TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in sensitivity to a plethora of irritating agents and endogenous mediators of oxidative stress. TRPA1 influences neuroinflammation and macrophage and lymphocyte functions, but its role is controversial in immune cells. We reported earlier a detectable, but orders-of-magnitude-lower level of Trpa1 mRNA in monocytes and lymphocytes than in sensory neurons by qRT-PCR analyses of cells from lymphoid organs of mice. Our present goals were to (a) further elucidate the expression of Trpa1 mRNA in immune cells by RNAscope in situ hybridization (ISH) and (b) test the role of TRPA1 in lymphocyte activation. RNAscope ISH confirmed that Trpa1 transcripts were detectable in CD14+ and CD4+ cells from the peritoneal cavity of mice. A selective TRPA1 agonist JT010 elevated Ca2+ levels in these cells only at high concentrations. However, a concentration-dependent inhibitory effect of JT010 was observed on T-cell receptor (TcR)-induced Ca2+ signals in CD4+ T lymphocytes, while JT010 neither modified B cell activation nor ionomycin-stimulated Ca2+ level. Based on our present and past findings, TRPA1 activation negatively modulates T lymphocyte activation, but it does not appear to be a key regulator of TcR-stimulated calcium signaling.

Co-localization of clusters of TCR-regulated genes with TAD rearrangements.

BACKGROUND: Gene expression has long been known to be influenced by the relative proximity of DNA regulatory elements. Topologically associating domains (TADs) are self-interacting genomic regions involved in regulating gene expression by controlling the proximity of these elements. Prior studies of TADs and their biological roles have revealed correlations between TAD changes and cellular differentiation. Here, we used Hi-C and RNA-seq data to correlate TCR-induced changes in TAD structure and gene expression in human CD4+ T cells. RESULTS: We developed a pipeline, Differentially Expressed Gene Enrichment Finder (DEGEF), that identifies regions of differentially expressed gene enrichment. Using DEGEF, we found that TCR-regulated genes cluster non-uniformly across the genome and that these clusters preferentially localized in regions of TAD rearrangement. Interestingly, clusters of upregulated genes preferentially formed new Hi-C contacts compared to downregulated clusters, suggesting that TCR-activated CD4+ T cells may regulate genes by changing stimulatory contacts rather than inhibitory contacts. CONCLUSIONS: Our observations support a significant relationship between TAD rearrangements and changes in local gene expression. These findings indicate potentially important roles for TAD rearrangements in shaping their local regulatory environments and thus driving differential expression of nearby genes during CD4+ T cell activation. Moreover, they provide new insights into global mechanisms that regulate gene expression.

TRPV4 regulates mitochondrial Ca2+-status and physiology in primary murine T cells based on their immunological state.

T cell activation process is critically affected by temperature and intracellular Ca2+-signalling. Yet, the nature and the key molecules involved in such complex Ca2+-signalling is poorly understood. It is mostly assumed that ion channels present in the plasma membrane primarily regulate the cytosolic Ca2+-levels exclusively. TRPV4 is a non-selective Ca2+ channel which can be activated at physiological temperature. TRPV4 is involved in several physiological, pathophysiological process as well as different forms of pain. Here we demonstrate that TRPV4 is endogenously expressed in T cell and is present in the mitochondria of T cells. TRPV4 activation increases mitochondrial Ca2+-levels, and alters mitochondrial temperature as well as specific metabolisms. The TRPV4-dependent increment in the mitochondrial Ca2+ is context-dependent and not just passively due to the increment in the cytosolic Ca2+. Our work also indicates that mitochondrial Ca2+-level correlates positively with a series of essential factors, such as mitochondrial membrane potential, mitochondrial ATP production and negatively correlates with certain factors such as mitochondrial temperature. We propose that TRPV4-mediated mitochondrial Ca2+-signalling and other metabolisms has implications in the immune activation process including immune synapse formation. Our data also endorse the re-evaluation of Ca2+-signalling in T cell, especially in the light of mitochondrial Ca2+-buffering and in higher body temperature, such as in case of fever. Presence of TRPV4 in the mitochondria of T cell is relevant for proper and optimum immune response and may provide evolutionary adaptive benefit. These findings may also have broad implications in different pathophysiological process, neuro-immune cross-talks, and channelopathies involving TRPV4.

