Nutrients: Signal 4 in T cell immunity.

T cells are integral in mediating adaptive immunity to infection, autoimmunity, and cancer. Upon immune challenge, T cells exit from a quiescent state, followed by clonal expansion and effector differentiation. These processes are shaped by three established immune signals, namely antigen stimulation (Signal 1), costimulation (Signal 2), and cytokines (Signal 3). Emerging findings reveal that nutrients, including glucose, amino acids, and lipids, are crucial regulators of T cell responses and interplay with Signals 1-3, highlighting nutrients as Signal 4 to license T cell immunity. Here, we first summarize the functional importance of Signal 4 and the underlying mechanisms of nutrient transport, sensing, and signaling in orchestrating T cell activation and quiescence exit. We also discuss the roles of nutrients in programming T cell differentiation and functional fitness and how nutrients can be targeted to improve disease therapy. Understanding how T cells respond to Signal 4 nutrients in microenvironments will provide insights into context-dependent functions of adaptive immunity and therapeutic interventions.

PTEN directs developmental and metabolic signaling for innate-like T cell fate and tissue homeostasis.

Phosphatase and tensin homologue (PTEN) is frequently mutated in human cancer, but its roles in lymphopoiesis and tissue homeostasis remain poorly defined. Here we show that PTEN orchestrates a two-step developmental process linking antigen receptor and IL-23-Stat3 signalling to type-17 innate-like T cell generation. Loss of PTEN leads to pronounced accumulation of mature IL-17-producing innate-like T cells in the thymus. IL-23 is essential for their accumulation, and ablation of IL-23 or IL-17 signalling rectifies the reduced survival of female PTEN-haploinsufficient mice that model human patients with PTEN mutations. Single-cell transcriptome and network analyses revealed the dynamic regulation of PTEN, mTOR and metabolic activities that accompanied type-17 cell programming. Furthermore, deletion of mTORC1 or mTORC2 blocks PTEN loss-driven type-17 cell accumulation, and this is further shaped by the Foxo1 and Stat3 pathways. Collectively, our study establishes developmental and metabolic signalling networks underpinning type-17 cell fate decisions and their functional effects at coordinating PTEN-dependent tissue homeostasis.

CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.

Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.

Investigating the Dynamic Changes in iNKT Cell Metabolic Profiles During Development.

Emerging research has highlighted the importance of metabolic pathways and metabolites in dictating immune cell lineage decisions during thymocyte development. Here, we discuss several complementary approaches, including flow cytometry, metabolic flux, and transcriptome analyses, to characterize the dynamic changes in metabolic profiles associated with invariant natural killer T cell development.

Metabolic Control of Memory T-Cell Generation and Stemness.

The formation of long-lived memory T cells is a critical feature of the adaptive immune response. T cells undergo metabolic reprogramming to establish a functional memory population. While initial studies characterized key metabolic pathways necessary for memory T-cell development, recent findings highlight that metabolic regulation of memory T-cell subsets is diverse. Here we describe the different requirements for metabolic programs and metabolism-related signaling pathways in memory T-cell development. We further discuss the contribution of cellular metabolism to memory T-cell functional reprogramming and stemness within acute and chronic inflammatory environments. Last, we highlight knowledge gaps and propose approaches to determine the roles of metabolites and metabolic enzymes in memory T-cell fate. Understanding how cellular metabolism regulates a functionally diverse memory population will undoubtedly provide new therapeutic insights to modulate protective T-cell immunity in human disease.

In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.

How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.

IL-10-producing Tfh cells accumulate with age and link inflammation with age-related immune suppression.

Aging results in profound immune dysfunction, resulting in the decline of vaccine responsiveness previously attributed to irreversible defects in the immune system. In addition to increased interleukin-6 (IL-6), we found aged mice exhibit increased systemic IL-10 that requires forkhead box P3-negative (FoxP3-), but not FoxP3+, CD4+T cells. Most IL-10-producing cells manifested a T follicular helper (Tfh) phenotype and required the Tfh cytokines IL-6 and IL-21 for their accrual, so we refer to them as Tfh10 cells. IL-21 was also required to maintain normal serum levels of IL-6 and IL-10. Notably, antigen-specific Tfh10 cells arose after immunization of aged mice, and neutralization of IL-10 receptor signaling significantly restored Tfh-dependent antibody responses, whereas depletion of FoxP3+ regulatory and follicular regulatory cells did not. Thus, these data demonstrate that immune suppression with age is reversible and implicate Tfh10 cells as an intriguing link between "inflammaging" and impaired immune responses with age.

Hippo/Mst signaling coordinates cellular quiescence with terminal maturation in iNKT cell development and fate decisions.

