L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity.

Metabolic activity is intimately linked to T cell fate and function. Using high-resolution mass spectrometry, we generated dynamic metabolome and proteome profiles of human primary naive T cells following activation. We discovered critical changes in the arginine metabolism that led to a drop in intracellular L-arginine concentration. Elevating L-arginine levels induced global metabolic changes including a shift from glycolysis to oxidative phosphorylation in activated T cells and promoted the generation of central memory-like cells endowed with higher survival capacity and, in a mouse model, anti-tumor activity. Proteome-wide probing of structural alterations, validated by the analysis of knockout T cell clones, identified three transcriptional regulators (BAZ1B, PSIP1, and TSN) that sensed L-arginine levels and promoted T cell survival. Thus, intracellular L-arginine concentrations directly impact the metabolic fitness and survival capacity of T cells that are crucial for anti-tumor responses.

IL-27 improves adoptive CD8+ T cells' antitumor activity via enhancing cell survival and memory T cell differentiation.

IL-27 is an anti-inflammatory cytokine that triggers enhanced antitumor immunity, particularly cytotoxic T lymphocyte responses. In the present study, we sought to develop IL-27 into a therapeutic adjutant for adoptive T cell therapy using our well-established models. We have found that IL-27 directly improved the survival status and cytotoxicity of adoptive OT-1 CD8+ T cells in vitro and in vivo. Meanwhile, IL-27 treatment programs memory T cell differentiation in CD8+ T cells, characterized by upregulation of genes associated with T cell memory differentiation (T-bet, Eomes, Blimp1, and Ly6C). Additionally, we engineered the adoptive OT-1 CD8+ T cells to deliver IL-27. In mice, the established tumors treated with OT-1 CD8+ T-IL-27 were completely rejected, which demonstrated that IL-27 delivered via tumor antigen-specific T cells enhances adoptive T cells' cancer immunity. To our knowledge, this is the first application of CD8+ T cells as a vehicle to deliver IL-27 to treat tumors. Thus, this study demonstrates IL-27 is a feasible approach for enhancing CD8+ T cells' antitumor immunity and can be used as a therapeutic adjutant for T cell adoptive transfer to treat cancer.

HIV p17 enhances T cell proliferation by suppressing autophagy through the p17-OLA1-GSK3ß axis under nutrient starvation.

Nutrient starvation is a common phenomenon that occurs during T cell activation. Upon pathogen infection, large amounts of immune cells migrate to infection sites, and antigen-specific T cells are activated; this is followed by rapid proliferation through clonal expansion. The dramatic expansion of cells will commonly lead to nutrient shortage. Cellular autophagy is often upregulated as a way to sustain the body's energy requirements. During infection, human immunodeficiency virus (HIV) co-opts a series of host cell metabolic pathways for replication. Several HIV proteins, such as Env, Nef, and Vpr, have already been reported as being involved in autophagy-related processes. In this report, we identified that the HIV p17 protein acts as a major factor in suppressing the autophagic process in T cells, especially under glucose starvation condition. HIV p17 interacts with Obg-like ATPase 1 (OLA1) and disrupts OLA1-glycogen synthase kinase-3 beta (GSK3ß) complex, leading to GSK3ß hyperactivation. Consequently, a prior proliferation of HIV-infected T cells under glucose starvation will occur. The inhibition of autophagy also aids HIV replication by antagonizing the antiviral effect of autophagy. Our study shows a new cellular pathway that HIV can hijack for viral spreading by a prior proliferation of HIV-loaded T cells and may provide new therapeutic targets for acquired immunodeficiency syndrome intervention.

CD8+ T cell survival in lethal fungal sepsis was ameliorated by T-cell-specific mTOR deletion.

Lethal fungal sepsis causes high morbidity and mortality in intensive care patients. Fungal infections have an immunological basis, and it has been shown in recent studies that decreased CD8+ T-cell count in fungal infections is related to prognosis, while the underlying mechanism is still unclear. Here, a lethal fungal sepsis model induced by candidemia was created and we found a decreased CD8+ T-cell count and exaggerated apoptosis. Simultaneously, expression of light chain (LC)3B in CD8+ T cells increased, along with increased autophagosomes and accumulation of p62 in infected mice. We regulated the activity of the mammalian target of rapamycin (mTOR) pathway using T-cell-specific mTOR/ TSC1 deletion mice. We observed increased number of autophagosomes and expression of LC3B in CD8+T cells after T-cell-specific mTOR knockout, while accumulation of p62 was not ameliorated, and there was no increase in the number of autolysosomes. Apoptosis rate and expression of BIM, a pro-apoptotic gene, decreased in CD8+ T cells in mTOR-deletion mice but increased in TSC1-deletion mice. Our results showed increased CD8+ T-cell death in spleen of lethal fungal sepsis mice, and decreased expression of mTOR ameliorated CD8+ T-cell survival. mTOR may be a possible target to reverse CD8+ T-cell immune dysfunction in lethal fungal sepsis.

The T-cell fingerprint of MALT1 paracaspase revealed by selective inhibition.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion.

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection.

Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrate's C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.

An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes.

CD43 is one of the most abundant co-stimulatory molecules on a T-cell surface; it transduces activation signals through its cytoplasmic domain, contributing to modulation of the outcome of T-cell responses. The aim of this study was to uncover new signalling pathways regulated by this sialomucin. Analysis of changes in protein abundance allowed us to identify pyruvate kinase isozyme M2 (PKM2), an enzyme of the glycolytic pathway, as an element potentially participating in the signalling cascade resulting from the engagement of CD43 and the T-cell receptor (TCR). We found that the glycolytic activity of this enzyme was not significantly increased in response to TCR+CD43 co-stimulation, but that PKM2 was tyrosine phosphorylated, suggesting that it was performing moonlight functions. We report that phosphorylation of both Y105 of PKM2 and of Y705 of signal transducer and activator of transcription 3 was induced in response to TCR+CD43 co-stimulation, resulting in activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway. ERK5 and the cAMP response element binding protein (CREB) were activated, and c-Myc and nuclear factor-κB (p65) nuclear localization, as well as Bad phosphorylation, were augmented. Consistent with this, expression of human CD43 in a murine T-cell hybridoma favoured cell survival. Altogether, our data highlight novel signalling pathways for the CD43 molecule in T lymphocytes, and underscore a role for CD43 in promoting cell survival through non-glycolytic functions of metabolic enzymes.

Gimap and T cells: a matter of life or death.

GTPase immune-associated proteins (Gimap) genes encode evolutionarily conserved GTP-binding proteins that are preferentially expressed in immune cells. Specific members have been shown to be involved in lymphocyte development, or are associated with inflammatory and autoimmune diseases. However, the function of these proteins remains poorly understood, both at the cellular and molecular levels. A new study in this issue of the European Journal of Immunology [Eur. J. Immunol. 2014. 44: 561-572] points to the distinct but partly overlapping functions of two members of this family, Gimap3 and Gimap5, and offers new insight into their potential functions in T cells.

Gimap3 and Gimap5 cooperate to maintain T-cell numbers in the mouse.

Gimap3 (IAN4) and Gimap5 (IAN5) are highly homologous GTP-binding proteins of the Gimap family. Gimap3 and Gimap5, whose transcripts are abundant in mature lymphocytes, can associate with antiapoptotic Bcl-2 family proteins. While it is established that Gimap5 regulates T-cell survival, the in vivo role of Gimap3 is unclear. Here we report the preparation and characteristics of mouse strains lacking Gimap3 and/or Gimap5. We found that the number of T cells was markedly reduced in mice deficient in both Gimap3 and Gimap5. The defects in T-cell cellularity were more severe in mice lacking both Gimap3 and Gimap5 than in mice lacking only Gimap5. No defects in the cellularity of T cells were detected in mice lacking only Gimap3, whereas bone marrow cells from Gimap3-deficient mice showed reduced T-cell production in a competitive hematopoietic environment. Moreover, retroviral overexpression and short hairpin RNAs-mediated silencing of Gimap3 in bone marrow cells elevated and reduced, respectively, the number of T cells produced in irradiated mice. These results suggest that Gimap3 is a regulator of T-cell numbers in the mouse and that multiple Gimap family proteins cooperate to maintain T-cell survival.

TRAF2 regulates T cell immunity by maintaining a Tpl2-ERK survival signaling axis in effector and memory CD8 T cells.

Generation and maintenance of antigen-specific effector and memory T cells are central events in immune responses against infections. We show that TNF receptor-associated factor 2 (TRAF2) maintains a survival signaling axis in effector and memory CD8 T cells required for immune responses against infections. This signaling axis involves activation of Tpl2 and its downstream kinase ERK by NF-κB-inducing kinase (NIK) and degradation of the proapoptotic factor Bim. NIK mediates Tpl2 activation by stimulating the phosphorylation and degradation of the Tpl2 inhibitor p105. Interestingly, while NIK is required for Tpl2-ERK signaling under normal conditions, uncontrolled NIK activation due to loss of its negative regulator, TRAF2, causes constitutive degradation of p105 and Tpl2, leading to severe defects in ERK activation and effector/memory CD8 T cell survival. Thus, TRAF2 controls a previously unappreciated signaling axis mediating effector/memory CD8 T cell survival and protective immunity.

PRMT5 Is Required for T Cell Survival and Proliferation by Maintaining Cytokine Signaling.

Arginine methylation is a post-translational modification that regulates many biological processes. However, the role of arginine methylation in immune cells is not well studied. Here we report an essential role of protein arginine methyltransferase 5 (PRMT5) in T cell homeostasis and activation-induced expansion. Using T cell-specific PRMT5 conditional knockout mice, we found that PRMT5 is required for natural killer T (NKT) cell but not for conventional or regulatory T (Treg) cell development after the double positive (DP) stage in the thymus. In contrast, PRMT5 was required for optimal peripheral T cell maintenance, for the transition of naïve T cells to effector/memory phenotype, and for early T cell development before the DP stage in a cell-intrinsic manner. Accordingly, PRMT5-deleted T cells showed impaired IL-7-mediated survival and TCR-induced proliferation in vitro. The latter was more pronounced and attributed to reduced responsiveness to IL-2. Acute deletion of PRMT5 revealed that not only naïve but also effector/memory T cells were impaired in TCR-induced proliferation in a development-independent manner. Reduced expression of common γ chain (γc), a shared receptor component for several cytokines including IL-7 and IL-2, on PRMT5-deleted T cells may be in part responsible for the defect. We further showed that PRMT5 was partially required for homeostatic T cell survival but absolutely required for lymphopenic T cell expansion in vivo. Thus, we propose that PRMT5 is required for T cell survival and proliferation by maintaining cytokine signaling, especially during proliferation. The inhibition of PRMT5 may provide a novel strategy for the treatment of diseases where uncontrolled T cell activation has a role, such as autoimmunity.

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells.

Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.

CD279 mediates the homeostasis and survival of regulatory T cells by enhancing T cell and macrophage interactions.

CD279 is a cell surface protein predominantly expressed on T cells. Its ligands CD273 and CD274 are expressed on antigen-presenting cells and tumors. CD279 has been shown to act as an important immune check point by inhibiting CD8 T cell activation, and antibodies against CD279 enhance T cell-mediated cytotoxic function. However, whether CD279 has other functions in CD4 T cell homeostasis or in mediating T cell interactions with antigen-presenting cells remains unclear. In the present study, we show that antibody-mediated inhibition of CD279 reduces T cell survival in bone marrow in vivo. Unexpectedly, CD279 blockade also compromised regulatory T cell and macrophage interactions by reducing their contact time. The observation that the CD273 antagonist had little effect suggests that CD274 (the second ligand of CD279) plays a more central role in contact between conventional T cells (Tcon) and macrophages. The results of the present study suggest that both CD279 ligands contribute to the interaction length between T cells and macrophages as a mechanism of maintaining Treg homeostasis. Furthermore, CD273 and CD274 are not redundant ligands because CD274 may have unique effects on Tcon in this complex immune axis. Therefore, ligand selection for check point blockade as a tool for cancer immunotherapy has important implications with respect to anti-tumor T cell activation and the avoidance of side effects.

IL-15 signaling promotes adoptive effector T-cell survival and memory formation in irradiation-induced lymphopenia.

BACKGROUND: Lymphopenia promotes naïve T-cell homeostatic proliferation and adoptive effector T-cell survival and memory formation. IL-7 plays a critical role in homeostatic proliferation, survival and memory formation of naïve T-cells in lymphopenia, and its underlying molecular mechanism has also been well studied. However, the mechanism for adoptively transferred effector T-cell survival and memory formation is not fully understood. Here, we transferred in vitro-activated transgenic OT-I CD8(+) effector T-cells into irradiation (600 rads)-induced lymphopenic C57BL/6, IL-7 knockout (KO) and IL-15 KO mice, and investigated the survival and memory formation of transferred T-cells in lymphopenia. RESULTS: We demonstrate that transferred T-cells prolong their survival and enhance their memory in lymphopenic mice, in a manner that depends on IL-15 signaling, but not IL-7. We determine that in vitro stimulation of naïve or effector T-cells with IL-7 and IL-15 reduces IL-7Rα, and increases and/or maintains IL-15Rß expression, respectively. Consistent with these findings, the expression of IL-7Rα and IL-15Rß is down- and up-regulated, respectively, in vivo on transferred T-cells in an early phase post T-cell transfer in lymphopenia. We further show that in vitro IL-15 restimulation-induced memory T-cells (compared to IL-2 restimulation-induced effector T-cells) and in vivo transferred T-cells in irradiated IL-15-sufficient C57BL/6 mice (compared to IL-15-deficient IL-15 KO mice) have increased mitochondrial content, but less NADH and lower mitochondrial potential (ΔΨm), and demonstrate greater phosphorylation of signal transducers and activators of transcription-5 (STAT5) and Unc-51-like kinase-1 (ULK1), and higher expression of B-cell leukemia/lymphoma-2 (Bcl2) and memory-, autophagy- and mitochondrial biogenesis-related molecules. CONCLUSION: Irradiation-induced lymphopenia promotes effector T-cell survival via IL-15 signaling the STAT5/Bcl2 pathway, enhances T-cell memory formation via IL-15 activation of the forkhead-box family of transcription factor (FOXO)/eomesodermin (Eomes) memory and ULK1/autophagy-related gene-7 (ATG7) autophagy pathways, and via IL-15 activation of the mitochondrial remodeling. Our data thus identify some important targets to consider when designing potent adoptive T-cell immunotherapies of cancer.

Deficiency of AMPK in CD8+ T cells suppresses their anti-tumor function by inducing protein phosphatase-mediated cell death.

A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression. However, the exact role of AMPK in tumor development is still controversial. Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo. While AMPK expression is dispensable for T cell development, genetic deletion of AMPK promotes T cell death during in vitro activation and in vivo tumor development. Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function. Altogether, our data suggest a novel mechanism by which AMPK regulates protein phosphatase activity in control of survival and function of CD8+ T cells, thereby enhancing their role in tumor immunosurveillance.

BTLA marks a less-differentiated tumor-infiltrating lymphocyte subset in melanoma with enhanced survival properties.

In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+ T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+ TIL subset and their CD8+BTLA- counterparts. We found that the CD8+ BTLA+ TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longer in vivo after infusion. In contrast, CD8+BTLA- TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+ TILs from apoptosis. Our results point to a new role of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+ T cells. These attributes may explain our previous observation that BTLA expression on CD8+ TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma.