FOXP3+ Regulatory T Cells Require TBET to Regulate Activated CD8+ T Cells During Recovery from Influenza Infection.

FOXP3+ regulatory T (Treg) cells are necessary to coordinate resolution of lung inflammation and a return to homeostasis after respiratory viral infections, but the specific molecular requirements for these functions and the cell types governed by Treg cells remain unclear. This question holds significance as clinical trials of Treg cell transfer therapy for respiratory viral infection are being planned and executed. Here, we report causal experiments in mice determining that Treg cells are necessary to control the numbers of activated CD8+ T cells during recovery from influenza infection. Using a genetic strategy paired with adoptive transfer techniques, we determined that Treg cells require the transcription factor TBET to regulate these potentially pro-inflammatory CD8+ T cells. Surprisingly, we found that Treg cells are dispensable for the generation of CD8+ lung tissue resident-memory T (Trm) cells yet similarly influence the transcriptional programming of CD8+ Trm and activated T cells. Our study highlights the role of Treg cells in regulating the CD8+ T cell response during recovery from influenza infection.

Reduced expression of mitochondrial complex I subunit Ndufs2 does not impact healthspan in mice.

Aging in mammals leads to reduction in genes encoding the 45-subunit mitochondrial electron transport chain complex I. It has been hypothesized that normal aging and age-related diseases such as Parkinson's disease are in part due to modest decrease in expression of mitochondrial complex I subunits. By contrast, diminishing expression of mitochondrial complex I genes in lower organisms increases lifespan. Furthermore, metformin, a putative complex I inhibitor, increases healthspan in mice and humans. In the present study, we investigated whether loss of one allele of Ndufs2, the catalytic subunit of mitochondrial complex I, impacts healthspan and lifespan in mice. Our results indicate that Ndufs2 hemizygous mice (Ndufs2+/-) show no overt impairment in aging-related motor function, learning, tissue histology, organismal metabolism, or sensitivity to metformin in a C57BL6/J background. Despite a significant reduction of Ndufs2 mRNA, the mice do not demonstrate a significant decrease in complex I function. However, there are detectable transcriptomic changes in individual cell types and tissues due to loss of one allele of Ndufs2. Our data indicate that a 50% decline in mRNA of the core mitochondrial complex I subunit Ndufs2 is neither beneficial nor detrimental to healthspan.

Mitochondrial Metabolism Regulation of T Cell-Mediated Immunity.

Recent evidence supports the notion that mitochondrial metabolism is necessary for T cell activation, proliferation, and function. Mitochondrial metabolism supports T cell anabolism by providing key metabolites for macromolecule synthesis and generating metabolites for T cell function. In this review, we focus on how mitochondrial metabolism controls conventional and regulatory T cell fates and function.

Expansible residence decentralizes immune homeostasis.

In metazoans, specific tasks are relegated to dedicated organs that are established early in development, occupy discrete locations and typically remain fixed in size. The adult immune system arises from a centralized haematopoietic niche that maintains self-renewing potential1,2, and-upon maturation-becomes distributed throughout the body to monitor environmental perturbations, regulate tissue homeostasis and mediate organism-wide defence. Here we examine how immunity is integrated within adult mouse tissues, and address issues of durability, expansibility and contributions to organ cellularity. Focusing on antiviral T cell immunity, we observed durable maintenance of resident memory T cells up to 450 days after infection. Once established, resident T cells did not require the T cell receptor for survival or retention of a poised, effector-like state. Although resident memory indefinitely dominated most mucosal organs, surgical separation of parabiotic mice revealed a tissue-resident provenance for blood-borne effector memory T cells, and circulating memory slowly made substantial contributions to tissue immunity in some organs. After serial immunizations or cohousing with pet-shop mice, we found that in most tissues, tissue pliancy (the capacity of tissues to vary their proportion of immune cells) enables the accretion of tissue-resident memory, without axiomatic erosion of pre-existing antiviral T cell immunity. Extending these findings, we demonstrate that tissue residence and organ pliancy are generalizable aspects that underlie homeostasis of innate and adaptive immunity. The immune system grows commensurate with microbial experience, reaching up to 25% of visceral organ cellularity. Regardless of the location, many populations of white blood cells adopted a tissue-residency program within nonlymphoid organs. Thus, residence-rather than renewal or recirculation-typifies nonlymphoid immune surveillance, and organs serve as pliant storage reservoirs that can accommodate continuous expansion of the cellular immune system throughout life. Although haematopoiesis restores some elements of the immune system, nonlymphoid organs sustain an accrual of durable tissue-autonomous cellular immunity that results in progressive decentralization of organismal immune homeostasis.

Metabolic determinants of cellular fitness dependent on mitochondrial reactive oxygen species.