Invariant natural killer T (iNKT) cells acquire effector functions during development by mechanisms that remain poorly understood. Here, we show that the Hippo kinases Mst1 and Mst2 act as molecular rheostats for the terminal maturation and effector differentiation programs of iNKT cells. Loss of Mst1 alone or together with Mst2 impedes iNKT cell development, associated with defective IL-15-dependent cell survival. Mechanistically, Mst1 enforces iNKT cellular and transcriptional quiescence associated with maturation and commitment to iNKT1 cells by suppressing proliferation and Opa1-related mitochondrial metabolism that are dynamically regulated during iNKT cell development. Furthermore, Mst1 shapes the reciprocal fate decisions between iNKT1 and iNKT17 effector cells, which respectively depend upon mitochondrial dynamics and ICOS-mTORC2 signaling. Collectively, these findings establish Mst1 as a crucial regulator of mitochondrial homeostasis and quiescence in iNKT cell development and effector lineage differentiation and highlight that establishment of quiescence programs underlies iNKT cell development and effector maturation.

Signaling networks in immunometabolism.

Adaptive immunity is essential for pathogen and tumor eradication, but may also trigger uncontrolled or pathological inflammation. T cell receptor, co-stimulatory and cytokine signals coordinately dictate specific signaling networks that trigger the activation and functional programming of T cells. In addition, cellular metabolism promotes T cell responses and is dynamically regulated through the interplay of serine/threonine kinases, immunological cues and nutrient signaling networks. In this review, we summarize the upstream regulators and signaling effectors of key serine/threonine kinase-mediated signaling networks, including PI3K-AGC kinases, mTOR and LKB1-AMPK pathways that regulate metabolism, especially in T cells. We also provide our perspectives about the pending questions and clinical applicability of immunometabolic signaling. Understanding the regulators and effectors of immunometabolic signaling networks may uncover therapeutic targets to modulate metabolic programming and T cell responses in human disease.

The Variable Genomic NK Cell Receptor Locus Is a Key Determinant of CD4+ T Cell Responses During Viral Infection.

Increasing evidence points to a key role for NK cells in controlling adaptive immune responses. In studies examining the role of CD1d on CD4+ T cell responses, we found that a line of CD1d-deficient mice on the C57BL/6J background had a homozygous 129 locus on chromosome 6 containing the entire NK cell gene cluster. Mice possessing this locus (C57BL/6.NKC129) displayed a >10-fold reduction in antigen-specific CD4+ T cell responses after intracranial infection with lymphocytic choriomeningitis virus (LCMV). Neither parental strain displayed defects in viral-specific CD4+ T cell responses. Interestingly, following infection, increased numbers of NK cells accumulated in the lymph nodes of C57BL/6.NKC129 mice and displayed enhanced in vivo functionality. Moreover, depletion of NK cells with anti-asialo-GM-1 antibody in C57BL/6.NKC129 mice resulted in a >20-fold increase in viral-specific CD4+ T cell responses. Mechanistically, we found that dendritic cell antigen presentation and early type I IFN production were significantly decreased in C57BL/6.NKC129 mice, but were restored in perforin-deficient C57BL/6.NKC129 mice or following NK depletion. Together, these data reveal that the variable genomic regions containing the activating/inhibitory NK cell receptors are key determinants of antigen-specific CD4+ T cell responses, controlling type I IFN production and the antigen-presenting capacity of dendritic cells.

Homeostasis and transitional activation of regulatory T cells require c-Myc.

Regulatory T cell (Treg) activation and expansion occur during neonatal life and inflammation to establish immunosuppression, yet the mechanisms governing these events are incompletely understood. We report that the transcriptional regulator c-Myc (Myc) controls immune homeostasis through regulation of Treg accumulation and functional activation. Myc activity is enriched in Tregs generated during neonatal life and responding to inflammation. Myc-deficient Tregs show defects in accumulation and ability to transition to an activated state. Consequently, loss of Myc in Tregs results in an early-onset autoimmune disorder accompanied by uncontrolled effector CD4+ and CD8+ T cell responses. Mechanistically, Myc regulates mitochondrial oxidative metabolism but is dispensable for fatty acid oxidation (FAO). Indeed, Treg-specific deletion of Cox10, which promotes oxidative phosphorylation, but not Cpt1a, the rate-limiting enzyme for FAO, results in impaired Treg function and maturation. Thus, Myc coordinates Treg accumulation, transitional activation, and metabolic programming to orchestrate immune homeostasis.

Tissue-specific control of latent CMV reactivation by regulatory T cells.

Cytomegalovirus (CMV) causes a persistent, lifelong infection. CMV persists in a latent state and undergoes intermittent subclinical viral reactivation that is quelled by ongoing T cell responses. While T cells are critical to maintain control of infection, the immunological factors that promote CMV persistence remain unclear. Here, we investigated the role of regulatory T cells (Treg) in a mouse model of latent CMV infection using Foxp3-diphtheria toxin receptor (Foxp3-DTR) mice. Eight months after infection, MCMV had established latency in the spleen, salivary gland, lung, and pancreas, which was accompanied by an increased frequency of Treg. Administration of diphtheria toxin (DT) after establishment of latency efficiently depleted Treg and drove a significant increase in the numbers of functional MCMV-specific CD4+ and CD8+ T cells. Strikingly, Treg depletion decreased the number of animals with reactivatable latent MCMV in the spleen. Unexpectedly, in the same animals, ablation of Treg drove a significant increase in viral reactivation in the salivary gland that was accompanied with augmented local IL-10 production by Foxp3-CD4+T cells. Further, neutralization of IL-10 after Treg depletion significantly decreased viral load in the salivary gland. Combined, these data show that Treg have divergent control of MCMV infection depending upon the tissue. In the spleen, Treg antagonize CD8+ effector function and promote viral persistence while in the salivary gland Treg prevent IL-10 production and limit viral reactivation and replication. These data provide new insights into the organ-specific roles of Treg in controlling the reactivation of latent MCMV infection.