Presence of TRPA1 Modifies CD4+/CD8+ T Lymphocyte Ratio and Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) has been reported to influence neuroinflammation and lymphocyte function. We analysed the immune phenotype and activation characteristics of TRPA1-deficient mice (knockout-KO) generated by targeted deletion of the pore-loop domain of the ion channel. We compared TRPA1 mRNA and protein expression in monocyte and lymphocyte subpopulations isolated from primary and secondary lymphatic organs of wild type (WT) and KO mice. qRT-PCR and flow cytometric studies indicated a higher level of TRPA1 in monocytes than in lymphocytes, but both were orders of magnitude lower than in sensory neurons. We found lower CD4+/CD8+ thymocyte ratios, diminished CD4/CD8 rates, and B cell numbers in the KO mice. Early activation marker CD69 was lower in CD4+ T cells of KO, while the level of CD8+/CD25+ cells was higher. In vitro TcR-mediated activation did not result in significant differences in CD69 level between WT and KO splenocytes, but lower cytokine (IL-1ß, IL-6, TNF-α, IL-17A, IL-22, and RANTES) secretion was observed in KO splenocytes. Basal intracellular Ca2+ level and TcR-induced Ca2+ signal in T lymphocytes did not differ significantly, but interestingly, imiquimod-induced Ca2+ level in KO thymocytes was higher. Our results support the role of TRPA1 in the regulation of activation, cytokine production, and T and B lymphocytes composition in mice.

Cav1.4 calcium channels control cytokine production by human peripheral TH17 cells and psoriatic skin-infiltrating T cells.

BACKGROUND: Type-17 inflammation characterizes psoriasis, a chronic skin disease. Because several inflammatory cytokines contribute to psoriasis pathogenesis, inhibiting the simultaneous production of these cytokines in TH17 cells may be beneficial in psoriasis. We found that Cav1.4, encoded by CACNA1F, was the only Cav1 calcium channel expressed in TH17 cells. OBJECTIVE: We sought to investigate the role of Cav1.4 expression in early TH17-activation events and effector functions, as well as its association with TH17 signature genes in lesional psoriatic (LP) skins. METHODS: Transcriptional gene signatures associated with CACNA1F expression were examined in LP skins by RT-PCR and in situ hybridization. Cav1 inhibitor and/or shRNA lentivectors were used to assess the contribution of Cav1.4 in TH17 activation and effector functions in a 3-dimensional skin reconstruction model. RESULTS: CACNA1F expression correlated with inflammatory cytokine expression that characterizes LP skins and was preferentially associated with RORC expression in CD4+ and CD4- cells from LP biopsies. Nicardipine, a Cav1 channel antagonist, markedly reduced inflammatory cytokine production by TH17 cells from blood or LP skin. This was associated with decreased TCR-induced early calcium events at cell membrane and proximal signaling events. The knockdown of Cav1.4 in TH17 cells impaired cytokine production. Finally, Cav1 inhibition reduced the expression of the keratinocyte genes characteristic of TH17-mediated psoriasis inflammation in human skin equivalents. CONCLUSIONS: Cav1.4 channels promote TH17-cell functions both at the periphery and in inflammatory psoriatic skin.

Calcium Imaging in T Lymphocytes: a Protocol for Use with Genetically Encoded or Chemical Ca2+ Indicators.

Elevations in cytosolic calcium (Ca2+) drive a wide array of immune cell functions, including cytokine production, gene expression, and cell motility. Live-cell imaging of cells loaded with ratiometric chemical Ca2+ indicators remains the gold standard for visualization and quantification of intracellular Ca2+ signals; ratiometric imaging can be accomplished with dyes such as Fura-2, the combination of Fluo-4 and Fura-Red, or, alternatively, by expressing genetically-encoded Ca2+ indicators (GECI) such as GCaMPs. Here, we describe a detailed protocol for Ca2+ imaging of T cells in vitro using genetically encoded or chemical indicators that can also be applied to a wide variety of cell types. The protocol addresses the challenge of facilitating T cell attachment on various substrates prepared on glass-bottom dishes to enable T cell imaging on an inverted microscope. The protocol also emphasizes cell preparation steps that ensure optimal cell viability - an essential requirement for recording dynamic changes in cytosolic Ca2+ levels - and that ensure reproducibility between multiple samples. Finally, we describe a simple algorithm to analyze single-cell Ca2+ signals over time using Fiji (ImageJ) software.

Lactate anions participate in T cell cytokine production and function.

