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Target of rapamycin complex 1 (TORC1) integrates nutrient signals to control cell growth and organismal homeostasis across eukaryotes1–4. The evolutionarily conserved GATOR complex regulates TORC1 signaling through Rag GTPases with GATOR1 displaying GTPase activating protein (GAP) activity towards RagA/B and GATOR2 proposed as an inhibitor of GATOR15,6. Furthermore, the metazoan-specific Sestrin proteins function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B, and interact with GATOR2 with unknown functions7–9. Here we show that SZT2, a metazoan-specific protein mutated in epilepsy10–13, recruits a fraction of mammalian GATOR1 and GATOR2 to form SZT2-Orchestrated GATOR (SOG) complex with an essential role in GATOR- and Sestrin-dependent nutrient sensing and mTORC1 regulation. SZT2 interaction with GATOR1 and GATOR2 was cooperative, and an integral SOG was required for its localization on the lysosome. SZT2 deficiency resulted in constitutive mTORC1 signaling in cells under nutrient deprivation conditions and neonatal lethality in mice associated with failed mTORC1 inactivation during fasting. mTORC1 hyperactivation in SZT2-deficient cells could be partially corrected by overexpression of the GATOR1 component DEPDC5, and by a lysosome-targeted GATOR2 component WDR59 or a lysosome-targeted Sestrin2. These findings demonstrate a central role of SZT2 in dictating GATOR-dependent nutrient sensing by promoting lysosomal localization of SOG, and reveal an unexpected function of lysosome-located GATOR2 in suppressing mTORC1 signaling via Sestrin recruitment.

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.

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.

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.

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.

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.

Tumors develop in a complex microenvironment alongside numerous cell types that impact their survival. Immune cells make up a large proportion of these accessory cells and many are known to promote tumor progression. Macrophages, in particular, are associated with poor patient prognosis and are therefore potential candidates for therapeutic targeting in cancer. However, to develop successful strategies to target macrophages, it is important to clarify whether these cells are derived from blood-borne precursors or a tissue-resident population. Parabiosis, or the surgical connection of two mice resulting in a shared blood circulation, allows the distinction between these two cellular sources. Here, we describe the use of parabiosis to define cell ontogeny in a mouse model of breast cancer.

Regulatory T cells (TReg cells), a specialized T cell lineage, have a pivotal function in the control of self tolerance and inflammatory responses. Recent studies have revealed a discrete mode of T cell receptor (TCR) signalling that regulates TReg cell differentiation, maintenance and function and that affects gene expression, metabolism, cell adhesion and migration of these cells. Here, we discuss the emerging understanding of TCR-guided differentiation of TReg cells in the context of their function in health and disease.

Macrophages are innate immune cells with evolutionarily conserved functions in tissue maintenance and host defense. As such, macrophages are among the first hematopoietic cells that seed developing tissues, and respond to inflammatory insults by in situ proliferation or de novo differentiation from monocytes. Recent studies have revealed that monocyte-derived tumor-induced macrophages represent a major tumor-associated macrophage population, which can further expand following their differentiation in tumors. Compared to tissue-resident tumor-associated macrophages, these newly differentiated cells are phenotypically distinct, and likely play a unique role in tissue dysregulation and immune modulation in cancer. These findings imply that tumor growth elicits a specific innate immune response. In this review, we explore the different routes of macrophage seeding and maintenance in tissues during steady state and inflammation and how these principles underlie the responses observed during tumor development. In addition, we highlight the relationship between the origin and function of macrophages in different settings and how this knowledge may be used to create new opportunities for cancer immunotherapy.

SummaryRegulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to preaccessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3-bound Treg cell enhancers lacking in Foxp3− CD4+ cells became accessible upon T cell receptor activation prior to Foxp3 expression, and only a small subset associated with several functionally important genes were exclusively Treg cell specific. Thus, in a late cellular differentiation process, Foxp3 defines Treg cell functionality in an “opportunistic” manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.

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.

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.

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.