Mitochondria-derived reactive oxygen species (mROS) are required for the survival, proliferation, and metastasis of cancer cells. The mechanism by which mitochondrial metabolism regulates mROS levels to support cancer cells is not fully understood. To address this, we conducted a metabolism-focused CRISPR-Cas9 genetic screen and uncovered that loss of genes encoding subunits of mitochondrial complex I was deleterious in the presence of the mitochondria-targeted antioxidant mito-vitamin E (MVE). Genetic or pharmacologic inhibition of mitochondrial complex I in combination with the mitochondria-targeted antioxidants, MVE or MitoTEMPO, induced a robust integrated stress response (ISR) and markedly diminished cell survival and proliferation in vitro. This was not observed following inhibition of mitochondrial complex III. Administration of MitoTEMPO in combination with the mitochondrial complex I inhibitor phenformin decreased the leukemic burden in a mouse model of T cell acute lymphoblastic leukemia. Thus, mitochondrial complex I is a dominant metabolic determinant of mROS-dependent cellular fitness.

Maintenance DNA methylation is essential for regulatory T cell development and stability of suppressive function.

Tregs require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Tregs persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet nonuniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg genome for both lineage establishment and stability of identity and suppressive function.

Mitochondrial ubiquinol oxidation is necessary for tumour growth.

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.

CD4+ resident memory T cells dominate immunosurveillance and orchestrate local recall responses.

This study examines the extent to which memory CD4+ T cells share immunosurveillance strategies with CD8+ resident memory T cells (TRM). After acute viral infection, memory CD4+ T cells predominantly used residence to survey nonlymphoid tissues, albeit not as stringently as observed for CD8+ T cells. In contrast, memory CD4+ T cells were more likely to be resident within lymphoid organs than CD8+ T cells. Migration properties of memory-phenotype CD4+ T cells in non-SPF parabionts were similar, generalizing these results to diverse infections and conditions. CD4+ and CD8+ TRM shared overlapping transcriptional signatures and location-specific features, such as granzyme B expression in the small intestine, revealing tissue-specific and migration property-specific, in addition to lineage-specific, differentiation programs. Functionally, mucosal CD4+ TRM reactivation locally triggered both chemokine expression and broad immune cell activation. Thus, residence provides a dominant mechanism for regionalizing CD4+ T cell immunity, and location enforces shared transcriptional, phenotypic, and functional properties with CD8+ T cells.

Mitochondrial complex III is essential for suppressive function of regulatory T cells.

Regulatory T cells (Treg cells), a distinct subset of CD4+ T cells, are necessary for the maintenance of immune self-tolerance and homeostasis1,2. Recent studies have demonstrated that Treg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4+ effector subsets3,4. Furthermore, the Treg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration5,6; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of Treg cells. Here we report that Treg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting Treg cell number. Mice that lack mitochondrial complex III specifically in Treg cells displayed a loss of T cell-suppression capacity without altering Treg cell proliferation and survival. Treg cells deficient in complex III showed decreased expression of genes associated with Treg function, whereas Foxp3 expression remained stable. Loss of complex III in Treg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases7. Thus, Treg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function.

Hexokinase 2 is dispensable for T cell-dependent immunity.

BACKGROUND: T cells and cancer cells utilize glycolysis for proliferation. The hexokinase (1-4) family of enzymes catalyze the first step of glycolysis. Hexokinase 2 (HK2) is one of the most highly upregulated metabolic enzymes in both cancer and activated T cells. HK2 is required for the development and/or growth of cancer in several cancer models, but the necessity of HK2 in T cells is not fully understood. The clinical applicability of HK2 inhibition in cancer may be significantly limited by any potential negative effects of HK2 inhibition on T cells. Therefore, we investigated the necessity of HK2 for T cell function. In order to identify additional therapeutic cancer targets, we performed RNA-seq to compare in vivo proliferating T cells to T cell leukemia. METHODS: HK2 was genetically ablated in mouse T cells using a floxed Hk2 allele crossed to CD4-Cre. CD4+ and CD8+ cells from mice were characterized metabolically and tested in vitro. T cell function in vivo was tested in a mouse model of colitis, Th2-mediated lung inflammation, and viral infection. Treg function was tested by crossing Hk2-floxed mice to FoxP3-Cre mice. Hematopoietic function was tested by deleting HK2 from bone marrow with Vav1-iCre. RNA-seq was used to compare T cells proliferating in response to virus with primary T-ALL leukemia induced with mutant Notch1 expression. RESULTS: We unexpectedly report that HK2 is largely dispensable for in vitro T cell activation, proliferation, and differentiation. Loss of HK2 does not impair in vivo viral immunity and causes only a small impairment in the development of pathological inflammation. HK2 is not required for Treg function or hematopoiesis in vivo. One hundred sixty-seven metabolic genes were identified as being differentially expressed between T cells and leukemia. CONCLUSIONS: HK2 is a highly upregulated enzyme in cancer and in T cells. The requirement for HK2 in various cancer models has been described previously. Our finding that T cells are able to withstand the loss of HK2 indicates that HK2 may be a promising candidate for cancer therapy. Furthermore, we identify several other potential metabolic targets in T-ALL leukemia that could spare T cell function.