After antigen stimulation, T cells preferentially increase aerobic glycolysis to meet the bioenergetic and biosynthetic demands of T cell activation, proliferation, and effector functions. Lactate, a by-product of glycolysis, has been reported to function as an important energy source and signaling molecule. Here, we found that lactate anions are involved in cytokine production in T cells after TCR activation. During ex vivo T cell activation, the addition of excess sodium lactate (NaL) increased the production of cytokines (such as IFNγ/IL-2/TNFα) more than the addition of sodium chloride (NaCl). This enhanced cytokine production was dependent on TCR/CD3 activation but not CD28 activation. In vivo, NaL treatment inhibited tumour growth in subcutaneously transplanted tumour models in a T cell-dependent manner, which was consistent with increased T cell cytokine production in the NaL treatment group compared to the NaCl treatment group. Furthermore, a mechanistic experiment showed that this enhanced cytokine production was regulated by GAPDH-mediated post-transcriptional regulation. Taken together, our findings indicate a new regulatory mechanism involved in glycolysis that promotes T cell function.

AP-1 and NF-κB synergize to transcriptionally activate latent HIV upon T-cell receptor activation.

Latent HIV-1 proviruses are capable of reactivating productive lytic infection, but the precise molecular mechanisms underlying emergence from latency are poorly understood. In this study, we determined the contribution of the transcription factors NF-κB, NFAT, and AP-1 in the reactivation of latent HIV following T-cell receptor (TCR) activation using Jurkat T-cell clones harboring single latent HIV proviruses. Our findings demonstrate that during reactivation from latency, NF-κB enhances HIV transcription while NFAT inhibits it by competing with NF-κB for overlapping binding sites on the HIV long terminal repeat (LTR). We have also demonstrated for the first time the molecular contribution of AP-1 in the reactivation of HIV from latency, whereby AP-1 synergizes with NF-κB to regulate HIV transcriptional elongation following TCR activation.

Human liver-derived MAIT cells differ from blood MAIT cells in their metabolism and response to TCR-independent activation.

Mucosal associated invariant T (MAIT) cells are anti-microbial innate-like T cells that are abundant in blood and liver. MAIT cells express a semi-invariant T-cell receptor (TCR) that recognizes a pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)-MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection. In this study, we compared the functional and transcriptomic response of human blood and ld-MAIT cells to TCR signals (Escherichia coli or the pyrimidine ligand) and cytokines (IL-12 + IL-18). While the response of blood and ld-MAIT cells to TCR signals were comparable, following cytokine stimulation ld-MAIT cells were more polyfunctional than blood MAIT cells. Transcriptomic analysis demonstrated different effector programmes of ld-MAIT cells with the two modes of activation, including the enrichment of a tissue repair signature in TCR-stimulated MAIT cells. Interestingly, we observed enhancement of IL-12 signaling and fatty acid metabolism in untreated ld-MAIT cells compared with blood MAIT cells. Additionally, MAIT cells from blood and liver were modulated similarly by TCR and cytokine signals. Therefore, we report that blood and ld-MAIT cells are fundamentally different but undergo conserved changes following activation via TCR or by cytokines.

Vγ9Vδ2 T Cells Activation Through Phosphoantigens Can Be Impaired by a RHOB Rerouting in Lung Cancer.

Vγ9Vδ2 T cells are known to be efficient anti-tumor effectors activated through phosphoantigens (PAg) that are naturally expressed by tumor cells or induced by amino bisphosphonates treatment. This PAg-activation which is TCR and butyrophilin BTN3A dependent can be modulated by NKG2D ligands, immune checkpoint ligands, adhesion molecules, and costimulatory molecules. This could explain the immune-resistance observed in certain clinical trials based on Vγ9Vδ2 T cells therapies. In NSCLC, encouraging responses were obtained with zoledronate administrations for 50% of patients. According to the in vivo results, we showed that the in vitro Vγ9Vδ2 T cell reactivity depends on the NSCLC cell line considered. If the PAg-pretreated KRAS mutated A549 is highly recognized and killed by Vγ9Vδ2 T cells, the EGFR mutated PC9 remains resistant to these killers despite a pre-treatment either with zoledronate or with exogenous BrHPP. The immune resistance of PC9 was shown not to be due to immune checkpoint ligands able to counterbalance NKG2D ligands or adhesion molecules such as ICAM-1 highly expressed by PC9. RHOB has been shown to be involved in the Vγ9Vδ2 TCR signaling against these NSCLC cell lines, in this study we therefore focused on its intracellular behavior. In comparison to a uniform distribution of RHOB in endosomes and at the plasma membrane in A549, the presence of large endosomal clusters of RHOB was visualized by a split-GFP system, suggesting that RHOB rerouting in the PC9 tumor cell could impair the reactivity of the immune response.

