The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Antigen-specific memory CD4+ T cells can persist and confer rapid and efficient protection from microbial reinfection. However, the mechanisms underlying the long-term maintenance of the memory CD4+ T cell pool remain largely unknown. Here, using a mouse model of acute infection with lymphocytic choriomeningitis virus (LCMV), we found that the serine/threonine kinase complex mammalian target of rapamycin complex 2 (mTORC2) is critical for the long-term persistence of virus-specific memory CD4+ T cells. The perturbation of mTORC2 signaling at memory phase led to an enormous loss of virus-specific memory CD4+ T cells by a unique form of regulated cell death (RCD), ferroptosis. Mechanistically, mTORC2 inactivation resulted in the impaired phosphorylation of downstream AKT and GSK3ß kinases, which induced aberrant mitochondrial reactive oxygen species (ROS) accumulation and ensuing ferroptosis-causative lipid peroxidation in virus-specific memory CD4+ T cells; furthermore, the disruption of this signaling cascade also inhibited glutathione peroxidase 4 (GPX4), a major scavenger of lipid peroxidation. Thus, the mTORC2-AKT-GSK3ß axis functions as a key signaling hub to promote the longevity of virus-specific memory CD4+ T cells by preventing ferroptosis.

Transcription factor EB coordinates environmental cues to regulate T regulatory cells' mitochondrial fitness and function.

T regulatory (Treg) cells are essential for self-tolerance whereas they are detrimental for dampening the host anti-tumor immunity. How Treg cells adapt to environmental signals to orchestrate their homeostasis and functions remains poorly understood. Here, we identified that transcription factor EB (TFEB) is induced by host nutrition deprivation or interleukin (IL)-2 in CD4+ T cells. The loss of TFEB in Treg cells leads to reduced Treg accumulation and impaired Treg function in mouse models of cancer and autoimmune disease. TFEB intrinsically regulates genes involved in Treg cell differentiation and mitochondria function while it suppresses expression of proinflammatory cytokines independently of its established roles in autophagy. This coordinated action is required for mitochondria integrity and appropriate lipid metabolism in Treg cells. These findings identify TFEB as a critical regulator for orchestrating Treg generation and function, which may contribute to the adaptive responses of T cells to local environmental cues.

Death-associated protein kinase 1 (DAPK1) controls CD8+ T cell activation, trafficking, and antitumor activity.

Appropriate migration of cytotoxic T effector cells into the tumors is crucial for their antitumor function. Despite the controversial role of PI3K-Akt in CD8+ T cell mTORC1 activation, a link between Akt-mTORC1 signaling and CD8+ trafficking has been demonstrated. We have recently discovered that TCR-induced calcineurin activates DAPK1, which interacts with TSC2 via its death domain and phosphorylates TSC2 via its kinase domain to mediate mTORC1 activation in CD8+ T cells. However, whether DAPK1 regulates CD8+ trafficking into tumors remains unclear. Here, using pharmacological inhibitor and genetic approaches, we found that like rapamycin, inhibition of DAPK1 activity led to enhanced expression of the homing receptors CD62L and CCR7. Deletion of either kinase domain or death domain in the T cell compartment reduced the T cell activation and maintained the expression of CD62L and CCR7. DAPK1-DD-deficient mice were more susceptible to tumor growth and deficiency of DAPK1 activity significantly reduced the migratory ability of CD8+ into the tumors. These data revealed a crucial role of DAPK1-mTORC1 in mediating CD8+ trafficking and antitumor function.

Alpha-lactose reverses liver injury via blockade of Tim-3-mediated CD8 apoptosis in sepsis.

In sepsis, the liver plays a crucial role in regulating immune responses and is also a target organ for immune-related injury. Despite the critical function of CD8+ T cells against opportunistic viral infections, the CD8 immune response in the liver during sepsis remains elusive. Here we found that Tim-3 is highly up-regulated in liver CD8+ T cells in a mouse cecal ligation and puncture model and in peripheral blood CD8+ T cells of human patients with sepsis. The expression of Tim-3 in liver CD8+ T cells displayed a bi-phasic pattern and deletion of Tim-3 led to reduction of CD8+ T cell apoptosis. Administration of α-lactose, a molecule with a similar structure to galactin-9, reduced Tim-3 expression and liver injury in sepsis. Our results demonstrate that targeting Tim-3 to boost CD8+ T cell immune response may offer an improved outcome in patients with sepsis.

Transcriptional Regulation of T Cell Metabolism Reprograming.

T cell activation, differentiation, and function are tightly regulated by a complex network of transcription factors, epigenetic modifications, and signaling pathways of both TCR and cytokines. Over the past decade, it is increasingly clear that T cell immune responses are also regulated by their associated metabolic reprograming. Compared with relatively well-understood transcriptional regulation of T cell activation, differentiation, and function, less is known about the transcriptional regulation of T cell metabolic reprograming during T cell immune responses. In this review, we first describe how signaling pathways of TCR and cytokines regulate metabolic reprograming and then focus on transcription factors that control metabolic pathways and outcomes of T cell immune responses. A better understanding of T cell metabolic regulation will provide new strategies and targets for the treatment of T cell-related diseases.

EZH2 restricts Tcf7 DNA methylation and promotes TFH differentiation during acute viral infection.

Follicular helper T (TFH) cells provide specialized help for B cells to ensure optimal humoral immunity. The histone methyltransferase EZH2, as a chromatin repressor, secures the TFH differentiation by promoting TFH lineage associated gene expression during acute viral infection, including Tcf7 and Bcl6. By using conditional deletion murine system, we observed that EZH2 ablation in CD4+ T cells was accompanied by aberrant accumulation of DNA methyltransferases (DNMTs) DNMT1 and DNMT3B in TFH cells. And the loss of EZH2 promoted aggravation of DNA methylation status at Tcf7 locus. Therefore, our findings suggested that EZH2 plays an important role in maintenance of hypomethylation at Tcf7 locus thus affecting TFH differentiation during acute viral infection.

CD4+ T-cell epitope-based heterologous prime-boost vaccination potentiates anti-tumor immunity and PD-1/PD-L1 immunotherapy.

BACKGROUND: Antitumor therapeutic vaccines are generally based on antigenic epitopes presented by major histocompatibility complex (MHC-I) molecules to induce tumor-specific CD8+ T cells. Paradoxically, continuous T cell receptor (TCR) stimulation from tumor-derived CD8+ T-cell epitopes can drive the functional exhaustion of tumor-specific CD8+ T cells. Tumor-specific type-I helper CD4+ T (TH1) cells play an important role in the population maintenance and cytotoxic function of exhausted tumor-specific CD8+ T cells in the tumor microenvironment. Nonetheless, whether the vaccination strategy targeting MHC-II-restricted CD4+ T-cell epitopes to induce tumor-specific TH1 responses can confer effective antitumor immunity to restrain tumor growth is not well studied. Here, we developed a heterologous prime-boost vaccination strategy to effectively induce tumor-specific TH1 cells and evaluated its antitumor efficacy and its capacity to potentiate PD-1/PD-L1 immunotherapy. METHODS: Listeria monocytogenes vector and influenza A virus (PR8 strain) vector stably expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein-specific I-Ab-restricted CD4+ T cell epitope (GP61-80) or ovalbumin-specific CD4+ T cell epitope (OVA323-339) were constructed and evaluated their efficacy against mouse models of melanoma and colorectal adenocarcinoma expressing lymphocytic choriomeningitis virus glycoprotein and ovalbumin. The impact of CD4+ T cell epitope-based heterologous prime-boost vaccination was detected by flow-cytometer, single-cell RNA sequencing and single-cell TCR sequencing. RESULTS: CD4+ T cell epitope-based heterologous prime-boost vaccination efficiently suppressed both mouse melanoma and colorectal adenocarcinoma. This vaccination primarily induced tumor-specific TH1 response, which in turn enhanced the expansion, effector function and clonal breadth of tumor-specific CD8+ T cells. Furthermore, this vaccination strategy synergized PD-L1 blockade mediated tumor suppression. Notably, prime-boost vaccination extended the duration of PD-L1 blockade induced antitumor effects by preventing the re-exhaustion of tumor-specific CD8+ T cells. CONCLUSION: CD4+ T cell epitope-based heterologous prime-boost vaccination elicited potent both tumor-specific TH1 and CTL response, leading to the efficient tumor control. This strategy can also potentiate PD-1/PD-L1 immune checkpoint blockade (ICB) against cancer.

ATP6V0d2 Suppresses Alveoli Macrophage Alternative Polarization and Allergic Asthma via Degradation of PU.1.

PURPOSE: Macrophages are important regulators of environmental allergen-induced airway inflammation and asthma. ATP6V0d2 is a subunit of vacuolar ATPase highly expressed in macrophages. However, the functions of ATP6V0d2 in the regulation of pathogenesis of allergic asthma remain unclear. The aim of this study is to determine the function and related molecular mechanisms of macrophage protein ATP6V0d2 in allergic asthma. METHODS: We compared the disease severity between female C57BL/6 wild-type and ATP6V0d2-/- mice in an ovalbumin (OVA)-induced asthma model. We also investigated the association of expression of ATP6V0d2, PU.1 and CCL17 with disease severity among asthmatic patients. RESULTS: The expression of ATP6V0d2 in sputum cells of asthmatic patients and in the lungs of OVA-challenged mice was enhanced compared to healthy subjects and their counterparts, respectively. However, ATP6V0d2-deficient mice exaggerated inflammatory cell infiltration as well as enhanced alternative activated macrophage (AAM) polarization and mucus production in an OVA-induced asthma model. Furthermore, we found that Atp6v0d2 promoted lysosomal degradation of Pu.1, which induced AAM polarization and Ccl17 production. Among asthma patients, ATP6V0d2 expression was inversely associated with disease severity, whereas PU.1 and CCL17 expression was positively associated with disease severity. CONCLUSIONS: Our results identify macrophage Atp6v0d2, as an induced feedback inhibitor of asthma disease severity by promoting Pu.1 lysosomal degradation, which may in turn leads to reduced AAM polarization and Ccl17 production.

DAPK1 (death associated protein kinase 1) mediates mTORC1 activation and antiviral activities in CD8+ T cells.

Mechanistic target of rapamycin complex 1 (mTORC1) regulates CD8+ T-cell differentiation and function. Despite the links between PI3K-AKT and mTORC1 activation in CD8+ T cells, the molecular mechanism underlying mTORC1 activation remains unclear. Here, we show that both the kinase activity and the death domain of DAPK1 are required for maximal mTOR activation and CD8+ T-cell function. We found that TCR-induced activation of calcineurin activates DAPK1, which subsequently interacts with TSC2 via its death domain and phosphorylates TSC2 to mediate mTORC1 activation. Furthermore, both the kinase domain and death domain of DAPK1 are required for CD8+ T-cell antiviral responses in an LCMV infection model. Together, our data reveal a novel mechanism of mTORC1 activation that mediates optimal CD8+ T-cell function and antiviral activity.

Regulation of T cell differentiation and function by epigenetic modification enzymes.

Peripheral naive CD4+ and CD8+ cells are developed in the thymus and proliferate and differentiate into various specialized T cell subsets upon activation by peptide-major histocompatibility complexes in periphery to execute different functions during immune responses. Cytokines, transcription factors, and a large number of intracellular molecules have been shown to affect T cell development, activation, and function. In addition, epigenetic modifications, such as histone modification and DNA methylation, regulate T cell biology. The epigenetic modifications are regulated by a range of DNA methyltransferases, DNA demethylation enzymes, and histone modification enzymes. Dysregulations of epigenetic modifications are closely associated with autoimmune diseases and tumorigenesis. Here, we review the current literature about the functions of DNA and histone modification enzymes in T cell development, activation, differentiation, and function.

TFEB Mediates Immune Evasion and Resistance to mTOR Inhibition of Renal Cell Carcinoma via Induction of PD-L1.

PURPOSE: Despite the FDA approval of mTOR inhibitors (mTORi) for the treatment of renal cell carcinoma (RCC), the benefits are relatively modest and the few responders usually develop resistance. We investigated whether the resistance to mTORi is due to upregulation of PD-L1 and the underlying molecular mechanism. EXPERIMENTAL DESIGN: The effects of transcription factor EB (TFEB) on RCC proliferation, apoptosis, and migration were evaluated. Correlation of TFEB with PD-L1 expression, as well as effects of mTOR inhibition on TFEB and PD-L1 expression, was assessed in human primary clear cell RCCs. The regulation of TFEB on PD-L1 was assessed by chromatin immunoprecipitation and luciferase reporter assay. The therapeutic efficacies of mTORi plus PD-L1 blockade were evaluated in a mouse model. The function of tumor-infiltrating CD8+ T cells was analyzed by flow cytometry. RESULTS: TFEB did not affect tumor cell proliferation, apoptosis, and migration. We found a positive correlation between TFEB and PD-L1 expression in RCC tumor tissues, primary tumor cells, and RCC cells. TFEB bound to PD-L1 promoter in RCCs and inhibition of mTOR led to enhanced TFEB nuclear translocation and PD-L1 expression. Simultaneous inhibition of mTOR and blockade of PD-L1 enhanced CD8+ cytolytic function and tumor suppression in a xenografted mouse model of RCC. CONCLUSIONS: These data revealed that TFEB mediates resistance to mTOR inhibition via induction of PD-L1 in human primary RCC tumors, RCC cells, and murine xenograft model. Our data provide a strong rationale to target mTOR and PD-L1 jointly as a novel immunotherapeutic approach for RCC treatment.

Lactate inhibits ATP6V0d2 expression in tumor-associated macrophages to promote HIF-2α-mediated tumor progression.

Macrophages perform key functions in tissue homeostasis that are influenced by the local tissue environment. Within the tumor microenvironment, tumor-associated macrophages can be altered to acquire properties that enhance tumor growth. Here, we found that lactate, a metabolite found in high concentration within the anaerobic tumor environment, activated mTORC1 that subsequently suppressed TFEB-mediated expression of the macrophage-specific vacuolar ATPase subunit ATP6V0d2. Atp6v0d2-/- mice were more susceptible to tumor growth, with enhanced HIF-2α-mediated VEGF production in macrophages that display a more protumoral phenotype. We found that ATP6V0d2 targeted HIF-2α but not HIF-1α for lysosome-mediated degradation. Blockade of HIF-2α transcriptional activity reversed the susceptibility of Atp6v0d2-/- mice to tumor development. Furthermore, in a cohort of patients with lung adenocarcinoma, expression of ATP6V0d2 and HIF-2α was positively and negatively correlated with survival, respectively, suggesting a critical role of the macrophage lactate/ATP6V0d2/HIF-2α axis in maintaining tumor growth in human patients. Together, our results highlight the ability of tumor cells to modify the function of tumor-infiltrating macrophages to optimize the microenvironment for tumor growth.

IL-2 Inhibition of Th17 Generation Rather Than Induction of Treg Cells Is Impaired in Primary Sjögren's Syndrome Patients.

Objective: To investigate the role of IL-2 in the balance of Th17 and Tregs and elucidate the underlying mechanisms of enhanced Th17 differentiation in primary Sjögren's syndrome (pSS) patients. Methods: This study involved 31 pSS patients, 7 Sicca patients, and 31 healthy subjects. Th17 and Treg cells were determined by flow cytometry, and IL-17A was detected by immunohistochemistry. IL-2 and IL-6 levels were assessed by ELISA and qPCR. p-STAT5 and p-STAT3 in salivary glands (SGs) were evaluated by immunohistochemistry and flow cytometry. The binding of STAT5 and STAT3 to the Il17a gene locus was measured by chromatin immunoprecipitation. Results: We found that the percentage of Th17 cells was increased in the periphery and SG of pSS patients when compared with healthy subjects, but the Treg cells was unchanged. Meanwhile, the IL-2 level was reduced, and the IL-6 and IL-17A level was increased in the plasma of pSS patients. The ratio of IL-2 and IL-6 level was also decreased and IL-2 level was negatively correlated with the level of IL-17A. The expression of Il6 and Il17a mRNA was significantly increased, whereas Foxp3, Tgfb1, Tnfa, and Ifng mRNA were comparable. Furthermore, the level of STAT5 phosphorylation (p-STAT5) was reduced and p-STAT3 was enhanced in the SGs and in peripheral CD4+ T cells of pSS patients. In vitro IL-2 treatment-induced STAT5 competed with STAT3 binding in human Il17a locus, leading to decreased Th17 differentiation, which was associated with the reduced transcription activation marker H3K4me3. Conclusion: Our findings demonstrated a Treg-independent upregulation of Th17 generation in pSS, which is likely due to a lack of IL-2-mediated suppression of Th17 differentiation. This study identified a novel mechanism of IL-2-mediated immune suppression in pSS.

α-Lactose Improves the Survival of Septic Mice by Blockade of TIM-3 Signaling to Prevent NKT Cell Apoptosis and Attenuate Cytokine Storm.

Sepsis is the leading cause of death among critically ill patients and natural killer T (NKT) cell activation is essential to induce inflammatory cytokine cascade in sepsis. However, little is known about what regulates the NKT cell function during sepsis. Herein, we showed that T-cell immunoglobulin and mucin domain 3 (Tim-3) expression in NKT cells is elevated in experimental mice during sepsis. Tim-3 expression was positively correlated with NKT cell activation and apoptosis. In sepsis, interleukin (IL)-12 secreted by dendritic cell exposure to lipopolysaccharide increased the expression of Tim-3 in NKT cells. Administration of α-lactose to block Tim-3 signaling pathway significantly improved the survival of septic mice, concomitant with reduced IL-12 production by dendritic cells, reduced Tim-3 expression, prevented NKT cell apoptosis, and attenuated production of inflammatory cytokines. Collectively, Tim-3 signaling in NKT cells plays a critical role in the immunopathogenesis of sepsis. Thus, α-lactose could be a promising immunomodulatory agent in the treatment of sepsis.