IκBζ is an essential mediator of immunity to oropharyngeal candidiasis.

Fungal infections are a global threat; yet, there are no licensed vaccines to any fungal pathogens. Th17 cells mediate immunity to Candida albicans, particularly oropharyngeal candidiasis (OPC), but essential downstream mechanisms remain unclear. In the murine model of OPC, IκBζ (Nfkbiz, a non-canonical NF-κB transcription factor) was upregulated in an interleukin (IL)-17-dependent manner and was essential to prevent candidiasis. Deletion of Nfkbiz rendered mice highly susceptible to OPC. IκBζ was dispensable in hematopoietic cells and acted partially in the suprabasal oral epithelium to control OPC. One prominent IκBζ-dependent gene target was ß-defensin 3 (BD3) (Defb3), an essential antimicrobial peptide. Human oral epithelial cells required IκBζ for IL-17-mediated induction of BD2 (DEFB4A, human ortholog of mouse Defb3) through binding to the DEFB4A promoter. Unexpectedly, IκBζ regulated the transcription factor Egr3, which was essential for C. albicans induction of BD2/DEFB4A. Accordingly, IκBζ and Egr3 comprise an antifungal signaling hub mediating mucosal defense against oral candidiasis.

To loop or not to loop? CNS-28 is the answer.

Regulation of interferon-γ is critical to constrain inflammation and mount effective responses to infection and anti-tumor immunity. In this issue of Immunity, Cui et al. identify a distal silencer element that disrupts promoter-enhancer looping, regulating IFN-γ expression and preventing inappropriate inflammation.

Excess Dietary Sugar Alters Colonocyte Metabolism and Impairs the Proliferative Response to Damage.

BACKGROUND & AIMS: The colonic epithelium requires continuous renewal by crypt resident intestinal stem cells (ISCs) and transit-amplifying (TA) cells to maintain barrier integrity, especially after inflammatory damage. The diet of high-income countries contains increasing amounts of sugar, such as sucrose. ISCs and TA cells are sensitive to dietary metabolites, but whether excess sugar affects their function directly is unknown. METHODS: Here, we used a combination of 3-dimensional colonoids and a mouse model of colon damage/repair (dextran sodium sulfate colitis) to show the direct effect of sugar on the transcriptional, metabolic, and regenerative functions of crypt ISCs and TA cells. RESULTS: We show that high-sugar conditions directly limit murine and human colonoid development, which is associated with a reduction in the expression of proliferative genes, adenosine triphosphate levels, and the accumulation of pyruvate. Treatment of colonoids with dichloroacetate, which forces pyruvate into the tricarboxylic acid cycle, restored their growth. In concert, dextran sodium sulfate treatment of mice fed a high-sugar diet led to massive irreparable damage that was independent of the colonic microbiota and its metabolites. Analyses on crypt cells from high-sucrose-fed mice showed a reduction in the expression of ISC genes, impeded proliferative potential, and increased glycolytic potential without a commensurate increase in aerobic respiration. CONCLUSIONS: Taken together, our results indicate that short-term, excess dietary sucrose can directly modulate intestinal crypt cell metabolism and inhibit ISC/TA cell regenerative proliferation. This knowledge may inform diets that better support the treatment of acute intestinal injury.

Regulation and Immunotherapeutic Targeting of the Epigenome in Exhausted CD8 T Cell Responses.

Exhaustion is a state of CD8 T cell differentiation that occurs in settings of chronic Ag such as tumors, chronic viral infection, and autoimmunity. Cellular differentiation is driven by a series of environmental signals that promote epigenetic landscapes that set transcriptomes needed for function. For CD8 T cells, the epigenome that underlies exhaustion is distinct from effector and memory cell differentiation, suggesting that signals early on set in motion a process where the epigenome is modified to promote a trajectory toward a dysfunctional state. Although we know many signals that promote exhaustion, putting this in the context of the epigenetic changes that occur during differentiation has been less clear. In this review, we aim to summarize the epigenetic changes associated with exhaustion in the context of signals that promote it, highlighting immunotherapeutic studies that support these observations or areas for future therapeutic opportunities.

Hypoxia drives CD39-dependent suppressor function in exhausted T cells to limit antitumor immunity.