Infiltrating T cells in the tumor microenvironment have crucial roles in the competing processes of pro-tumor and anti-tumor immune response. However, the infiltration level of distinct T cell subsets and the signals that draw them into a tumor, such as the expression of antigen presenting machinery (APM) genes, remain poorly characterized across human cancers. Here, we define a novel mRNA-based T cell infiltration score (TIS) and profile infiltration levels in 19 tumor types. We find that clear cell renal cell carcinoma (ccRCC) is the highest for TIS and among the highest for the correlation between TIS and APM expression, despite a modest mutation burden. This finding is contrary to the expectation that immune infiltration and mutation burden are linked. To further characterize the immune infiltration in ccRCC, we use RNA-seq data to computationally infer the infiltration levels of 24 immune cell types in a discovery cohort of 415 ccRCC patients and validate our findings in an independent cohort of 101 ccRCC patients. We find three clusters of tumors that are primarily separated by levels of T cell infiltration and APM gene expression. In ccRCC, the levels of Th17 cells and the ratio of CD8+ T/Treg levels are associated with improved survival whereas the levels of Th2 cells and Tregs are associated with negative clinical outcome. Our analysis illustrates the utility of computational immune cell decomposition for solid tumors, and the potential of this method to guide clinical decision-making.

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.

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06249.001, eLife digest Cells contain thousands of genes that are encoded by molecules of DNA. In yeast and other eukaryotic organisms, this DNA is wrapped around proteins called histones to make structures called nucleosomes. This compacts the DNA and allows it to fit inside the tiny nucleus within the cell. The positioning of the nucleosomes influences how tightly packed the DNA is, which in turn influences the activity of genes. Less active genes tend to be found within regions of DNA that are tightly packed, while more active genes are found in less tightly packed regions. To activate a gene, proteins called transcription factors bind to a section of DNA within the gene called the promoter. Enzymes known as ‘chromatin remodelers’ can alter the locations of nucleosomes on DNA to allow the transcription factors access to the promoters of particular genes. In yeast, the SWI/SNF family of chromatin remodelers can disassemble nucleosomes to promote gene activity, while the ISW1 family organises nucleosomes into closely spaced groups to repress gene activity. However, it is not clear if, or how, chromatin remodelers can influence transcription factors that are already bound to DNA. Here, Li et al. studied the interactions between a transcription factor and the chromatin remodelers in yeast. The experiment used a piece of DNA that contained a bound transcription factor and a single nucleosome. Li et al. used a technique called ‘single molecule DNA unzipping’, which enabled them to precisely locate the position of the nucleosome and transcription factor before and after the nucleosome was remodeled. The experiments found that a chromatin remodeler called ISW1a moved the nucleosome away from the transcription factor, while a SWI/SNF chromatin remodeler moved the nucleosome towards it. Significantly, Li et al. also found that a transcription factor is a major barrier to ISW1a's remodeling activity, suggesting that ISW1a may use transcription factors as reference points to position nucleosomes. In contrast, SWI/SNF was able to slide a nucleosome past the transcription factor, which led to the transcription factor falling off the DNA. Therefore, SWI/SNF is able to move transcription factors out of the way to deactivate genes. Li et al. propose a new model for how chromatin remodelers can move nucleosomes and regulate transcription factors to alter gene activity. A future challenge will be to observe these types of activities in living cells. DOI: http://dx.doi.org/10.7554/eLife.06249.002

We have previously isolated insulin-reactive Tregs from diabetic NOD mice designated 2H6, from which TCR transgenic mice were generated. The T cells from these 2H6 transgenic mice recognize insulin but have suppressive properties in vitro. They protect NOD mice in vivo from spontaneous development of diabetes and adoptive transfer of disease caused by polyclonal diabetogenic spleen cells as well as the highly diabetogenic monoclonal BDC2.5 TCR transgenic T cells that recognize an islet granule antigen. Using cells from both NOD and BDC2.5 mice that express a dominant-negative TGF-β receptor type II (TGF-βDNRII), we show that 2H6 T cells protected from disease by producing TGF-β and that the ability of the target diabetogenic T cells to respond to TGF-β was crucial. We further demonstrate that TGF-β signaling in 2H6 cells was important for their protective properties, as 2H6 cells were unable to protect from adoptive transfer–induced diabetes if they were unable to respond to TGF-β. Thus, our data demonstrate that insulin-specific regulatory cells protect from diabetes by virtue of their production of TGF-β1 that acts in an autocrine manner to maintain their regulatory function and acts in a paracrine manner on the target cells.

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.

Transforming growth factor (TGF)-beta is a potent suppressive cytokine with an essential role in T-cell tolerance. TGF-beta acts on T cells to inhibit T-cell activation and effector T-cell differentiation and promote peripheral regulatory T cells. Recently there has been growing interest in targeting the TGF-beta pathway for the treatment of autoimmune diseases and cancer. Anti-CD3 antibody therapy of autoimmune diabetes leads to T-cell production of TGF-beta and the consequent inhibition of autoaggressive T cells. Inhibition of TGF-beta2 expression in gliomas restores the antitumor immune response, which appears to be a promising strategy to treat these tumors. Future studies will elucidate the cellular and molecular mechanisms of TGF-beta regulation of T-cell responses in autoimmune diseases and cancer, which will be crucial for the optimization of immunotherapeutic strategies and for the identification of novel drug targets.

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.

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.

TGF-β1 deficient mice develop multifocal inflammatory autoimmune disease and serve as a valuable animal model of autoimmunity. Transgenic expression of a dominant negative form of TGF-β receptor type II in T cells have enabled the study of cell lineage specific effects of TGF-β providing clues to the potential etiology of autoimmunity. These studies suggest that TGF-β deficiency may induce autoimmune disease by influencing a number of immunological phenomena including lymphocyte activation and differentiation, cell adhesion molecule expression, regulatory T cell function, the expression of MHC molecules and cytokines, and cell apoptosis. The spectrum of effects appears to be significant in mucosal immunity and may contribute to the pathogenesis of inflammatory bowel disease.

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.

Asymmetric division in CD8 T cells produces two daughter cells expressing different levels of c-Myc with distinct metabolic profiles. Manipulating this asymmetric partitioning of c-Myc skews T cell responses and potentially allows the development of more effective vaccines and cancer immunotherapies.

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.

511 Background: Clear cell renal cell carcinoma (ccRCC) tumors develop mechanisms that impair function and/or prevent entry of the host infiltrating immune cells (immune exclusion) within the tumor microenvironment. The goal of immunotherapy is to overcome this immune resistance. We aim to characterize the T−cell populations in a cohort of largely untreated high risk patients with ccRCC. Methods: We prospectively collected ccRCC tumor and adjacent normal kidney (NK) from patients undergoing surgical resection at our institution from 6/2015-8/2016. Immune cell phenotyping was performed by immune cell staining of single cell suspensions. Analysis of immune cell populations were determined by CD45+ staining and corresponding proportions of different T−cell populations (CD3+, CD4+, CD4+Treg, and CD8+ T cells). Staining for CD4+Treg was not available for two patients. Student t−test was utilized to compare the immune populations between tumor and adjacent NK tissue. Analysis was also conducted by stratifying patients who presented with localized versus metastatic disease. Results: A total of 31 tumor and adjacent NK were analyzed. Median tumor pathological size was 8.5cm (2.9cm−18cm), 27(87%) had pT3a−pT3b and 13(42%) presented with metastatic disease. Overall 84% of tumors had higher immune infiltrate with an average ratio of four-fold increase compared to adjacent NK as determined by CD45+ cells. Intriguingly, the other 16% presented with metastatic (4) or rapidly metastatic disease (1). Orthogonal validation with inferred immune populations using RNAseq data from the The Cancer Genome Atlas (TCGA) demonstrated similar aggressive behavior in tumors with lower immune infiltrate compared to NK. Comparison of immune cell populations of tumor and NK tissue is shown in table 1. No specific T-cell subtype was associated with specific clinical outcomes in this cohort. Conclusions: Our data shows a general trend of immune infiltration in ccRCC when compared to adjacent NK with a diversity of T-cell subsets and possible evidence of immune exclusion. Further genomic characterization of these tumors is currently underway. [Table: see text]

Clinical and experimental models have identified macrophages as potential targets for cancer therapy, however, the nature of macrophage differentiation and function in the context of malignant disease remain largely uncharacterized. This commentary provides the author's perspective on the recently published article “The cellular and molecular origin of tumor-associated macrophages,” which demonstrated that tumor growth elicits a specific macrophage differentiation pathway.

Upon pathogen encounter, naive CD8+ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8+ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian’s group to generate large number of CD44hiCD62Lhi antigen-specific memory T cells in vitro.

