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It is poorly understood how the tumor immune microenvironment influences disease recurrence in localized clear-cell renal cell carcinoma (ccRCC). Here we performed whole-transcriptomic profiling of 236 tumors from patients assigned to the placebo-only arm of a randomized, adjuvant clinical trial for high-risk localized ccRCC. Unbiased pathway analysis identified myeloid-derived IL6 as a key mediator. Furthermore, a novel myeloid gene signature strongly correlated with disease recurrence and overall survival on uni- and multivariate analyses and is linked to TP53 inactivation across multiple data sets. Strikingly, effector T-cell gene signatures, infiltration patterns, and exhaustion markers were not associated with disease recurrence. Targeting immunosuppressive myeloid inflammation with an adenosine A2A receptor antagonist in a novel, immunocompetent, Tp53-inactivated mouse model significantly reduced metastatic development. Our findings suggest that myeloid inflammation promotes disease recurrence in ccRCC and is targetable as well as provide a potential biomarker-based framework for the design of future immuno-oncology trials in ccRCC. Significance: Improved understanding of factors that influence metastatic development in localized ccRCC is greatly needed to aid accurate prediction of disease recurrence, clinical decision-making, and future adjuvant clinical trial design. Our analysis implicates intratumoral myeloid inflammation as a key driver of metastasis in patients and a novel immunocompetent mouse model. This article is highlighted in the In This Issue feature, p. 2221

Cellular transformation induces phenotypically diverse populations of tumour-infiltrating T cells

The mTORC1 pathway coordinates nutrient and growth factor signals to maintain organismal homeostasis. Whether nutrient signaling to mTORC1 regulates stem cell function remains unknown. Here, we show that SZT2 - a protein required for mTORC1 downregulation upon nutrient deprivation - is critical for hematopoietic stem cell (HSC) homeostasis. Ablation of SZT2 in HSCs decreased the reserve and impaired the repopulating capacity of HSCs. Furthermore, ablation of both SZT2 and TSC1 - 2 repressors of mTORC1 on the nutrient and growth factor arms, respectively - led to rapid HSC depletion, pancytopenia, and premature death of the mice. Mechanistically, loss of either SZT2 or TSC1 in HSCs led to only mild elevation of mTORC1 activity and reactive oxygen species (ROS) production. Loss of both SZT2 and TSC1, on the other hand, simultaneously produced a dramatic synergistic effect, with an approximately 10-fold increase of mTORC1 activity and approximately 100-fold increase of ROS production, which rapidly depleted HSCs. These data demonstrate a critical role of nutrient mTORC1 signaling in HSC homeostasis and uncover a strong synergistic effect between nutrient- and growth factor-mediated mTORC1 regulation in stem cells.

Malignancy can be suppressed by the immune system. However, the classes of immunosurveillance responses and their mode of tumor sensing remain incompletely understood. Here, we show that while clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8(+) T cells which negatively correlated with patient prognosis, chromophobe RCC had abundant infiltration of granzyme A-expressing intraepithelial type 1 innate lymphoid cells (ILC1s) that positively associated with patient survival. Interleukin-15 (IL-15) promoted ILC1 granzyme A expression and cytotoxicity, and IL-15 expression in chRCC tumor tissue positively tracked with the ILC1 response. An ILC1 gene signature also predicted survival of a subset of breast cancer patients in association with IL-15 expression. Notably, ILC1s directly interacted with cancer cells, and IL-15 produced by cancer cells supported the expansion and anti-tumor function of ILC1s in a murine breast cancer model. Thus, ILC1 sensing of cancer cell IL-15 defines an immunosurveillance mechanism of epithelial malignancies.

Background & Aims Sestrin 1/2/3 (Sesn1/2/3) belong to a small family of proteins that have been implicated in the regulation of metabolic homeostasis and oxidative stress. However, the underlying mechanisms remain incompletely understood. The aim of this work was to illustrate the collective function of Sesn1/2/3 in the protection against hepatic lipotoxicity. Methods We used Sesn1/2/3 triple knockout (TKO) mouse and cell models to characterize oxidative stress and signal transduction under lipotoxic conditions. Biochemical, histologic, and physiological approaches were applied to illustrate the related processes. Results After feeding with a Western diet for 8 weeks, TKO mice developed remarkable metabolic associated fatty liver disease that was manifested by exacerbated hepatic steatosis, inflammation, and fibrosis compared with wild-type counterparts. Moreover, TKO mice exhibited higher levels of hepatic lipotoxicity and oxidative stress. Our biochemical data revealed a critical signaling node from sestrins to c-Jun N-terminal kinases (JNKs) in that sestrins interact with JNKs and mitogen-activated protein kinase kinase 7 and suppress the JNK phosphorylation and activity. In doing so, sestrins markedly reduced palmitate-induced lipotoxicity and oxidative stress in both mouse and human hepatocytes. Conclusions The data from this study suggest that Sesn1/2/3 play an important role in the protection against lipotoxicity-associated oxidative stress and related pathology in the liver., Graphical abstract

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C–expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C–expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor–β (TGF-β) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C–expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond “helper-like” lymphocytes.

Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.

Summary The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b., Graphical abstract, Highlights • Isolation, activation, and differentiation of mouse primary T cells • SLO-mediated cell membrane permeabilization protocol • SLO-mediated intracellular small molecule delivery protocol, The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects.

Summary Aerobic glycolysis—the Warburg effect—converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic β-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness., Ovarian cancer cells often metastasize to the peritoneal cavity, forming spheroid-like structures and promoting a highly immunosuppressive tumor microenvironment. Here, the authors show that the ubiquitin ligase UBR5 is required for ovarian cancer growth and metastasis, sustaining spheroid formation and the infiltration of immunosuppressive tumor associated macrophages.

A metabolic circuit in T cell immunity Naïve T cells are metabolically reprogrammed when they differentiate into T effector (T eff ) cells, transitioning from a reliance on mitochondrial oxidative phosphorylation to aerobic glycolysis. Xu et al. found that lactate dehydrogenase A (LDHA), a glycolytic enzyme that converts pyruvate to lactate, is a key player in this process. T eff cells that differentiate in mice infected with the bacterium Listeria monocytogenes turned on LDHA through phosphoinositide 3-kinase (PI3K) signaling. By promoting adenosine triphosphate (ATP) production, LDHA in turn facilitated PI3K-dependent inactivation of the transcription factor Foxo1 needed for effective T eff cell responses. Thus, glycolytic ATP acts like a rheostat that both gauges and regulates PI3K-dependent signaling. This type of positive feedback circuit may also provide a mechanistic explanation for the Warburg effect observed in cancer cells. Science , this issue p. 405

Cancer arises from malignant cells that exist in dynamic multilevel interactions with the host tissue. Cancer therapies aiming to directly kill cancer cells, including oncogene-targeted therapy and immune-checkpoint therapy that revives tumour-reactive cytotoxic T lymphocytes, are effective in some patients

Foxp3+ regulatory T (T reg) cells are pivotal regulators of immune tolerance, with T cell receptor (TCR)-driven activated T reg (aT reg) cells playing a central role; yet how TCR signaling propagates to control aT reg cell responses remains poorly understood. Here we show that TCR signaling induces expression of amino acid transporters, and renders amino acid-induced activation of mTORC1 in aT reg cells. T reg cell-specific ablation of the Rag family small GTPases RagA and RagB impairs amino acid-induced mTORC1 signaling, causing defective amino acid anabolism, reduced T reg cell proliferation, and a rampant autoimmune disorder similar in severity to that triggered by T reg cell-specific TCR deficiency. Notably, T reg cells in peripheral tissues, including tumors, are more sensitive to Rag GTPase-dependent nutrient sensing. Ablation of RagA alone impairs T reg cell accumulation in the tumor, resulting in enhanced antitumor immunity. Thus, nutrient mTORC1 signaling is an essential component of TCR-initiated T reg cell reprogramming, and Rag GTPase activities may be titrated to break tumor immune tolerance., Memorial Sloan-Kettering Cancer Center (Support Grant/Core Grant P30CA08748)

SUMMARY Fascin protein is the main actin-bundling protein in filopodia and invadopodia, which are critical for tumor cell migration, invasion, and metastasis. Small-molecule fascin inhibitors block tumor invasion and metastasis and increase the overall survival of tumor-bearing mice. Here, we report a finding that fascin blockade additionally reinvigorates anti-tumor immune response in syngeneic mouse models of various cancers. Fascin protein levels are increased in conventional dendritic cells (cDCs) in the tumor microenvironment. Mechanistically, fascin inhibitor NP-G2–044 increases the number of intratumoral-activated cDCs and enhances the antigen uptake by cDCs. Furthermore, together with PD-1 blocking antibody, NP-G2–044 markedly increases the number of activated CD8+ T cells in the otherwise anti-PD-1 refractory tumors. Reduction of fascin levels in cDCs, but not fascin gene knockout in tumor cells, mimics the anti-tumor immune effect of NP-G2–044. These data demonstrate that fascin inhibitor NP-G2–044 simultaneously limits tumor metastasis and reinvigorates anti-tumor immune responses., Graphical Abstract, In brief Wang et al. report that fascin inhibitors act on intratumoral dendritic cells to block the migration and increase the antigen uptake. Together with anti-PD-1 antibody, fascin inhibitors increase the number of intratumoral proliferating and activated CD8+ T cells and the overall survival of mice bearing the otherwise anti-PD-1 refractory tumors.

Aerobic glycolysis (the Warburg effect) is a metabolic hallmark of activated T cells and has been implicated in augmenting effector T cell responses, including expression of the proinflammatory cytokine interferon-γ (IFN-γ), via 3' untranslated region (3'UTR)-mediated mechanisms. Here, we show that lactate dehydrogenase A (LDHA) is induced in activated T cells to support aerobic glycolysis but promotes IFN-γ expression independently of its 3'UTR. Instead, LDHA maintains high concentrations of acetyl-coenzyme A to enhance histone acetylation and transcription of Ifng Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells. These findings reveal an epigenetic mechanism by which aerobic glycolysis promotes effector T cell differentiation and suggest that LDHA may be targeted therapeutically in autoinflammatory diseases.

Innate lymphocytes play an important role in maintaining tissue homeostasis at steady state and during inflammation. The population of innate lymphocytes is incredibly diverse and heterogeneous with the successive identification of new subsets including innate lymphoid cells that arise from progenitors distinct from those of natural killer cells. Although generally considered as T helper-like lymphocytes, innate lymphoid cells with cytotoxic potential can be identified in many tissues. The tissue-resident cytotoxic innate lymphocytes derived from innate lymphoid cell and/or natural killer cell lineages are well positioned in sensing malignant transformation and initiating antitumor immunity. This review provides an overview of innate lymphocyte biology and discuss their roles in tumor immunosurveillance.

Innate lymphocytes are a diverse population of cells that carry out specialized functions in steady-state homeostasis and during immune challenge. While circulating cytotoxic natural killer (NK) cells have been studied for decades, tissue-resident innate lymphoid cells (ILCs) have only been characterized and studied over the past few years. As ILCs have been largely viewed in the context of helper T-cell biology, models of ILC lineage and function have been founded within this perspective. Notably, tissue-resident innate lymphocytes with cytotoxic potential have been described in an array of tissues, yet whether they are derived from the NK or ILC lineage is only beginning to be elucidated. In this review, we aim to shed light on the identities of innate lymphocytes through the lenses of cell lineage, localization, and timing of differentiation.

Lymphocytes are an integral component of the immune system. Classically, all lymphocytes were thought to perpetually recirculate between secondary lymphoid organs and only traffic to non-lymphoid tissues upon activation. In recent years, a diverse family of non-circulating lymphocytes have been identified. These include innate lymphocytes, innate-like T cells and a subset of conventional T cells. Spanning the innate-adaptive spectrum, these tissue-resident lymphocytes carry out specialized functions and cross-talk with other immune cell types to maintain tissue integrity and homeostasis both at the steady state and during pathological conditions. In this review, we provide an overview of the heterogeneous tissue-resident lymphocyte populations, discuss their development, and highlight their functions both in the context of microbial infection and cancer.

Cross-talk in the mammary gland Macrophages engulf damaged and dead cells to clear infection, but they also participate in tissue regeneration. Chakrabarti et al. expand the macrophage repertoire for mammary gland development (see the Perspective by Kannan and Eaves). Mammary gland stem cells secrete the Notch ligand Dll1 and activate Notch signaling, which promotes survival of adjacent macrophages. This stimulates production of Wnt ligands, which signal back to the mammary gland stem cells. This cross-talk plays an important role in coordinating mammary gland development, tissue homeostasis, and, not least, breast cancer. Science , this issue p. eaan4153 ; see also p. 1401

Innate lymphocytes play critical roles in maintaining tissue homeostasis and integrity of the host at steady state and during pathogenic insults. The successive identification of new innate lymphocyte subsets has revealed an incredible diversity within the family. While this heterogeneous population can be grouped based on their cytotoxic potential into exclusively cytokine-producing helpers and cytolytic killers, the exact developmental relationships between the subsets are not fully understood. The former group is enriched at mucosal surfaces, whereas innate lymphocytes with cytotoxic potential can be identified in a wider array of tissues, including tumors. Although their cytotoxicity suggests an antitumor role, the nature of tumor-elicited innate lymphocyte responses has only begun to be investigated, and the identities of participating subsets still remain contentious. In this review, we provide a brief overview of innate lymphocyte biology, review the current knowledge on their ontogeny, and discuss their roles in tumor immunosurveillance. Cancer Immunol Res; 6(4); 372–7. ©2018 AACR.

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remain obscure. Here we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, TCRαβ and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a and CD103, these cells share a gene expression signature distinct from those of conventional NK cells, T cells and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15, but not Nfil3, deficiency results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type 1-like innate lymphoid cells and type 1 innate-like T cells.

Mammalian cells possess two amino acid-sensing kinases: general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). Their combined effects orchestrate cellular adaptation to amino acid levels, but how their activities are coordinated remains poorly understood. Here, we demonstrate an important link between GCN2 and mTORC1 signaling. Upon deprivation of various amino acids, activated GCN2 up-regulates ATF4 to induce expression of the stress response protein Sestrin2, which is required to sustain repression of mTORC1 by blocking its lysosomal localization. Moreover, Sestrin2 induction is necessary for cell survival during glutamine deprivation, indicating that Sestrin2 is a critical effector of GCN2 signaling that regulates amino acid homeostasis through mTORC1 suppression.

Regulatory T cells (Treg cells) deficient in the transcription factor Foxp3 lack suppressor function and manifest an effector T (Teff) cell–like phenotype. We demonstrate that Foxp3 deficiency dysregulates metabolic checkpoint kinase mammalian target of rapamycin (mTOR) complex 2 (mTORC2) signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Specific deletion of the mTORC2 adaptor gene Rictor in Foxp3-deficient Treg cells ameliorated disease in a Foxo1 transcription factor–dependent manner. Rictor deficiency re-established a subset of Treg cell genetic circuits and suppressed the Teff cell–like glycolytic and respiratory programs, which contributed to immune dysregulation. Treatment of Treg cells from patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their Teff cell–like program and restored suppressive function. Thus, regulatory function can be re-established in Foxp3-deficient Treg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in Treg cell disorders. Loss of the transcription factor Foxp3 impairs Treg cell development and immune homeostasis. Chatila and colleagues use a series of Foxp3 mutants to show that its disruption results in altered Treg cell metabolism and loss of regulation, which are rescuable by metabolic manipulation.

Treatment of type 2 diabetes mellitus continues to pose an important clinical challenge, with most existing therapies lacking demonstrable ability to improve cardiovascular outcomes. The atheroprotective peptide apelin (APLN) enhances glucose utilization and improves insulin sensitivity. However, the mechanism of these effects remains poorly defined. We demonstrate that the expression of APLNR (APJ/AGTRL1), the only known receptor for apelin, is predominantly restricted to the endothelial cells (ECs) of multiple adult metabolic organs, including skeletal muscle and adipose tissue. Conditional endothelial-specific deletion of Aplnr (AplnrECKO) resulted in markedly impaired glucose utilization and abrogation of apelin-induced glucose lowering. Furthermore, we identified in-activation of Forkhead box protein O1 (FOXO1) and inhibition of endothelial expression of fatty acid (FA) binding protein 4 (FABP4) as key downstream signaling targets of apelin/APLNR signaling. Both the Apln−/− and AplnrECKO mice demonstrated increased endothelial FABP4 expression and excess tissue FA accumulation, whereas concurrent endothelial Foxo1 deletion or pharmacologic FABP4 inhibition rescued the excess FA accumulation phenotype of the Apln−/− mice. The impaired glucose utilization in the AplnrECKO mice was associated with excess FA accumulation in the skeletal muscle. Treatment of these mice with an FABP4 inhibitor abrogated these metabolic phenotypes. These findings provide mechanistic insights that could greatly expand the therapeutic repertoire for type 2 diabetes and related metabolic disorders.

Innate lymphoid cells (ILCs) are critical components of tissues in the body, providing a first line of defense against challenges to host integrity. In contrast to strictly cytokine-producing helper ILCs, resident innate lymphocyte populations with cytolytic potential have been identified in multiple tissues in both mouse and human. These cells express the transcription factor Tbet, NK cell receptors, granzymes, perforin, and death receptors, and can directly kill tumor cells. Signals in the tumor microenvironment may promote this response, including the cytokine IL-15 and stress-associated ligands for activating NK receptors. Although there is evidence that these cells are tissue and tumor resident, their lineage remains unclear. Whether they are derived from the NK or helper ILC lineages or represent a third differentiation pathway remains to be determined. A better understanding of their lineage will help clarify their regulation and function in the context of antitumor immunity.

Transforming growth factor β (TGF-β) is a pleiotropic cytokine involved in both suppressive and inflammatory immune responses. After 30 years of intense study, we have only begun to elucidate how TGF-β alters immunity under various conditions. Under steady-state conditions, TGF-β regulates thymic T-cell selection and maintains homeostasis of the naive T-cell pool. TGF-β inhibits cytotoxic T lymphocyte (CTL), Th1-, and Th2-cell differentiation while promoting peripheral (p)Treg-, Th17-, Th9-, and Tfh-cell generation, and T-cell tissue residence in response to immune challenges. Similarly, TGF-β controls the proliferation, survival, activation, and differentiation of B cells, as well as the development and functions of innate cells, including natural killer (NK) cells, macrophages, dendritic cells, and granulocytes. Collectively, TGF-β plays a pivotal role in maintaining peripheral tolerance against self- and innocuous antigens, such as food, commensal bacteria, and fetal alloantigens, and in controlling immune responses to pathogens.

SummaryMechanistic target of rapamycin complex 1 (mTORC1) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. Here, we show that Sestrins bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino-acid-induced Rag guanine nucleotide exchange and mTORC1 translocation to the lysosome. Mutation of the conserved GDI motif creates a dominant-negative form of Sestrin that renders mTORC1 activation insensitive to amino acid deprivation, whereas a cell-permeable peptide containing the GDI motif inhibits mTORC1 signaling. Mice deficient in all Sestrins exhibit reduced postnatal survival associated with defective mTORC1 inactivation in multiple organs during neonatal fasting. These findings reveal a nonredundant mechanism by which the Sestrin family of GDIs regulates the nutrient-sensing Rag GTPases to control mTORC1 signaling.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-β (TGF-β) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

Regulatory T (Treg) cells are essential for maintenance of immune homeostasis. Here we found that hydrogen sulfide (H2S) was required for Foxp3+ Treg cell differentiation and function, and that H2S deficiency led to systemic autoimmune disease. H2S maintained expression of methylcytosine dioxygenases Tet1 and Tet2 by sulfhydrating nuclear transcription factor Y subunit beta (NFYB) to facilitate its binding to Tet1 and Tet2 promoters. Transforming growth factor-β (TGF-β)-activated Smad3 and interleukin-2 (IL-2)-activated Stat5 facilitated Tet1 and Tet2 binding to Foxp3. Tet1 and Tet2 catalyzed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in Foxp3 to establish a Treg cell-specific hypomethylation pattern and stable Foxp3 expression. Consequently, Tet1 and Tet2 deletion led to Foxp3 hypermethylation, impaired Treg cell differentiation and function, and autoimmune disease. Thus, H2S promotes Tet1 and Tet2 expression, which are recruited to Foxp3 by TGF-β and IL-2 signaling to maintain Foxp3 demethylation and Treg cell-associated immune homeostasis.

Highlights • An intermediate amount of T cell stimulation induces Foxp3 transcription. • Treg cell lineage factor Foxp3 cooperates with its partners to promote Treg cell function. • Cell signaling-regulated Foxo1 is indispensable for Treg cell function., Regulatory T (Treg) cells differentiate from thymocytes or peripheral T cells in response to host and environmental cues, culminating in induction of the transcription factor forkhead box P3 (Foxp3) and the Treg cell-specific epigenome. An intermediate amount of antigen stimulation is required to induce Foxp3 expression by engaging T cell receptor (TCR)-activated [e.g., nuclear factor (NF)-κB] and TCR-inhibited (e.g., Foxo) transcription factors. Furthermore, Treg cell differentiation is associated with attenuated Akt signaling, resulting in enhanced nuclear retention of Foxo1, which is indispensable for Treg cell function. These findings reveal that Treg cell lineage commitment is not only controlled by genetic and epigenetic imprinting, but also modulated by transcriptional programs responding to extracellular signals.

SummaryInterleukin-7 receptor α chain (IL-7Rα) is induced upon T cell positive selection and controls thymic CD8-lineage specification and peripheral naive T cell homeostasis. How IL-7Rα expression is regulated in developing thymocytes is unclear. Here, we show that transforming growth factor β (TGF-β) signaling promoted IL-7Rα expression and CD8+ T cell differentiation. In addition, TGF-β signaling was required for high IL-7Rα expression in CD4+ T cells bearing low-affinity T cell receptors, and the abrogation of TGF-β receptor expression led to failed maintenance of peripheral CD4+ T cells. Compromised IL-7Rα expression in TGF-β-receptor-deficient T cells was associated with increased expression of the Il7ra transcriptional repressor, Gfi-1. IL-7Rα transgenesis or T-cell-specific ablation of Gfi-1 restored IL-7Rα expression and largely ameliorated the development and homeostasis defects of TGF-β-receptor-deficient T cells. These findings reveal functions for TGF-β signaling in controlling IL-7Rα expression and in promoting T cell repertoire diversification.

Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.

Blockade of C3aR/C5aR signaling in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival., Thymus-derived (natural) CD4+ FoxP3+ regulatory T cells (nT reg cells) are required for immune homeostasis and self-tolerance, but must be stringently controlled to permit expansion of protective immunity. Previous findings linking signals transmitted through T cell–expressed C5a receptor (C5aR) and C3a receptor (C3aR) to activation, differentiation, and expansion of conventional CD4+CD25− T cells (T conv cells), raised the possibility that C3aR/C5aR signaling on nT reg cells could physiologically modulate nT reg cell function and thereby further impact the induced strength of T cell immune responses. In this study, we demonstrate that nT reg cells express C3aR and C5aR, and that signaling through these receptors inhibits nT reg cell function. Genetic and pharmacological blockade of C3aR/C5aR signal transduction in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival. Mechanisms involve C3a/C5a-induced phosphorylation of AKT and, as a consequence, phosphorylation of the transcription factor Foxo1, which results in lowered nT reg cell Foxp3 expression. The documentation that C3a/C3aR and C5a/C5aR modulate nT reg cell function via controlling Foxp3 expression suggests targeting this pathway could be exploited to manipulate pathogenic or protective T cell responses.

Regulatory T (T-reg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses(1-4). Foxp3 operates as a late-acting differentiation factor controlling T-reg cell homeostasis and function(5), whereas the early T-reg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors(6-10). However, whether Foxo proteins act beyond the T-reg-cell-commitment stage to control T-reg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulator of T-reg cell function. T-reg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with T-reg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of T-reg cells. Genome-wide analysis of Foxo1 binding sites reveals similar to 300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt-Foxo1 signalling module controls a novel genetic program indispensable for T-reg cell function.

SummaryTGF-β1 is a regulatory cytokine that has an important role in controlling T cell differentiation. T cell-produced TGF-β1 acts on T cells to promote Th17 cell differentiation and the development of experimental autoimmune encephalomyelitis (EAE). However, the exact TGF-β1-producing T cell subset required for Th17 cell generation and its cellular mechanism of action remain unknown. Here we showed that deletion of the Tgfb1 gene from activated T cells and Treg cells, but not Treg cells alone, abrogated Th17 cell differentiation, resulting in almost complete protection from EAE. Furthermore, differentiation of T cells both in vitro and in vivo demonstrated that TGF-β1 was highly expressed by Th17 cells and acted in a predominantly autocrine manner to maintain Th17 cells in vivo. These findings reveal an essential role for activated T cell-produced TGF-β1 in promoting the differentiation of Th17 cells and controlling inflammatory diseases.

Summary T helper 17 (Th17) cell development is driven by cytokines including transforming growth factor-β (TGF-β), interleukin-6 (IL-6), IL-1, and IL-23. Regulatory T (Treg) cells can provide the TGF-β in vitro, but their role in vivo remains unclear, particularly because Treg cells inhibit inflammation in many models of Th17 cell-associated autoimmunity. We used mice expressing Diphtheria toxin receptor under control of the Foxp3 promoter to deplete Foxp3 + Treg cells in adult mice during in vivo Th17 cell priming. Treg cell depletion resulted in a reduced frequency of antigen-specific IL-17 producers in draining lymph nodes and blood, correlating with reduced inflammatory skin responses. In contrast, Treg cells did not promote IL-17 secretion after initial activation stages. Treg cell production of TGF-β was not required for Th17 cell promotion, and neither was suppression of Th1 cell-associated cytokines. Rather, regulation of IL-2 availability and resultant signaling through CD25 by Treg cells was found to play an important role.

CD4(+) regulatory T cells (T(reg) cells) characterized by expression of the transcription factor Foxp3 have a pivotal role in maintaining immunological tolerance. Here we show that mice with T cell-specific deletion of both the Foxo1 and Foxo3 transcription factors (collectively called 'Foxo proteins' here) developed a fatal multifocal inflammatory disorder due in part to T(reg) cell defects. Foxo proteins functioned in a T(reg) cell-intrinsic manner to regulate thymic and transforming growth factor-beta (TGF-beta)-induced Foxp3 expression, in line with the ability of Foxo proteins to bind to Foxp3 locus and control Foxp3 promoter activity. Transcriptome analyses showed that Foxo proteins regulated the expression of additional T(reg) cell-associated genes and were essential for inhibiting the acquisition of effector T cell characteristics by T(reg) cells. Thus, Foxo proteins have crucial roles in specifying the T(reg) cell lineage.

Cilia are present on nearly all cell types in mammals and perform remarkably diverse functions. However, the mechanisms underlying ciliogenesis are unclear. Here, we cloned a previously uncharacterized highly conserved gene, stumpy , located on mouse chromosome 7. Stumpy was ubiquitously expressed, and conditional loss in mouse resulted in complete penetrance of perinatal hydrocephalus (HC) and severe polycystic kidney disease (PKD). We found that cilia in stumpy mutant brain and kidney cells were absent or markedly deformed, resulting in defective flow of cerebrospinal fluid. Stumpy colocalized with ciliary basal bodies, physically interacted with γ-tubulin, and was present along ciliary axonemes, suggesting that stumpy plays a role in ciliary axoneme extension. Therefore, stumpy is essential for ciliogenesis and may be involved in the pathogenesis of human congenital malformations such as HC and PKD.

Langerhans cells (LCs) are bone marrow (BM)-derived epidermal dendritic cells (DCs) that develop from precursors found in the dermis. Epidermal LCs are absent in transforming growth factor (TGF) beta1-deficient mice. It is not clear whether TGFbeta1 acts directly on LC precursors to promote maturation or whether it acts on accessory cells, which in turn affect LC precursors. In addition, the physiologic source of TGFbeta1 is uncertain because BM chimera experiments showed that neither hematopoietic nor nonhematopoietic-derived TGFbeta1 is required for LC development. To address these issues, we created mice transgenic for a bacterial artificial chromosome (BAC) containing the gene for human Langerin into which Cre recombinase had been inserted by homologous recombination (Langerin-Cre). These mice express Cre selectively in LCs, and they were bred to floxed TGFbetaRII and TGFbeta1 mice, thereby generating mice with LCs that either cannot respond to or generate TGFbeta1, respectively. Langerin-Cre TGFbetaRII mice had substantially reduced numbers of epidermal LCs, demonstrating that TGFbeta1 acts directly on LCs in vivo. Interestingly, Langerin-Cre TGFbeta1 mice also had very few LCs both in the steady state and after BM transplantation. Thus, TGFbeta1 derived from LCs acts directly on LCs through an autocrine/paracrine loop, and it is required for LC development and/or survival.

TGF-beta1 is a regulatory cytokine with a pleiotropic role in immune responses. TGF-beta1 is widely expressed in leukocytes and stromal cells. However, the functions of TGF-beta1 expressed by specific lineages of cells remain unknown in vivo. Here, we show that mice with a T cell-specific deletion of the Tgfb1 gene developed lethal immunopathology in multiple organs, and this development was associated with enhanced T cell proliferation, activation, and CD4+ T cell differentiation into T helper 1 (Th1) and Th2 cells. TGF-beta1 produced by Foxp3-expressing regulatory T cells was required to inhibit Th1-cell differentiation and inflammatory-bowel disease in a transfer model. In addition, T cell-produced TGF-beta1 promoted Th17-cell differentiation and was indispensable for the induction of experimental autoimmune encephalomyelitis. These findings reveal essential roles for T cell-produced TGF-beta1 in controlling differentiation of T helper cells and controlling inflammatory diseases.

Summary Follicular helper T (Tfh) cells are crucial for germinal center (GC) formation and humoral adaptive immunity. Mechanisms underlying Tfh cell differentiation in peripheral and mucosal lymphoid organs are incompletely understood. We report here that mTOR kinase complexes 1 and 2 (mTORC1 and mTORC2) are essential for Tfh cell differentiation and GC reaction under steady state and after antigen immunization and viral infection. Loss of mTORC1 and mTORC2 in T cells exerted distinct effects on Tfh cell signature gene expression, whereas increased mTOR activity promoted Tfh responses. Deficiency of mTORC2 impaired CD4 + T cell accumulation and immunoglobulin A production and aberrantly induced the transcription factor Foxo1. Mechanistically, the costimulatory molecule ICOS activated mTORC1 and mTORC2 to drive glycolysis and lipogenesis, and glucose transporter 1-mediated glucose metabolism promoted Tfh cell responses. Altogether, mTOR acts as a central node in Tfh cells by linking immune signals to anabolic metabolism and transcriptional activity.

Mechanistic target of rapamycin complex 1 (TORC1) integrates nutrient signals to control cell growth and organismal homeostasis across eukaryotes. The evolutionarily conserved GATOR complex regulates mTORC1 signalling through Rag GTPases, and GATOR1 displays GTPase activating protein (GAP) activity for RAGA and RAGB (RAGA/B) and GATOR2 has been proposed to be an inhibitor of GATOR1. Furthermore, the metazoan-specific SESN proteins function as guanine nucleotide dissociation inhibitors (GDIs) for RAGA/B, and interact with GATOR2 with unknown effects. Here we show that SZT2 (seizure threshold 2), a metazoan-specific protein mutated in epilepsy, recruits a fraction of mammalian GATOR1 and GATOR2 to form a SZT2-orchestrated GATOR (SOG) complex with an essential role in GATOR- and SESN-dependent nutrient sensing and mTORC1 regulation. The interaction of SZT2 with GATOR1 and GATOR2 was synergistic, and an intact SOG complex was required for its localization at the lysosome. SZT2 deficiency resulted in constitutive mTORC1 signalling in cells under nutrient-deprived conditions and neonatal lethality in mice, which was associated with failure to inactivate mTORC1 during fasting. Hyperactivation of mTORC1 in SZT2-deficient cells could be partially corrected by overexpression of the GATOR1 component DEPDC5, and by the lysosome-targeted GATOR2 component WDR59 or lysosome-targeted SESN2. These findings demonstrate that SZT2 has a central role in dictating GATOR-dependent nutrient sensing by promoting lysosomal localization of SOG, and reveal an unexpected function of lysosome-located GATOR2 in suppressing mTORC1 signalling through SESN recruitment.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 have a pivotal role in maintaining immunological self-tolerance1-5; yet, excessive Treg cell activities suppress anti-tumor immune responses6-8. Compared to resting phenotype Treg (rTreg) cells in the secondary lymphoid organs, Treg cells in non-lymphoid tissues including solid tumors exhibit an activated Treg (aTreg) cell phenotype9-11. However, aTreg cell function and whether its generation can be manipulated to promote tumor immunity without evoking autoimmunity are largely unexplored. Here we show that the transcription factor Foxo1, previously demonstrated to promote Treg cell suppression of lymphoproliferative diseases12,13, has an unexpected function in inhibiting aTreg cell-mediated immune tolerance. We found that aTreg cells turned over at a slower rate than rTreg cells, but were not locally maintained in tissues. Transcriptome analysis revealed that aTreg cell differentiation was associated with repression of Foxo1-dependent gene transcription, concomitant with reduced Foxo1 expression and enhanced Foxo1 phosphorylation at sites of the Akt kinase. Treg cell-specific expression of an Akt-insensitive Foxo1 mutant prevented downregulation of lymphoid organ homing molecules, and depleted aTreg cells, causing CD8+ T cell-mediated autoimmune diseases. Compared to Treg cells from healthy tissues, tumor-infiltrating Treg cells downregulated Foxo1 target genes more substantially. Expression of the Foxo1 mutant at a lower dose was sufficient to deplete tumor-associated Treg cells, activate effector CD8+ T cells, and inhibit tumor growth without inflicting autoimmunity. Thus, Foxo1 inactivation is essential for the generation of aTreg cells that have a crucial function in suppressing CD8+ T cell responses; and the Foxo signaling pathway in Treg cells can be titrated to preferentially break tumor immune tolerance.

Transforming growth factor-beta (TGF-beta) is a potent regulatory cytokine with diverse effects on hemopoietic cells. The pivotal function of TGF-beta in the immune system is to maintain tolerance via the regulation of lymphocyte proliferation, differentiation, and survival. In addition, TGF-beta controls the initiation and resolution of inflammatory responses through the regulation of chemotaxis, activation, and survival of lymphocytes, natural killer cells, dendritic cells, macrophages, mast cells, and granulocytes. The regulatory activity of TGF-beta is modulated by the cell differentiation state and by the presence of inflammatory cytokines and costimulatory molecules. Collectively, TGF-beta inhibits the development of immunopathology to self or nonharmful antigens without compromising immune responses to pathogens. This review highlights the findings that have advanced our understanding of TGF-beta in the immune system and in disease.

Transforming growth factor-beta (TGF-beta) is a key regulator of immune tolerance. In this paper, we will focus on T cells and natural killer (NK) cells, which are directly regulated by TGF-beta in vivo. TGF-beta controls T-cell activation and differentiation, and is involved in the suppressive function and generation of regulatory T cells. Recently, TGF-beta has also been shown to directly inhibit NK cell activity. These studies demonstrate that TGF-beta utilizes multiple mechanisms to ensure immune tolerance, which is critical in a variety of autoimmune and inflammatory disorders. We will also discuss recent advances on the role of TGF-beta in immune-mediated diabetes, inflammatory bowel disease, arthritis, and systemic lupus erythematosus.

JNK1 has divergent roles in regulating the effector functions of CD4+ and CD8+ T cells. However, the function of JNK1 in tumor immune surveillance is unknown. In this study, we show that similar to IFN-γ−/− mice, JNK1−/− mice are highly susceptible to tumor development after inoculation of both melanoma cell line B16 and lymphoma cell line EL-4. Using T cell depletion and reconstitution approaches, we show that CD8+ T cells, but not CD4+ T cells, from JNK1−/− mice are responsible for tumor susceptibility. JNK1−/− CD8+ T cells have an intrinsic defect in early IFN-γ gene transcription and production after activation by either anti-CD3/anti-CD28 Abs or dendritic cells loaded with specific Ag in vitro. The impaired IFN-γ production in JNK1−/− CD8+ T cells is associated with reduced expression of both T-bet and Eomesodermin, indicating that JNK1 regulates the transcription program of CD8+ T cells. Finally, JNK1−/− CD8+ T cells showed reduced perforin expression and impaired CTL function. Taken together, our results demonstrate that JNK1 plays an important role in tumor immune surveillance through regulating the effector functions of CD8+ T cells.

CD4+CD25+regulatory T cells are essential in the protection from organ-specific autoimmune diseases. In the pancreas, they inhibit actions of autoreactive T cells and thereby prevent diabetes progression. The signals that control the generation, the maintenance, or the expansion of regulatory T cell poolin vivoremain poorly understood. Here we show that a transient pulse of transforming growth factor β (TGF-β) in the islets during the priming phase of diabetes is sufficient to inhibit disease onset by promoting the expansion of intraislet CD4+CD25+T cell pool. Approximately 40–50% of intraislet CD4+T cells expressed the CD25 marker and exhibited characteristics of regulatory T cells including small size, high level of intracellular CTLA-4, expression of Foxp3, and transfer of protection against diabetes. Results fromin vivoincorporation of BrdUrd revealed that the generation of a high frequency of regulatory T cells in the islets is due toin situexpansion upon TGF-β expression. Thus, these findings demonstrate a previously uncharacterized mechanism by which TGF-β inhibits autoimmune diseases via regulation of the size of the CD4+CD25+regulatory T cell poolin vivo.

Cells undergoing apoptosis during development are removed by phagocytes, but the underlying mechanisms of this process are not fully understood. Phagocytes lacking the phosphatidylserine receptor (PSR) were defective in removing apoptotic cells. Consequently, in PSR-deficient mice, dead cells accumulated in the lung and brain, causing abnormal development and leading to neonatal lethality. A fraction of PSR knockout mice manifested a hyperplasic brain phenotype resembling that of mice deficient in the cell death–associated genes encoding Apaf-1, caspase-3, and caspase-9, which suggests that phagocytes may also be involved in promoting apoptosis. These data demonstrate a critical role for PSR in early stages of mammalian organogenesis and suggest that this receptor may be involved in respiratory distress syndromes and congenital brain malformations.

SUMMARY Protein kinase B (also known as AKT) and the mechanistic target of rapamycin (mTOR) are central regulators of T cell differentiation, proliferation, metabolism, and survival. Here, we show that during chronic murine lymphocytic choriomeningitis virus infection, activation of AKT and mTOR are impaired in antiviral cytotoxic T lymphocytes (CTLs), resulting in enhanced activity of the transcription factor FoxO1. Blockade of inhibitory receptor programmed cell death protein 1 (PD-1) in vivo increased mTOR activity in virus-specific CTLs, and its therapeutic effects were abrogated by the mTOR inhibitor rapamycin. FoxO1 functioned as a transcriptional activator of PD-1 that promoted the differentiation of terminally exhausted CTLs. Importantly, FoxO1-null CTLs failed to persist and control chronic viral infection. Collectively, this study shows that CTLs adapt to persistent infection through a positive feedback pathway (PD-1/FoxO1/PD-1) that functions to both desensitize virus-specific CTLs to antigen and support their survival during chronic viral infection.