CD8+ T cells are critical for elimination of cancer cells. Factors within the tumor microenvironment (TME) can drive these cells to a hypofunctional state known as exhaustion. The most terminally exhausted T (tTex) cells are resistant to checkpoint blockade immunotherapy and might instead limit immunotherapeutic efficacy. Here we show that intratumoral CD8+ tTex cells possess transcriptional features of CD4+Foxp3+ regulatory T cells and are similarly capable of directly suppressing T cell proliferation ex vivo. tTex cell suppression requires CD39, which generates immunosuppressive adenosine. Restricted deletion of CD39 in endogenous CD8+ T cells resulted in slowed tumor progression, improved immunotherapy responsiveness and enhanced infiltration of transferred tumor-specific T cells. CD39 is induced on tTex cells by tumor hypoxia, thus mitigation of hypoxia limits tTex suppression. Together, these data suggest tTex cells are an important regulatory population in cancer and strategies to limit their generation, reprogram their immunosuppressive state or remove them from the TME might potentiate immunotherapy.

Tumor microenvironmental signals reshape chromatin landscapes to limit the functional potential of exhausted T cells.

Response rates to immunotherapy in solid tumors remain low due in part to the elevated prevalence of terminally exhausted T cells, a hypofunctional differentiation state induced through persistent antigen and stress signaling. However, the mechanisms promoting progression to terminal exhaustion in the tumor remain undefined. Using the low-input chromatin immunoprecipitation sequencing method CUT&RUN, we profiled the histone modification landscape of tumor-infiltrating CD8+ T cells throughout differentiation. We found that terminally exhausted T cells had unexpected chromatin features that limit their transcriptional potential. Terminally exhausted T cells had a substantial fraction of active chromatin, including active enhancers enriched for bZIP/AP-1 transcription factor motifs that lacked correlated gene expression, which was restored by immunotherapeutic costimulatory signaling. Reduced transcriptional potential was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Enforced expression of the hypoxia-insensitive histone demethylase Kdm6b was sufficient to overcome hypoxia, increase function, and promote antitumor immunity. Our study reveals the specific epigenetic changes mediated by histone modifications during T cell differentiation that support exhaustion in cancer, highlighting that their altered function is driven by improper costimulatory signals and environmental factors. These data suggest that even terminally exhausted T cells may remain competent for transcription in settings of increased costimulatory signaling and reduced hypoxia.

IL-33 acts as a costimulatory signal to generate alloreactive Th1 cells in graft-versus-host disease.

Antigen-presenting cells (APCs) integrate signals emanating from local pathology and program appropriate T cell responses. In allogeneic hematopoietic stem cell transplantation (alloHCT), recipient conditioning releases damage-associated molecular patterns (DAMPs) that generate proinflammatory APCs that secrete IL-12, which is a driver of donor Th1 responses, causing graft-versus-host disease (GVHD). Nevertheless, other mechanisms exist to initiate alloreactive T cell responses, as recipients with disrupted DAMP signaling or lacking IL-12 develop GVHD. We established that tissue damage signals are perceived directly by donor CD4+ T cells and promoted T cell expansion and differentiation. Specifically, the fibroblastic reticular cell-derived DAMP IL-33 is increased by recipient conditioning and is critical for the initial activation, proliferation, and differentiation of alloreactive Th1 cells. IL-33 stimulation of CD4+ T cells was not required for lymphopenia-induced expansion, however. IL-33 promoted IL-12-independent expression of Tbet and generation of Th1 cells that infiltrated GVHD target tissues. Mechanistically, IL-33 augmented CD4+ T cell TCR-associated signaling pathways in response to alloantigen. This enhanced T cell expansion and Th1 polarization, but inhibited the expression of regulatory molecules such as IL-10 and Foxp3. These data establish an unappreciated role for IL-33 as a costimulatory signal for donor Th1 generation after alloHCT.

CAR T cells are in it [CD]4 the long haul.

In a recent issue of Nature, Melenhorst et al. perform an extensive analysis of CAR T cells that persist for ten years in CLL patients. They find a dominant cytotoxic CD4+ population, raising the possibility that CD4+ T cells play an important role in durable CAR T cell therapy.

The m6A reader IMP2 directs autoimmune inflammation through an IL-17- and TNFα-dependent C/EBP transcription factor axis.

Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor-α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional "epitranscriptomic" mRNA modification [N6-methyladenosine (m6A)] in regulating C/EBPß and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPß/δ-encoding transcripts contain m6A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m6A modification. Induction of C/EBPs is enhanced by an m6A methylase "writer" and suppressed by a demethylase "eraser." The only m6A "reader" found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m6A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPß/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17-induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPß/δ. Imp2-/- mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2 Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m6A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.