CD4+ T-Cell Differentiation In Vitro.

CD4+ T helper cells play crucial roles in adaptive immune response against pathogens, as well as in host immune homeostasis. Upon TCR activation, naïve CD4+ T cells differentiate into one of several lineages of Th cells, with hallmark transcription factors, cytokine production, and functions in vivo, according to the particular cytokine milieu. To study the regulating mechanism and function of Th cells, in vitro CD4+ T-cell differentiation is crucial. The following protocols describe the methods to induce naïve CD4+ T-cell differentiate into Th1, Th2, Th17 and Treg by activating TCR, together with the different cytokines and blocking antibodies in vitro. The efficiency of T helper cell differentiation is examined by detecting the expression of hallmark cytokines and transcription factors.

Type I interferons are important co-stimulatory signals during T cell receptor mediated human MAIT cell activation.

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that can be stimulated either via their TCR or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as TLR agonists, that can modulate TCR-mediated activation. Recently, type I interferons (T1-IFNs) have been identified as contributing to cytokine-mediated MAIT cell activation. However, it is unknown whether T1-IFNs also have a role during TCR-mediated MAIT cell activation. In this study, we investigated the co-stimulatory role of T1-IFNs during TCR-mediated activation of MAIT cells by the MR1 ligand 5-amino-6-d-ribitylaminouracil/methylglyoxal. We found that T1-IFNs were able to boost interferon-γ and granzyme B production in 5-amino-6-d-ribitylaminouracil/methylglyoxal-stimulated MAIT cells. Similarly, influenza virus-induced T1-IFNs enhanced TCR-mediated MAIT cell activation. An essential role of T1-IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co-stimulatory role of T1-IFNs was also evident in liver-derived MAIT cells. T1-IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1-IFNs during TCR-mediated MAIT cell activation.

Fcµ receptor as a Costimulatory Molecule for T Cells.

Fc receptor for IgM (FcµR)-deficient mice display dysregulated function of neutrophils, dendritic cells, and B cells. The relevance of FcµR to human T cells is still unknown. We show that FcµR is mostly stored inside the cell and that surface expression is tightly regulated. Decreased surface expression on T cells from elderly individuals is associated with alterations in the methylation pattern of the FCMR gene. Binding and internalization of IgM stimulate transport of FcµR to the cell surface to ensure sustained IgM uptake. Concurrently, IgM accumulates within the cell, and the surface expression of other receptors increases, among them the T cell receptor (TCR) and costimulatory molecules. This leads to enhanced TCR signaling, proliferation, and cytokine release, in response to low, but not high, doses of antigen. Our findings indicate that FcµR is an important regulator of T cell function and reveal an additional mode of interaction between B and T cells.

Analysis of Thymocyte Migration, Cellular Interactions, and Activation by Multiphoton Fluorescence Microscopy of Live Thymic Slices.

Thymocytes migrate through discrete compartments within the thymus, engaging in cellular interactions essential for their differentiation into functional and self-tolerant T cells. Thus, understanding the temporal and spatial behavior of thymocytes within an intact thymic microenvironment is critical for elucidating processes governing T cell development. Towards this end, we describe methods for preparing thymic explant slices, in which the migration of thymocytes through three-dimensional space can be probed using time-lapse, multiphoton fluorescence microscopy. Thymocytes, enriched for developmental subsets of interest, are labeled with cytoplasmic fluorescent dyes, and seeded onto live thymic slices that express an endogenous, stromal cell-specific fluorescent reporter. In response to chemotactic cues produced by thymic stromal cells, the labeled thymocytes migrate withinthymic microenvironments and engage in cellular interactions that recapitulate a physiological system, whichcan be readily imaged. Here we describe specimen preparation that maintains the integrity of thymic structures. We also describe imaging protocols for acquiring multiple fluorochrome channels to enable detection of thymocyte:stromal cell interactions and quantification of relative intracellular calcium levels to monitor T cell receptor activation. Parameters for quantifying motility and interaction behaviors during data analysis are also briefly described. The thymic slice is a versatile tool for probing live cell behaviors and developing novel hypotheses not readily apparent by static experimental methods.

Defects in CD4+ T cell LFA-1 integrin-dependent adhesion and proliferation protect Cd47-/- mice from EAE.

CD47 is known to play an important role in CD4+ T cell homeostasis. We recently reported a reduction in mice deficient in the Cd47 gene (Cd47-/-) CD4+ T cell adhesion and transendothelial migration (TEM) in vivo and in vitro as a result of impaired expression of high-affinity forms of LFA-1 and VLA-4 integrins. A prior study concluded that Cd47-/- mice were resistant to experimental autoimmune encephalomyelitis (EAE) as a result of complete failure in CD4+ T cell activation after myelin oligodendrocyte glycoprotein peptide 35-55 aa (MOG35-55) immunization. As the prior EAE study was published before our report, authors could not have accounted for defects in T cell integrin function as a mechanism to protect Cd47-/- in EAE. Thus, we hypothesized that failure of T cell activation involved defects in LFA-1 and VLA-4 integrins. We confirmed that Cd47-/- mice were resistant to MOG35-55-induced EAE. Our data, however, supported a different mechanism that was not a result of failure of CD4+ T cell activation. Instead, we found that CD4+ T cells in MOG35-55-immunized Cd47-/- mice were activated, but clonal expansion contracted within 72 h after immunization. We used TCR crosslinking and mitogen activation in vitro to investigate the underlying mechanism. We found that naïve Cd47-/- CD4+ T cells exhibited a premature block in proliferation and survival because of impaired activation of LFA-1, despite effective TCR-induced activation. These results identify CD47 as an important regulator of LFA-1 and VLA-4 integrin-adhesive functions in T cell proliferation, as well as recruitment, and clarify the roles played by CD47 in MOG35-55-induced EAE.

TCR/pMHC Interaction: Phenotypic Model for an Unsolved Enigma.

TCR-pMHC interaction is the keystone of the adaptive immune response. This process exhibits an impressive capacity of speed, sensitivity, and discrimination that allows detecting foreign pMHCs at very low concentration among much more abundant self-pMHC ligands. However, and despite over three decades of intensive research, the mechanisms by which this remarkable discrimination and sensitivity is attained remain controversial. In kinetic proofreading mechanisms (KPR), an increase of specificity occurs by reducing the sensitivity. To overcome this difficulty, more elaborate models including feedback processes or induced rebinding have been incorporated into the KPR scheme. Here a new approach based on the assumption that the proofreading chain behaves differently for foreign- and self-pMHC complexes has been integrated into a phenotypic model in which the complexes responsible for T cell activation stabilize (for foreign peptides) or weaken (for foreign peptides), resulting in a dramatic increase in sensitivity and specificity. Stabilization and destabilization of complexes may be caused by conformational changes, rebinding, or any other process leading to variations in the dissociation rate constants of the complexes transmitting the activation. The numerical solution and the analytical expression for the steady-state response as a function of koff(i) (i = 0, 1, …, N, where C0, C1, …, CN are the complexes in the proofreading chain) are provided. The activation chain speeds up, and larger increases in sensitivity and discrimination are obtained if the rate of activation along the proofreading chain increases for foreign pMHCs and decreases for self-ligands. Experimental implications and comparison with current models are discussed.

Innate immune defects in HIV permissive cell lines.

BACKGROUND: Primary CD4+ T cells and cell lines differ in their permissiveness to HIV infection. Impaired innate immunity may contribute to this different phenotype. FINDINGS: We used transcriptome profiling of 1503 innate immunity genes in primary CD4+ T cells and permissive cell lines. Two clusters of differentially expressed genes were identified: a set of 249 genes that were highly expressed in primary cells and minimally expressed in cell lines and a set of 110 genes with the opposite pattern. Specific to HIV, HEK293T, Jurkat, SupT1 and CEM cell lines displayed unique patterns of downregulation of genes involved in viral sensing and restriction. Activation of primary CD4+ T cells resulted in reversal of the pattern of expression of those sets of innate immunity genes. Functional analysis of prototypical innate immunity pathways of permissive cell lines confirmed impaired responses identified in transcriptome analyses. CONCLUSION: Integrity of innate immunity genes and pathways needs to be considered in designing gain/loss functional genomic screens of viral infection.

Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming.

Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures.