Tsyn-Seq: a T-cell Synapse-Based Antigen Identification Platform.

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

PD-1+ CD4 T cell immune response is mediated by HIF-1α/NFATc1 pathway after P. yoelii infection.

The morbidity and mortality of malaria are still high. Programmed cell death-1(PD-1) is an important co-inhibitory factor and CD8 T cells with PD-1 were reported to be exhausted cells. It remains unknown what the role of CD4 T cells expressing PD-1 is and what the upstream regulating molecules of PD-1 in CD4 T cells are. The C57BL/6 mice were injected with Plasmodium yoelii (P. yoelii) in this study. Expressions of PD-1, activation markers, and cytokines were tested. The differentially expressed genes between PD-1+/- CD4 T cells were detected by microarray sequencing. Western blot, chromatin immunoprecipitation (ChIP), siRNA, hypoxia inducible factor-1α (HIF-1α) inducer and inhibitor were used to explore PD-1's upstream molecules, respectively. The proportions of PD-1+ CD4 T cells increased post P. yoelii infection. PD-1+ CD4 T cells expressed more activated surface markers and could produce more cytokines. Nuclear factor of activated T cells 1 (NFATc1) was found to be a key transcription factor to induce PD-1 expression after infection. Both the inducer and the inhibitor of HIF-1α could change the expressions of NFATc1 and PD-1 in vivo and in vitro, respectively. Taken together, P. yoelii infection induced NFATc1 expression by HIF-1α. The highly expressed NFATc1 entered the nucleus and initiated PD-1 expression. PD-1+ CD4 T cells appeared to be more activated and could secrete more cytokines to regulate the host's immune responses against malaria.

Intestinal Microbiome Inhibits CD8+ T Cells via the Calcineurin-NFAT Axis.

Bacterial sensing activates the calcineurin-NFAT axis, inhibiting CD8+ T cells in colon cancer.

NFAT-dependent and -independent exhaustion circuits program maternal CD8 T cell hypofunction in pregnancy.

Pregnancy is a common immunization event, but the molecular mechanisms and immunological consequences provoked by pregnancy remain largely unknown. We used mouse models and human transplant registry data to reveal that pregnancy induced exhausted CD8 T cells (Preg-TEX), which associated with prolonged allograft survival. Maternal CD8 T cells shared features of exhaustion with CD8 T cells from cancer and chronic infection, including transcriptional down-regulation of ribosomal proteins and up-regulation of TOX and inhibitory receptors. Similar to other models of T cell exhaustion, NFAT-dependent elements of the exhaustion program were induced by fetal antigen in pregnancy, whereas NFAT-independent elements did not require fetal antigen. Despite using conserved molecular circuitry, Preg-TEX cells differed from TEX cells in chronic viral infection with respect to magnitude and dependency of T cell hypofunction on NFAT-independent signals. Altogether, these data reveal the molecular mechanisms and clinical consequences of maternal CD8 T cell hypofunction and identify pregnancy as a previously unappreciated context in which T cell exhaustion may occur.

Generation of an NFκB-Driven Alpharetroviral "All-in-One" Vector Construct as a Potent Tool for CAR NK Cell Therapy.

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen-so-called "T cells redirected for universal cytokine-mediated killing" (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2+ target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies.

Immunosuppression Affects Neutrophil Functions: Does Calcineurin-NFAT Signaling Matter?

Neutrophils are innate immune cells with important roles in antimicrobial defense. However, impaired or dysregulated neutrophil function can result in host tissue damage, loss of homeostasis, hyperinflammation or pathological immunosuppression. A central link between neutrophil activation and immune outcomes is emerging to be the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, which is activated by neutrophil detection of a microbial threat via pattern recognition receptors and results in inflammatory cytokine production. This potent pro-inflammatory pathway is also the target of several immunosuppressive drugs used for the treatment of autoimmune disorders, during solid organ and hematopoietic cell transplantations, and as a part of anti-cancer therapy: but what effects these drugs have on neutrophil function, and their broader consequences for immune homeostasis and microbial defense are not yet known. Here, we bring together the emerging literature describing pathology- and drug- induced neutrophil impairment, with particular focus on their effects on calcineurin-NFAT signaling in the innate immune compartment.

The role of NFAT2/miR-20a-5p signaling pathway in the regulation of CD8+ naïve T cells activation and differentiation.

T cell dysfunction is a common characteristic in leukemia patients that significantly impacts clinical treatment and prognosis. However, the mechanism underlying T cell dysfunction and its reversal remains unclear. In this study, in accordance with our previous findings, we found that the expression of NFAT2 and pri-miR-17 ~ 92 are lower in peripheral blood CD3+ T cells from chronic myelogenous leukemia (CML) patients by gene expression analysis. We further demonstrate that the NFAT2-induced activation, differentiation, and expression of cytokines in human umbilical cord blood CD8+ naïve T cells are miR-20a-5p dependent. We also preliminarily explored the relationship between NFAT2 and miR-20a-5p in naive T cells. These results suggest that NFAT2 and miR-20a are crucial for regulating functional CD8+ T cells. Additionally, their alteration may be related to CD8+ T cell dysfunction in CML patients; thus, NFAT2 and miR-20a-5p may be considered potential targets for revising T cell function in leukemia immunotherapy.

Propofol maintains Th17/Treg cell balance and reduces inflammation in rats with traumatic brain injury via the miR­145­3p/NFATc2/NF­κB axis.

Propofol is a commonly used intravenous anesthetic. The aim of the study was to examine the mechanism of propofol in traumatic brain injury (TBI) by regulating interleukin (IL)­17 activity and maintaining the Th17/Treg balance. A rat model with moderate TBI was established using the weight­drop method. Rats with TBI were regularly injected with propofol and their brain injuries were monitored. The peripheral blood of rats was collected to measure the Th17/Treg ratio. MicroRNA (miR)­145­3p expression was detected in the brain tissues of rats and antagomiR­145­3p was injected into the lateral ventricles of their brains to verify the effect of miR­145­3p on brain injury. The downstream target of miR­145­3p was predicted. The targeting relationship between miR­145­3p and nuclear factor of activated T cells c2 (NFATc2) was confirmed. NFATC2 expression and phosphorylation of NF­κB pathway­related proteins were measured. Propofol alleviated brain injury in rats with TBI and maintained the Th17/Treg balance. Propofol upregulated miR­145­3p expression in rat brains, while the inhibition of miR­145­3p reversed the effect of propofol on brain injury. A binding relationship was observed between miR­145­3p and NFATc2. Furthermore, propofol decreased the phosphorylation of p65 and IκBα, and inhibited activation of the NF­κB pathway in the brains of rats with TBI. In conclusion, propofol maintained Th17/Treg balance and reduced inflammation in the rats with TBI via the miR­145­3p/NFATc2/NF­κB axis.

Isoform-Selective NFAT Inhibitor: Potential Usefulness and Development.

Nuclear factor of activated T cells (NFAT), which is the pharmacological target of immunosuppressants cyclosporine and tacrolimus, has been shown to play an important role not only in T cells (immune system), from which their name is derived, but also in many biological events. Therefore, functional and/or structural abnormalities of NFAT are linked to the pathogenesis of diseases in various organs. The NFAT protein family consists of five isoforms, and each isoform performs diverse functions and has unique expression patterns in the target tissues. This diversity has made it difficult to obtain ideal pharmacological output for immunosuppressants that inhibit the activity of almost all NFAT family members, causing serious and wide-ranging side effects. Moreover, it remains unclear whether isoform-selective NFAT regulation can be achieved by targeting the structural differences among NFAT isoforms and whether this strategy can lead to the development of better drugs than the existing ones. This review summarizes the role of the NFAT family members in biological events, including the development of various diseases, as well as the usefulness of and problems associated with NFAT-targeting therapies, including those dependent on current immunosuppressants. Finally, we propose a novel therapeutic strategy based on the molecular mechanisms that enable selective regulation of specific NFAT isoforms.

Calcineurin inhibitors suppress acute graft-versus-host disease via NFAT-independent inhibition of T cell receptor signaling.

Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and acute graft-versus-host disease (aGVHD), for which inhibition of gene expression dependent on nuclear factor of activated T cells (NFAT) is the mechanistic paradigm. We recently reported that CNIs inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of LckS59, an inhibitory modification, raising the possibility of another mechanism by which CNIs suppress immune responses. Here we used T cells from mice that express LckS59A, which cannot accept a phosphate at residue 59, to initiate aGVHD. Although CsA inhibited NFAT-dependent gene upregulation in allo-aggressive T cells expressing either LckWT or LckS59A, it was ineffective in treating disease when the T cells expressed LckS59A. Two important NFAT-independent T cell functions were found to be CsA-resistant in LckS59A T cells: upregulation of the cytolytic protein perforin in tissue-infiltrating CD8+ T cells and antigen-specific T/DC adhesion and clustering in lymph nodes. These results demonstrate that effective treatment of aGVHD by CsA requires NFAT-independent inhibition of TCR signaling. Given that NFATs are widely expressed and off-target effects are a major limitation in CNI use, it is possible that targeting TCR-associated calcineurin directly may provide effective therapies with less toxicity.

DNA hypomethylating agents increase activation and cytolytic activity of CD8+ T cells.

We demonstrate that DNA hypomethylating agent (HMA) treatment can directly modulate the anti-tumor response and effector function of CD8+ T cells. In vivo HMA treatment promotes CD8+ T cell tumor infiltration and suppresses tumor growth via CD8+ T cell-dependent activity. Ex vivo, HMAs enhance primary human CD8+ T cell activation markers, effector cytokine production, and anti-tumor cytolytic activity. Epigenomic and transcriptomic profiling shows that HMAs vastly regulate T cell activation-related transcriptional networks, culminating with over-activation of NFATc1 short isoforms. Mechanistically, demethylation of an intragenic CpG island immediately downstream to the 3' UTR of the short isoform was associated with antisense transcription and alternative polyadenylation of NFATc1 short isoforms. High-dimensional single-cell mass cytometry analyses reveal a selective effect of HMAs on a subset of human CD8+ T cell subpopulations, increasing both the number and abundance of a granzyme Bhigh, perforinhigh effector subpopulation. Overall, our findings support the use of HMAs as a therapeutic strategy to boost anti-tumor immune response.

NFATc1/αA and Blimp-1 Support the Follicular and Effector Phenotype of Tregs.

CD4+CXCR5+Foxp3+ T-follicular regulatory (TFR) cells control the germinal center responses. Like T-follicular helper cells, they express high levels of Nuclear Factor of Activated T-cells c1, predominantly its short isoform NFATc1/αA. Ablation of NFATc1 in Tregs prevents upregulation of CXCR5 and migration of TFR cells into B-cell follicles. By contrast, constitutive active NFATc1/αA defines the surface density of CXCR5, whose level determines how deep a TFR migrates into the GC and how effectively it controls antibody production. As one type of effector Treg, TFR cells express B lymphocyte-induced maturation protein-1 (Blimp-1). Blimp-1 can directly repress Cxcr5 and NFATc1/αA is necessary to overcome this Blimp-1-mediated repression. Interestingly, Blimp-1 even reinforces the recruitment of NFATc1 to Cxcr5 by protein-protein interaction and by those means cooperates with NFATc1 for Cxcr5 transactivation. On the contrary, Blimp-1 is necessary to counterbalance NFATc1/αA and preserve the Treg identity. This is because although NFATc1/αA strengthens the follicular development of Tregs, it bears the inherent risk of causing an ex-Treg phenotype.

Lack of NFATc1 SUMOylation prevents autoimmunity and alloreactivity.

Posttranslational modification with SUMO is known to regulate the activity of transcription factors, but how SUMOylation of individual proteins might influence immunity is largely unexplored. The NFAT transcription factors play an essential role in antigen receptor-mediated gene regulation. SUMOylation of NFATc1 represses IL-2 in vitro, but its role in T cell-mediated immune responses in vivo is unclear. To this end, we generated a novel transgenic mouse in which SUMO modification of NFATc1 is prevented. Avoidance of NFATc1 SUMOylation ameliorated experimental autoimmune encephalomyelitis as well as graft-versus-host disease. Elevated IL-2 production in T cells promoted T reg expansion and suppressed autoreactive or alloreactive immune responses. Mechanistically, increased IL-2 secretion counteracted IL-17 and IFN-γ expression through STAT5 and Blimp-1 induction. Then, Blimp-1 repressed IL-2 itself, as well as the induced, proliferation-associated survival factor Bcl2A1. Collectively, these data demonstrate that prevention of NFATc1 SUMOylation fine-tunes T cell responses toward lasting tolerance. Thus, targeting NFATc1 SUMOylation presents a novel and promising strategy to treat T cell-mediated inflammatory diseases.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

All-trans-retinoic acid shifts Th1 towards Th2 cell differentiation by targeting NFAT1 signalling to ameliorate immune-mediated aplastic anaemia.

Severe acquired aplastic anaemia (AA) is a serious disease characterised by autoreactive T cells attacking haematopoietic stem cells, leading to marrow hypoplasia and pancytopenia. Immunosuppressive therapy combined with antithymocyte globulin and ciclosporin can rescue most patients with AA. However, the relapse after ciclosporin withdrawal and the severe side effects of long-term ciclosporin administration remain unresolved. As such, new strategies should be developed to supplement current therapeutics and treat AA. In this study, the possibility of all-trans-retinoic acid (ATRA) as an alternative AA treatment was tested by using an immune-mediated mouse model of AA. Results revealed that ATRA inhibited T-cell proliferation, activation and effector function. It also restrained the Fas/Fasl pathway, shifted Th1 towards Th2 cell development, rebalanced T-cell subsets at a relatively high level and corrected the Th1/Th2 ratio by targeting NFAT1 signalling. In addition, ATRA inhibited Th17 cell differentiation and promoted regulatory T-cell development. Therefore, ATRA was an effective agent to improve AA treatment outcomes.

Mechanosensing through YAP controls T cell activation and metabolism.

Upon immunogenic challenge, lymph nodes become mechanically stiff as immune cells activate and proliferate within their encapsulated environments, and with resolution, they reestablish a soft baseline state. Here we show that sensing these mechanical changes in the microenvironment requires the mechanosensor YAP. YAP is induced upon activation and suppresses metabolic reprogramming of effector T cells. Unlike in other cell types in which YAP promotes proliferation, YAP in T cells suppresses proliferation in a stiffness-dependent manner by directly restricting the translocation of NFAT1 into the nucleus. YAP slows T cell responses in systemic viral infections and retards effector T cells in autoimmune diabetes. Our work reveals a paradigm whereby tissue mechanics fine-tune adaptive immune responses in health and disease.

Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune CTL Functions.

Adenomatous polyposis coli (Apc) is a cell polarity regulator and a tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Apc involvement in T lymphocyte functions and antitumor immunity remains poorly understood. Investigating Apc-depleted human CD8 T cells and CD8 T cells from ApcMin/+ mutant mice, we found that Apc regulates actin and microtubule cytoskeleton remodeling at the immunological synapse, controlling synapse morphology and stability and lytic granule dynamics, including targeting and fusion at the synapse. Ultimately, Apc tunes cytotoxic T cell activity, leading to tumor cell killing. Furthermore, Apc modulates early TCR signaling and nuclear translocation of the NFAT transcription factor with mild consequences on the expression of some differentiation markers. In contrast, no differences in the production of effector cytokines were observed. These results, together with our previous findings on Apc function in regulatory T cells, indicate that Apc mutations may cause a dual damage, first unbalancing epithelial cell differentiation and growth driving epithelial neoplasms and, second, impairing T cell-mediated antitumor immunity at several levels.