Long recognized as an evolutionarily ancient cell type involved in tissue homeostasis and immune defense against pathogens, macrophages are being rediscovered as regulators of several diseases, including cancer. Here we show that in mice, mammary tumor growth induces the accumulation of tumor-associated macrophages (TAMs) that are phenotypically and functionally distinct from mammary tissue macrophages (MTMs). TAMs express the adhesion molecule Vcam1 and proliferate upon their differentiation from inflammatory monocytes, but do not exhibit an “alternatively activated” phenotype. TAM terminal differentiation depends on the transcriptional regulator of Notch signaling, RBPJ; and TAM, but not MTM, depletion restores tumor-infiltrating cytotoxic T cell responses and suppresses tumor growth. These findings reveal the ontogeny of TAMs and a discrete tumor-elicited inflammatory response, which may provide new opportunities for cancer immunotherapy.

The 1980s witnessed the identification of numerous secreted proteins that participate in intercellular communication. For immunologists, much attention was paid to the soluble proteins produced specifically by activated lymphocytes or myeloid cells. These factors belong to several cytokine families

The G1 kinase CDK4 is amplified or overexpressed in some human tumors and promotes tumorigenesis by inhibiting known tumor suppressors. Here, we report that CDK4 deficiency markedly accelerated lymphoma development in the Eμ-Myc transgenic mouse model of B lymphoma and that silencing or loss of CDK4 augmented the tumorigenic potential of Myc-driven mouse and human B cell lymphoma in transplant models. Accelerated disease in CDK4-deficient Eμ-Myc transgenic mice was associated with rampant genomic instability that was provoked by dysregulation of a FOXO1/RAG1/RAG2 pathway. Specifically, CDK4 phosphorylated and inactivated FOXO1, which prevented FOXO1-dependent induction of Rag1 and Rag2 transcription. CDK4-deficient Eμ-Myc B cells had high levels of the active form of FOXO1 and elevated RAG1 and RAG2. Furthermore, overexpression of RAG1 and RAG2 accelerated lymphoma development in a transplant model, with RAG1/2-expressing tumors exhibiting hallmarks of genomic instability. Evaluation of human tumor samples revealed that CDK4 expression was markedly suppressed, while FOXO1 expression was elevated, in several subtypes of human non-Hodgkin B cell lymphoma. Collectively, these findings establish a context-specific tumor suppressor function for CDK4 that prevents genomic instability, which contributes to B cell lymphoma. Furthermore, our data suggest that targeting CDK4 may increase the risk for the development and/or progression of lymphoma.

Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSElow vs. unpulsed target cells-CFSEhi. The frequencies between antigen-pulsed target cells and control target cells are used as readouts of antigen-specific killing.

Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-κB and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-κB activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-κB/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.

Double-stranded RNA (dsRNA) molecules generated during virus infection can initiate a host antiviral response to limit further infection. Such a response involves induction of antiviral gene expression by the dsRNA-activated protein kinase (PKR) and the NF-kappaB transcription factor. In addition, dsRNA can also induce apoptosis by an incompletely understood mechanism that may serve to further limit viral replication. Here we demonstrate a novel role for the RelA subunit of NF-kappaB in inhibiting dsRNA-induced cell death. dsRNA treatment resulted in caspase 3 activation and apoptotic morphological transformations in mouse embryonic fibroblasts (MEFs) derived from RelA-/- mice but not from RelA+/+ mice. Such dsRNA-induced killing could be inhibited by expression of either a dominant-negative mutant of PKR or wild-type RelA. Interestingly, caspase 3 activated following dsRNA treatment of RelA-/- MEFs was essential for apoptotic nuclear changes but dispensable for cytotoxicity. A broader specificity caspase inhibitor was also unable to inhibit dsRNA-induced cytotoxicity, suggesting that caspase activation is not essential for the induction of cell death by dsRNA in MEFs. However, combined inhibition of caspase 3 and reactive oxygen species production resulted in complete inhibition of dsRNA-induced cytotoxicity. These results demonstrate an essential role for NF-kappaB in protecting cells from dsRNA-induced apoptosis and suggest that NF-kappaB may inhibit both caspase-dependent and reactive oxygen species-dependent cytotoxic pathways.

A fundamental aspect of the adaptive immune system is the generation and maintenance of a diverse and self-tolerant T cell repertoire. Through its regulation of T cell development, homeostasis, tolerance, and differentiation, the highly evolutionarily conserved cytokine TGF-β critically supports a functional T cell pool. The pleiotropic nature of this regulation is likely due to the elaborate control of TGF-β production and activation in the immune system, and the intricacy of TGF-β signaling pathways. In this review we discuss the current understanding of TGF-β regulation of T cells.