Nutrient and Metabolic Sensing in T Cell Responses.

T cells play pivotal roles in shaping host immune responses in infectious diseases, autoimmunity, and cancer. The activation of T cells requires immune and growth factor-derived signals. However, alterations in nutrients and metabolic signals tune T cell responses by impinging upon T cell fates and immune functions. In this review, we summarize how key nutrients, including glucose, amino acids, and lipids, and their sensors and transporters shape T cell responses. We also briefly discuss regulation of T cell responses by oxygen and energy sensing mechanisms.

IL-6 and ICOS Antagonize Bim and Promote Regulatory T Cell Accrual with Age.

Regulatory T cells (Tregs), a subset of CD4(+) T cells, dramatically accumulate with age in humans and mice and contribute to age-related immune suppression. Recently, we showed that a majority of accumulating Tregs in aged mice expressed low levels of CD25, and their accrual is associated with declining levels of IL-2 in aged mice. In this study, we further investigated the origin of CD25(lo) Tregs in aged mice. First, aged Tregs had high expression of neuropilin-1 and Helios, and had a broad Vß repertoire. Next, we analyzed the gene expression profile of Tregs, naive T cells, and memory T cells in aged mice. We found that the gene expression profile of aged CD25(lo) Tregs were more related to young CD25(lo) Tregs than to either naive or memory T cells. Further, the gene expression profile of aged Tregs was consistent with recently described "effector" Tregs (eTregs). Additional analysis revealed that nearly all Tregs in aged mice were of an effector phenotype (CD44(hi)CD62L(lo)) and could be further characterized by high levels of ICOS and CD69. ICOS contributed to Treg maintenance in aged mice, because in vivo Ab blockade of ICOSL led to a loss of eTregs, and this loss was rescued in Bim-deficient mice. Further, serum levels of IL-6 increased with age and contributed to elevated expression of ICOS on aged Tregs. Finally, Treg accrual was significantly blunted in aged IL-6-deficient mice. Together, our data show a role for IL-6 in promoting eTreg accrual with age likely through maintenance of ICOS expression.

IL-15 Fosters Age-Driven Regulatory T Cell Accrual in the Face of Declining IL-2 Levels.

We and others have shown that regulatory T cells (Treg) accumulate dramatically with age in both humans and mice. Such Treg accrual contributes to age-related immunosenescence as they reduce the response to tumors and parasite infection. While we reported earlier that aged Treg have decreased expression of the pro-apoptotic molecule Bim and germline deletion of Bim promoted earlier accumulation of Treg, it remains unclear whether the effects of Bim are: (i) Treg intrinsic and (ii) dominant to other BH3-only pro-apoptotic molecules. Further, the mechanism(s) controlling Bim expression in aged Treg remain unclear. Here we show that Treg-specific loss of Bim is sufficient to drive Treg accrual with age and that additional loss of the downstream apoptotic effectors Bax and Bak did not exacerbate Treg accumulation. Further, our results demonstrate that a subpopulation of Treg expands with age and is characterized by lower expression of CD25 (IL-2Rα) and Bim. Mechanistically, we found that IL-2 levels decline with age and likely explain the emergence of CD25(lo)Bim(lo) Treg because Treg in IL-2(-/-) mice are almost entirely comprised of CD25(lo)Bim(lo) cells, and IL-2 neutralization increases CD25(lo)Bim(lo) Treg in both young and middle-aged mice. Interestingly, the Treg population in aged mice had increased expression of CD122 (IL-2/IL-15Rß) and neutralization or genetic loss of IL-15 led to less Treg accrual with age. Further, the decreased Treg accrual in middle-aged IL-15(-/-) mice was restored by the additional loss of Bim (IL-15(-/-)Bim(-/-)). Together, our data show that aging favors the accrual of CD25(lo) Treg whose homeostasis is supported by IL-15 as IL-2 levels become limiting. These data have implications for manipulating Treg to improve immune responses in the elderly.

Homeostasis and function of regulatory T cells in aging.

A hallmark of aging is the progressive deterioration of immune function. Age-related immune suppression increases susceptibility to infectious diseases and cancer, significant causes of morbidity and mortality in the elderly. In particular, age-related T cell dysfunction is a major contributor to 'immune-senescence'. Recently, it has become clear that the frequency of regulatory T cells (Treg) significantly increases in aged mice and humans. As Treg control the intensity of T cell responses, their accrual probably contributes to age-related immune dysfunction. This review will focus on mechanisms underlying Treg homeostasis and function in aging.

A major role for Bim in regulatory T cell homeostasis.

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4(+) T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bim(lo) Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.