Cutting Edge: Evidence for Nonvascular Route of Visceral Organ Immunosurveillance by T Cells.

Lymphocytes enter tissues from blood vessels through a well-characterized three-step process of extravasation. To our knowledge, nonvascular routes of lymphocyte entry have not been described. In this article, we report that Ag-experienced CD8 T cells in mice recirculate from blood through the peritoneal cavity. In the event of infection, Ag-experienced CD8 T cell subsets adhered to visceral organs, indicating potential transcapsular immunosurveillance. Focusing on the male genital tract (MGT), we observed Ag-experienced CD8 T cell migration from the peritoneal cavity directly to the infected MGT across the capsule, which was dependent on the extracellular matrix receptor CD44. We also observed that, following clearance of infection, the MGT retained functional resident memory CD8 T cells. These data suggest that recirculation through body cavities may provide T cells with opportunities for broad immunosurveillance and potential nonvascular mechanisms of entry.

Mitochondria-ER Pas de Deux Controls Memory T Cell Function.

Memory CD8+ T cells mediate protective secondary immune responses. In this issue, Bantug et al. (2018) demonstrate that mTORC2-AKT-GSK3ß signaling at mitochondria-ER contact sites enables the TCA cycle flux that is necessary for memory CD8+ T cells to produce IFN-γ.

Quantifying Memory CD8 T Cells Reveals Regionalization of Immunosurveillance.

Memory CD8 T cells protect against intracellular pathogens by scanning host cell surfaces; thus, infection detection rates depend on memory cell number and distribution. Population analyses rely on cell isolation from whole organs, and interpretation is predicated on presumptions of near complete cell recovery. Paradigmatically, memory is parsed into central, effector, and resident subsets, ostensibly defined by immunosurveillance patterns but in practice identified by phenotypic markers. Because isolation methods ultimately inform models of memory T cell differentiation, protection, and vaccine translation, we tested their validity via parabiosis and quantitative immunofluorescence microscopy of a mouse memory CD8 T cell population. We report three major findings: lymphocyte isolation fails to recover most cells and biases against certain subsets, residents greatly outnumber recirculating cells within non-lymphoid tissues, and memory subset homing to inflammation does not conform to previously hypothesized migration patterns. These results indicate that most host cells are surveyed for reinfection by segregated residents rather than by recirculating cells that migrate throughout the blood and body.

At low precursor frequencies, the T-cell response to chronic self-antigen results in anergy without deletion.

The behavior of self-reactive T cells in the peripheral immune system has often been studied by following the fate of adoptively transferred antigen-specific T cells in antigen expressing mice. In most cases, after a period of expansion, such cells undergo a slow clonal deletion, accompanied by the onset of anergy and/or suppression in the remaining cells. Here, we demonstrate that at initial frequencies approaching those found in normal repertoires, it is possible to completely avoid deletion and still maintain peripheral tolerance. At starting numbers of <1000 T cells, stimulation by chronic self-antigens resulted in a period of robust clonal expansion, followed by a steady plateau phase extending beyond 4 months. Despite their stable persistence, the self-reactive T cells did not convert to a Foxp3⁺ fate. However, they displayed a considerable block in their ability to make IL-2, consistent with the onset of anergy - in a precursor frequency or deletion independent fashion.

Incomplete TCR-ß allelic exclusion accelerates spontaneous autoimmune arthritis in K/BxN TCR transgenic mice.

Allelic exclusion of antigen receptor loci is a fundamental mechanism of immunological self-tolerance. Incomplete allelic exclusion leads to dual T-cell receptor (TCR) expression and can allow developing autoreactive αß T lymphocytes to escape clonal deletion. Because allelic exclusion at the TCR-ß locus is more stringent than at the TCR-α locus, dual TCR-ß expression has not been considered a likely contributor to autoimmunity. We show here that incomplete TCR-ß allelic exclusion permits developing thymocytes bearing the autoreactive, transgene-encoded KRN TCR to be positively selected more efficiently, thereby accelerating the onset of spontaneous autoimmune arthritis. Our findings highlight dual TCR-ß expression as a mechanism that can enhance the maturation of autoreactive pathogenic T cells and lead to more rapid development of autoimmune disease.