functional properties of <i>in vitro</i>-generated Tc17 cells and elucidate their potential to become long lasting memory cells. Our results show that <i>in vitro</i>-generated Tc17 cells display a greater <i>in vivo</i> persistence and expansion in response to secondary antigen stimulation compared to Tc1 cells. When transferred into recipient mice, Tc17 cells persist in secondary lymphoid organs, present a recirculation behavior consistent with central memory T cells, and can shift to a Tc1 phenotype. Accordingly, Tc17 cells are endowed with a higher mitochondrial spare respiratory capacity than Tc1 cells and express higher levels of memory-related molecules than Tc1 cells. Together, these results demonstrate that <i>in vitro</i>-generated Tc17 cells acquire a central memory program and provide a lasting reservoir of Tc1 cells <i>in vivo</i>, thus supporting the use of Tc17 lymphocytes in the design of novel and more effective therapies.
Project description:The prevailing 'division of labor' concept in cellular immunity is that CD8<sup>+</sup> T cells primarily utilize cytotoxic functions to kill target cells, while CD4<sup>+</sup> T cells exert helper/inducer functions. Multiple subsets of CD4<sup>+</sup> memory T cells have been characterized by distinct chemokine receptor expression. Here, we demonstrate that analogous CD8<sup>+</sup> memory T-cell subsets exist, characterized by identical chemokine receptor expression signatures and controlled by similar generic programs. Among them, Tc2, Tc17 and Tc22 cells, in contrast to Tc1 and Tc17?+?1 cells, express IL-6R but not SLAMF7, completely lack cytotoxicity and instead display helper functions including CD40L expression. CD8<sup>+</sup> helper T cells exhibit a unique TCR repertoire, express genes related to skin resident memory T cells (T<sub>RM</sub>) and are altered in the inflammatory skin disease psoriasis. Our findings reveal that the conventional view of CD4<sup>+</sup> and CD8<sup>+</sup> T cell capabilities and functions in human health and disease needs to be revised.
Project description:Enrichment of CD103<sup>+</sup> tumor-infiltrating T lymphocytes (TIL) is associated with improved outcomes in patients. However, the characteristics of human CD103<sup>+</sup> cytotoxic CD8<sup>+</sup> T cells (CTL) and their role in tumor control remain unclear. We investigated the features and antitumor mechanisms of CD103<sup>+</sup> CTLs by assessing T-cell receptor (TCR)-matched CD103<sup>+</sup> and CD103<sup>-</sup> cancer-specific CTL immunity <i>in vitro</i> and its immunophenotype <i>ex vivo</i> Interestingly, we found that differentiated CD103<sup>+</sup> cancer-specific CTLs expressed the active form of TGFβ1 to continually self-regulate CD103 expression, without relying on external TGFβ1-producing cells. The presence of CD103 on CTLs improved TCR antigen sensitivity, which enabled faster cancer recognition and rapid antitumor cytotoxicity. These CD103<sup>+</sup> CTLs had elevated energetic potential and faster migration capacity. However, they had increased inhibitory receptor coexpression and elevated T-cell apoptosis following prolonged cancer exposure. Our data provide fundamental insights into the properties of matured human CD103<sup>+</sup> cancer-specific CTLs, which could have important implications for future designs of tissue-localized cancer immunotherapy strategies.
Project description:Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8<sup>+</sup> T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8<sup>+</sup> T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8<sup>+</sup> T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8<sup>+</sup> T cells by alleviating the suppressive effect on CD8<sup>+</sup> T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.