BCR-Induced Ca2+ Signals Dynamically Tune Survival, Metabolic Reprogramming, and Proliferation of Naive B Cells.

B cell receptor (BCR) engagement induces naive B cells to differentiate and perform critical immune-regulatory functions. Acquisition of functional specificity requires that a cell survive, enter the cell cycle, and proliferate. We establish that quantitatively distinct Ca2+ signals triggered by variations in the extent of BCR engagement dynamically regulate these transitions by controlling nuclear factor κB (NF-κB), NFAT, and mTORC1 activity. Weak BCR engagement induces apoptosis by failing to activate NF-κB-driven anti-apoptotic gene expression. Stronger signals that trigger more robust Ca2+ signals promote NF-κB-dependent survival and NFAT-, mTORC1-, and c-Myc-dependent cell-cycle entry and proliferation. Finally, we establish that CD40 or TLR9 costimulation circumvents these Ca2+-regulated checkpoints of B cell activation and proliferation. As altered BCR signaling is linked to autoimmunity and B cell malignancies, these results have important implications for understanding the pathogenesis of aberrant B cell activation and differentiation and therapeutic approaches to target these responses.

NFAT1 Regulates Ly6Chi Monocyte Recruitment to the CNS and Plays an Essential Role in Resistance to Toxoplasma gondii Infection.

Monocytes play key roles in the maintenance of homeostasis and in the control of the infection. Monocytes are recruited from the bone marrow to inflammatory sites and are essential for antimicrobial activity to limit tissue damage and promote adaptive T cell responses. Here, we investigated the role of Nuclear Factor of Activated T cells 1 (NFAT1) in the regulation of Ly6Chi inflammatory monocyte recruitment to the CNS upon T. gondii infection. We show that NFAT-1-deficient monocytes are unable to migrate to the CNS of T. gondii-infected mice. Moreover, NFAT1-/- mice are highly susceptible to chronic T. gondii infection due to a failure to control parasite replication in the CNS. The inhibition of Ly6Chi inflammatory monocyte recruitment to the CNS severely blocked CXCL10 production and consequently the migration of IFN-γ-producing CD4+ T cells. Moreover, the transfer of Ly6Chi monocytes to infected NFAT1-/- mice favored CD4+ T cell migration to the CNS and resulted in the inhibition of parasite replication and host defense. Together, these results demonstrated for the first time the contribution of NFAT1 to the regulation of Ly6Chi monocyte recruitment to the CNS and to resistance during chronic T. gondii infection.

Fas signaling-mediated TH9 cell differentiation favors bowel inflammation and antitumor functions.

Fas induces apoptosis in activated T cell to maintain immune homeostasis, but the effects of non-apoptotic Fas signaling on T cells remain unclear. Here we show that Fas promotes TH9 cell differentiation by activating NF-κB via Ca2+-dependent PKC-ß activation. In addition, PKC-ß also phosphorylates p38 to inactivate NFAT1 and reduce NFAT1-NF-κB synergy to promote the Fas-induced TH9 transcription program. Fas ligation exacerbates inflammatory bowel disease by increasing TH9 cell differentiation, and promotes antitumor activity in p38 inhibitor-treated TH9 cells. Furthermore, low-dose p38 inhibitor suppresses tumor growth without inducing systemic adverse effects. In patients with tumor, relatively high TH9 cell numbers are associated with good prognosis. Our study thus implicates Fas in CD4+ T cells as a target for inflammatory bowel disease therapy. Furthermore, simultaneous Fas ligation and low-dose p38 inhibition may be an effective approach for TH9 cell induction and cancer therapy.