MicroRNA-221 and -222 modulate intestinal inflammatory Th17 cell response as negative feedback regulators downstream of interleukin-23.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion.

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.

Noncanonical STAT3 activity sustains pathogenic Th17 proliferation and cytokine response to antigen.

The STAT3 signaling pathway is required for early Th17 cell development, and therapies targeting this pathway are used for autoimmune disease. However, the role of STAT3 in maintaining inflammatory effector Th17 cell function has been unexplored. Th17ΔSTAT3 mice, which delete STAT3 in effector Th17 cells, were resistant to experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Th17 cell numbers declined after STAT3 deletion, corresponding to reduced cell cycle. Th17ΔSTAT3 cells had increased IL-6-mediated phosphorylation of STAT1, known to have antiproliferative functions. Th17ΔSTAT3 cells also had reduced mitochondrial membrane potential, which can regulate intracellular Ca2+. Accordingly, Th17ΔSTAT3 cells had reduced production of proinflammatory cytokines when stimulated with myelin antigen but normal production of cytokines when TCR-induced Ca2+ flux was bypassed with ionomycin. Thus, early transcriptional roles of STAT3 in developing Th17 cells are later complimented by noncanonical STAT3 functions that sustain pathogenic Th17 cell proliferation and cytokine production.

Blimp-1 is essential for allergen-induced asthma and Th2 cell development in the lung.

A Th2 immune response is central to allergic airway inflammation, which afflicts millions worldwide. However, the mechanisms that augment GATA3 expression in an antigen-primed developing Th2 cell are not well understood. Here, we describe an unexpected role for Blimp-1, a transcriptional repressor that constrains autoimmunity, as an upstream promoter of GATA3 expression that is critical for Th2 cell development in the lung to inhaled but not systemically delivered allergens but is dispensable for TFH function and IgE production. Mechanistically, Blimp-1 acts through Bcl6, leading to increased GATA3 expression in lung Th2 cells. Surprisingly, the anti-inflammatory cytokine IL-10, but not the pro-inflammatory cytokines IL-6 or IL-21, is required via STAT3 activation to up-regulate Blimp-1 and promote Th2 cell development. These data reveal a hitherto unappreciated role for an IL-10-STAT3-Blimp-1 circuit as an initiator of an inflammatory Th2 response in the lung to allergens. Thus, Blimp-1 in a context-dependent fashion can drive inflammation by promoting rather than terminating effector T cell responses.

Shear stress and oxygen availability drive differential changes in opossum kidney proximal tubule cell metabolism and endocytosis.

Kidney proximal tubule (PT) cells have high-metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT functions are impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96 hours showed significant time- and culture-dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis vs development of ion transport and endocytic capacities in our culture system that highlight the complexity of O2 -dependent and mechanosensitive crosstalk to regulate PT cell function.

Adaptive plasticity of IL-10+ and IL-35+ Treg cells cooperatively promotes tumor T cell exhaustion.

Regulatory T cells (Treg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. Treg cells subvert beneficial anti-tumor immunity by modulating inhibitory receptor expression on tumor-infiltrating lymphocytes (TILs); however, the underlying mediators and mechanisms have remained elusive. Here, we found that the cytokines IL-10 and IL-35 (Ebi3-IL-12α heterodimer) were divergently expressed by Treg cell subpopulations in the tumor microenvironment (TME) and cooperatively promoted intratumoral T cell exhaustion by modulating several inhibitory receptor expression and exhaustion-associated transcriptomic signature of CD8+ TILs. While expression of BLIMP1 (encoded by Prdm1) was a common target, IL-10 and IL-35 differentially affected effector T cell versus memory T cell fates, respectively, highlighting their differential, partially overlapping but non-redundant regulation of anti-tumor immunity. Our results reveal previously unappreciated cooperative roles for Treg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8+ TILs that limits effective anti-tumor immunity.

IL-23 and IL-1ß Drive Human Th17 Cell Differentiation and Metabolic Reprogramming in Absence of CD28 Costimulation.

Th17 cells drive autoimmune disease but also control commensal microbes. A common link among antigens from self-proteins or commensal microbiota is relatively low activation of T cell receptor (TCR) and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naive T cells, but not effector/memory cells. CD28 suppressed the classical Th17 transcriptional program, while inducing known Th17 regulators, and acted through an Akt-dependent mechanism. Th17 cells differentiated without CD28 were not anergic: they showed robust proliferation and maintained Th17 cytokine production following restimulation. Interleukin (IL)-23 and IL-1ß promoted glucose uptake and increased glycolysis. Although modestly increased compared to CD28 costimulation, glycolysis was necessary to support Th17 differentiation, indicating that cytokine-mediated metabolic shifts were sufficient to obviate the classical requirement for CD28 in Th17 differentiation. Together, these data propose that, in humans, strength of TCR/CD28/Akt activation serves as a rheostat tuning the magnitude of Th17 development driven by IL-23 and IL-1ß.

B cells in T follicular helper cell development and function: separable roles in delivery of ICOS ligand and antigen.

B cells are required for follicular Th (Tfh) cell development, as is the ICOS ligand (ICOS-L); however, the separable contributions of Ag and ICOS-L delivery by cognate B cells to Tfh cell development and function are unknown. We find that Tfh cell and germinal center differentiation are dependent on cognate B cell display of ICOS-L, but only when Ag presentation by the latter is limiting, with the requirement for B cell expression of ICOS-L overcome by robust Ag delivery. These findings demonstrate that Ag-specific B cells provide different, yet compensatory, signals for Tfh cell differentiation, while reconciling conflicting data indicating a requirement for ICOS-L expression on cognate B cells for Tfh cell development with those demonstrating that the latter requirement could be bypassed in lieu of that tendered by noncognate B cells. Our findings clarify the separable roles of delivery of Ag and ICOS-L by cognate B cells for Tfh cell maturation and function, and have implications for using therapeutic ICOS blockade in settings of abundantly available Ag, such as in systemic autoimmunity.

Type I IFN induces binding of STAT1 to Bcl6: divergent roles of STAT family transcription factors in the T follicular helper cell genetic program.

CD4(+) T follicular helper cells (TFH) are critical for the formation and function of B cell responses to infection or immunization, but also play an important role in autoimmunity. The factors that contribute to the differentiation of this helper cell subset are incompletely understood, although several cytokines including IL-6, IL-21, and IL-12 can promote TFH cell formation. Yet, none of these factors, nor their downstream cognate STATs, have emerged as nonredundant, essential drivers of TFH cells. This suggests a model in which multiple factors can contribute to the phenotypic characteristics of TFH cells. Because type I IFNs are often generated in immune responses, we set out to investigate whether these factors are relevant to TFH cell differentiation. Type I IFNs promote Th1 responses, thus one possibility was these factors antagonized TFH-expressed genes. However, we show that type I IFNs (IFN-α/ß) induced B cell lymphoma 6 (Bcl6) expression, the master regulator transcription factor for TFH cells, and CXCR5 and programmed cell death-1 (encoded by Pdcd1), key surface molecules expressed by TFH cells. In contrast, type I IFNs failed to induce IL-21, the signature cytokine for TFH cells. The induction of Bcl6 was regulated directly by STAT1, which bound to the Bcl6, Cxcr5, and Pdcd1 loci. These data suggest that type I IFNs (IFN-α/ß) and STAT1 can contribute to some features of TFH cells but are inadequate in inducing complete programming of this subset.

In vivo regulation of Bcl6 and T follicular helper cell development.

Follicular helper T (T(FH)) cells, defined by expression of the surface markers CXCR5 and programmed death receptor-1 (PD-1) and synthesis of IL-21, require upregulation of the transcriptional repressor Bcl6 for their development and function in B cell maturation in germinal centers. We have explored the role of B cells and the cytokines IL-6 and IL-21 in the in vivo regulation of Bcl6 expression and T(FH) cell development. We found that T(FH) cells are characterized by a Bcl6-dependent downregulation of P-selectin glycoprotein ligand 1 (PSGL1, a CCL19- and CCL21-binding protein), indicating that, like CXCR5 and PD-1 upregulation, modulation of PSGL1 expression is part of the T(FH) cell program of differentiation. B cells were neither required for initial upregulation of Bcl6 nor PSGL1 downregulation, suggesting these events preceded T-B cell interactions, although they were required for full development of the T(FH) cell phenotype, including CXCR5 and PD-1 upregulation, and IL-21 synthesis. Bcl6 upregulation and T(FH) cell differentiation were independent of IL-6 and IL-21, revealing that either cytokine is not absolutely required for development of Bcl6(+) T(FH) cells in vivo. These data increase our understanding of Bcl6 regulation in T(FH) cells and their differentiation in vivo and identifies a new surface marker that may be functionally relevant in this subset.