Interruption of the intratumor CD8+ T cell:Treg crosstalk improves the efficacy of PD-1 immunotherapy.

PD-1 blockade unleashes potent antitumor activity in CD8+ T cells but can also promote immunosuppressive T regulatory (Treg) cells, which may worsen the response to immunotherapy. Tumor-Treg inhibition is a promising strategy to improve the efficacy of checkpoint blockade immunotherapy; however, our understanding of the mechanisms supporting tumor-Tregs during PD-1 immunotherapy is incomplete. Here, we show that PD-1 blockade increases tumor-Tregs in mouse models of melanoma and metastatic melanoma patients. Mechanistically, Treg accumulation is not caused by Treg-intrinsic inhibition of PD-1 signaling but depends on an indirect effect of activated CD8+ T cells. CD8+ T cells produce IL-2 and colocalize with Tregs in mouse and human melanomas. IL-2 upregulates the anti-apoptotic protein ICOS on tumor-Tregs, promoting their accumulation. Inhibition of ICOS signaling before PD-1 immunotherapy improves control over immunogenic melanoma. Thus, interrupting the intratumor CD8+ T cell:Treg crosstalk represents a strategy to enhance the therapeutic efficacy of PD-1 immunotherapy.

Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence.

Interleukin 22 (IL-22) has a non-redundant role in immune defence of the intestinal barrier1-3. T cells, but not innate lymphoid cells, have an indispensable role in sustaining the IL-22 signalling that is required for the protection of colonic crypts against invasion during infection by the enteropathogen Citrobacter rodentium4 (Cr). However, the intestinal epithelial cell (IEC) subsets targeted by T cell-derived IL-22, and how T cell-derived IL-22 sustains activation in IECs, remain undefined. Here we identify a subset of absorptive IECs in the mid-distal colon that are specifically targeted by Cr and are differentially responsive to IL-22 signalling. Major histocompatibility complex class II (MHCII) expression by these colonocytes was required to elicit sustained IL-22 signalling from Cr-specific T cells, which was required to restrain Cr invasion. Our findings explain the basis for the regionalization of the host response to Cr and demonstrate that epithelial cells must elicit MHCII-dependent help from IL-22-producing T cells to orchestrate immune protection in the intestine.

Regulatory T cells in skin mediate immune privilege of the hair follicle stem cell niche.

Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (Tregs) only in skin. Selective depletion of skin Tregs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin Tregs, functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin Tregs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of Tregs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.

Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation.

Intestinal colonization by antigenically foreign microbes necessitates expanded peripheral immune tolerance. Here we show commensal microbiota prime expansion of CD4 T cells unified by the Kruppel-like factor 2 (KLF2) transcriptional regulator and an essential role for KLF2+ CD4 cells in averting microbiota-driven intestinal inflammation. CD4 cells with commensal specificity in secondary lymphoid organs and intestinal tissues are enriched for KLF2 expression, and distinct from FOXP3+ regulatory T cells or other differentiation lineages. Mice with conditional KLF2 deficiency in T cells develop spontaneous rectal prolapse and intestinal inflammation, phenotypes overturned by eliminating microbiota or reconstituting with donor KLF2+ cells. Activated KLF2+ cells selectively produce IL-10, and eliminating IL-10 overrides their suppressive function in vitro and protection against intestinal inflammation in vivo. Together with reduced KLF2+ CD4 cell accumulation in Crohn's disease, a necessity for the KLF2+ subpopulation of T regulatory type 1 (Tr1) cells in sustaining commensal tolerance is demonstrated.

IL-2-induced Stat3 Signaling is Critical for Effector Treg Cell Programming.

Maintenance of immune homeostasis to the intestinal mictrobiota is dependent on a population of effector regulatory T (eTreg) cells that develop from microbiota-reactive induced (i)Treg cells. A cardinal feature of eTreg cells is their production of IL-10, which plays a non-redundant role in immune tolerance of commensal microbes. Here, we identify an unexpected role for IL-2-induced Stat3 signaling to program iTreg cells for eTreg cell differentiation and Il10 transcriptional competency. IL-2 proved to be both necessary and sufficient for eTreg cell development - contingent on Stat3 output of the IL-2 receptor coordinate with IL-2 signaling during early Treg cell commitment. Induction of iTreg cell programming in absence of IL-2-induced Stat3 signaling resulted in impaired eTreg cell differentiation and a failure to produce IL-10. An IL-2 mutein with reduced affinity for the IL-2Rγ (γ c ) chain was found to have blunted IL-2R Stat3 output, resulting in a deficiency of Il10 transcriptional programming that could not be fully rescued by Stat3 signaling subsequent to an initial window of iTreg cell differentiation. These findings expose a heretofore unappreciated role of IL-2 signaling that acts early to program subsequent production of IL-10 by developing eTreg cells, with broad implications for IL-2-based therapeutic interventions in immune-mediated diseases.

Batf stabilizes Th17 cell development via impaired Stat5 recruitment of Ets1-Runx1 complexes.

Although the activator protein-1 (AP-1) factor Batf is required for Th17 cell development, its mechanisms of action to underpin the Th17 program are incompletely understood. Here, we find that Batf ensures Th17 cell identity in part by restricting alternative gene programs through its actions to restrain IL-2 expression and IL-2-induced Stat5 activation. This, in turn, limits Stat5-dependent recruitment of Ets1-Runx1 factors to Th1- and Treg-cell-specific gene loci. Thus, in addition to pioneering regulatory elements in Th17-specific loci, Batf acts indirectly to inhibit the assembly of a Stat5-Ets1-Runx1 complex that enhances the transcription of Th1- and Treg-cell-specific genes. These findings unveil an important role for Stat5-Ets1-Runx1 interactions in transcriptional networks that define alternate T cell fates and indicate that Batf plays an indispensable role in both inducing and maintaining the Th17 program through its actions to regulate the competing actions of Stat5-assembled enhanceosomes that promote Th1- and Treg-cell developmental programs.

Local IL-23 is required for proliferation and retention of skin-resident memory TH17 cells.

The cytokine interleukin-23 (IL-23) is critical for development and maintenance of autoimmune inflammation in nonlymphoid tissues; however, the mechanism through which IL-23 supports tissue-specific immunity remains unclear. In mice, we found that circulating memory T cells were dispensable for anamnestic protection from Candida albicans skin infection, and tissue-resident memory (TRM) cell-mediated protection from C. albicans reinfection required IL-23. Administration of anti-IL-23 receptor antibody to mice after resolution of primary C. albicans infection resulted in loss of CD69+ CD103+ tissue-resident memory T helper 17 (TRM17) cells from skin, and clinical anti-IL-23 therapy depleted TRM17 cells from skin of patients with psoriasis. IL-23 receptor blockade impaired TRM17 cell proliferation but did not affect apoptosis susceptibility or tissue egress. IL-23 produced by CD301b+ myeloid cells was required for TRM17 maintenance in skin after C. albicans infection, and CD301b+ cells were necessary for TRM17 expansion during the development of imiquimod dermatitis. This study demonstrates that locally produced IL-23 promotes in situ proliferation of cutaneous TRM17 cells to support their longevity and function and provides mechanistic insight into the durable efficacy of IL-23 blockade in the treatment of psoriasis.

A nonredundant role for T cell-derived interleukin 22 in antibacterial defense of colonic crypts.

Interleukin (IL)-22 is central to immune defense at barrier sites. We examined the contributions of innate lymphoid cell (ILC) and T cell-derived IL-22 during Citrobacter rodentium (C.r) infection using mice that both report Il22 expression and allow lineage-specific deletion. ILC-derived IL-22 activated STAT3 in C.r-colonized surface intestinal epithelial cells (IECs) but only temporally restrained bacterial growth. T cell-derived IL-22 induced a more robust and extensive activation of STAT3 in IECs, including IECs lining colonic crypts, and T cell-specific deficiency of IL-22 led to pathogen invasion of the crypts and increased mortality. This reflected a requirement for T cell-derived IL-22 for the expression of a host-protective transcriptomic program that included AMPs, neutrophil-recruiting chemokines, and mucin-related molecules, and it restricted IFNγ-induced proinflammatory genes. Our findings demonstrate spatiotemporal differences in the production and action of IL-22 by ILCs and T cells during infection and reveal an indispensable role for IL-22-producing T cells in the protection of the intestinal crypts.

Intrinsic IL-2 production by effector CD8 T cells affects IL-2 signaling and promotes fate decisions, stemness, and protection.

Here, we show that the capacity to manufacture IL-2 identifies constituents of the expanded CD8 T cell effector pool that display stem-like features, preferentially survive, rapidly attain memory traits, resist exhaustion, and control chronic viral challenges. The cell-intrinsic synthesis of IL-2 by CD8 T cells attenuates the ability to receive IL-2-dependent STAT5 signals, thereby limiting terminal effector formation, endowing the IL-2-producing effector subset with superior protective powers. In contrast, the non-IL-2-producing effector cells respond to IL-2 signals and gain effector traits at the expense of memory formation. Despite having distinct properties during the effector phase, IL-2-producing and nonproducing CD8 T cells appear to converge transcriptionally as memory matures to form populations with equal recall abilities. Therefore, the potential to produce IL-2 during the effector, but not memory stage, is a consequential feature that dictates the protective capabilities of the response.

CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

Experimental evidence of pathogenic role of IgG autoantibodies in IgA nephropathy.

BACKGROUND: IgA nephropathy is thought to be an autoimmune disease wherein galactose-deficient IgA1 (Gd-IgA1) is recognized by IgG autoantibodies, resulting in formation and renal accumulation of nephritogenic immune complexes. Although this hypothesis is supported by recent findings that, in renal immunodeposits of IgA nephropathy patients, IgG is enriched for Gd-IgA1-specific autoantibodies, experimental proof is still lacking. METHODS: IgG isolated from sera of IgA nephropathy patients or produced as a recombinant IgG (rIgG) was mixed with human Gd-IgA1 to form immune complexes. IgG from healthy individuals served as a control. Nude and SCID mice were injected with human IgG and Gd-IgA1, in immune complexes or individually, and their presence in kidneys was ascertained by immunofluorescence. Pathologic changes in the glomeruli were evaluated by quantitative morphometry and exploratory transcriptomic profiling was performed by RNA-Seq. RESULTS: Immunodeficient mice injected with Gd-IgA1 mixed with IgG autoantibodies from patients with IgA nephropathy, but not Gd-IgA1 mixed with IgG from healthy individuals, displayed IgA, IgG, and mouse complement C3 glomerular deposits and mesangioproliferative glomerular injury with hematuria and proteinuria. Un-complexed Gd-IgA1 or IgG did not induce pathological changes. Moreover, Gd-IgA1-rIgG immune complexes injected into immunodeficient mice induced histopathological changes characteristic of human disease. Exploratory transcriptome profiling of mouse kidney tissues indicated that these immune complexes altered gene expression of multiple pathways, in concordance with the changes observed in kidney biopsies of patients with IgA nephropathy. CONCLUSIONS: This study provides the first in vivo evidence for a pathogenic role of IgG autoantibodies specific for Gd-IgA1 in the pathogenesis of IgA nephropathy.

Bone marrow Tregs mediate stromal cell function and support hematopoiesis via IL-10.

The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis.

TH17 cells require ongoing classic IL-6 receptor signaling to retain transcriptional and functional identity.

Acting in concert with TGF-ß, interleukin-6 (IL-6) signaling induces T helper 17 (TH17) cell development by programming TH17-related genes via signal transducers and activators of transcription 3 (STAT3). A role for IL-6 signaling beyond the inductive phase of TH17 cell development has not been defined because IL-23 signaling downstream of TH17 cell induction also activates STAT3 and is thought responsible for TH17 cell maintenance. Here, we find that IL-6 signaling is required for both induction and maintenance of mouse TH17 cells; IL-6Rα-deficient TH17 cells rapidly lost their TH17 phenotype and did not cause disease in two models of colitis. Cotransfer of wild-type TH17 cells with IL-6Rα-deficient TH17 cells induced colitis but was unable to rescue phenotype loss of the latter. High IL-6 expression in the colon promoted classic, or cis, rather than transreceptor signaling that was required for maintenance of TH17 cells. Thus, ongoing classic IL-6 signaling underpins the TH17 program and is required for TH17 cell maintenance and function.

Trigger-dependent differences determine therapeutic outcome in murine primary hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory syndrome affecting patients with genetic cytotoxicity defects. Perforin-deficient (PKO) mice recapitulate the full clinical picture of FHL after infection with lymphocytic choriomeningitis virus (LCMV). Hyperactivated CD8+ T cells and IFN-γ have been identified as the key drivers of FHL and represent targets for therapeutic interventions. However, the response of patients is variable. This could be due to trigger-dependent differences in pathogenesis, which is difficult to address in FHL patients, since the trigger frequently escapes detection. We established an alternative FHL model using intravenous infection of PKO mice with murine CMV (MCMV)Smith . PKO mice developed acute FHL after both infections and fulfilled HLH diagnostic criteria accompanied by excessive IFN-γ production by disease-inducing T cells, that enrich in the BM. However, direct comparison of the two infection models disclosed trigger-dependence of FHL progression and revealed a higher contribution of CD4 T cells and NK cells to IFN-γ production after MCMV infection. Importantly, therapeutic intervention by IFN-γ neutralization or CD8+ T-cell depletion had less benefit in MCMV-triggered FHL compared to LCMV-triggered FHL, likely due to MCMV-induced cytopathology. Thus, the context of the specific triggering viral infection can impact the success of targeted immunotherapeutic HLH control.

Insulin-Like Growth Factors Are Key Regulators of T Helper 17 Regulatory T Cell Balance in Autoimmunity.

Appropriate balance of T helper 17 (Th17) and regulatory T (Treg) cells maintains immune tolerance and host defense. Disruption of Th17-Treg cell balance is implicated in a number of immune-mediated diseases, many of which display dysregulation of the insulin-like growth factor (IGF) system. Here, we show that, among effector T cell subsets, Th17 and Treg cells selectively expressed multiple components of the IGF system. Signaling through IGF receptor (IGF1R) activated the protein kinase B-mammalian target of rapamycin (AKT-mTOR) pathway, increased aerobic glycolysis, favored Th17 cell differentiation over that of Treg cells, and promoted a heightened pro-inflammatory gene expression signature. Group 3 innate lymphoid cells (ILC3s), but not ILC1s or ILC2s, were similarly responsive to IGF signaling. Mice with deficiency of IGF1R targeted to T cells failed to fully develop disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Thus, the IGF system represents a previously unappreciated pathway by which type 3 immunity is modulated and immune-mediated pathogenesis controlled.

Reduction of AMPA receptor activity on mature oligodendrocytes attenuates loss of myelinated axons in autoimmune neuroinflammation.

Glutamate dysregulation occurs in multiple sclerosis (MS), but whether excitotoxic mechanisms in mature oligodendrocytes contribute to demyelination and axonal injury is unexplored. Although current treatments modulate the immune system, long-term disability ensues, highlighting the need for neuroprotection. Glutamate is elevated before T2-visible white matter lesions appear in MS. We previously reported that myelin-reactive T cells provoke microglia to release glutamate from the system xc - transporter promoting myelin degradation in experimental autoimmune encephalomyelitis (EAE). Here, we explore the target for glutamate in mature oligodendrocytes. Most glutamate-stimulated calcium influx into oligodendrocyte somas is AMPA receptor (AMPAR)-mediated, and genetic deletion of AMPAR subunit GluA4 decreased intracellular calcium responses. Inducible deletion of GluA4 on mature oligodendrocytes attenuated EAE and loss of myelinated axons was selectively reduced compared to unmyelinated axons. These data link AMPAR signaling in mature oligodendrocytes to the pathophysiology of myelinated axons, demonstrating glutamate regulation as a potential neuroprotective strategy in MS.

Preventing dysbiosis of the neonatal mouse intestinal microbiome protects against late-onset sepsis.

Late-onset sepsis (LOS) is thought to result from systemic spread of commensal microbes from the intestines of premature infants. Clinical use of probiotics for LOS prophylaxis has varied owing to limited efficacy, reflecting an incomplete understanding of relationships between development of the intestinal microbiome, neonatal dysbiosis and LOS. Using a model of LOS, we found that components of the developing microbiome were both necessary and sufficient to prevent LOS. Maternal antibiotic exposure that eradicated or enriched transmission of Lactobacillus murinus exacerbated and prevented disease, respectively. Prophylactic administration of some, but not all Lactobacillus spp. was protective, as was administration of Escherichia coli. Intestinal oxygen level was a major driver of colonization dynamics, albeit via mechanisms distinct from those in adults. These results establish a link between neonatal dysbiosis and LOS, and provide a basis for rational selection of probiotics that modulate primary succession of the microbiome to prevent disease.

Natural Tr1-like cells do not confer long-term tolerogenic memory.

IL-10-producing Tr1 cells promote tolerance but their contributions to tolerogenic memory are unclear. Using 10BiT mice that carry a Foxp3-eGFP reporter and stably express CD90.1 following IL-10 production, we characterized the spatiotemporal dynamics of Tr1 cells in a house dust mite model of allergic airway inflammation. CD90.1+Foxp3-IL-10+ Tr1 cells arise from memory cells and rejoin the tissue-resident memory T-cell pool after cessation of IL-10 production. Persistent antigenic stimulation is necessary to sustain IL-10 production and Irf1 and Batf expression distinguishes CD90.1+Foxp3-IL-10+ Tr1 cells from CD90.1+Foxp3-IL-10- 'former' Tr1. Depletion of Tr1-like cells after primary sensitization exacerbates allergic airway inflammation. However, neither transfer nor depletion of former Tr1 cells influences either Tr1 numbers or the inflammatory response during subsequent allergen memory re-challenge weeks later. Together these data suggest that naturally-arising Tr1 cells do not necessarily give rise to more Tr1 upon allergen re-challenge or contribute to tolerogenic memory. This phenotypic instability may limit efforts to re-establish tolerance by expanding Tr1 in vivo.

Batf Pioneers the Reorganization of Chromatin in Developing Effector T Cells via Ets1-Dependent Recruitment of Ctcf.

The basic leucine zipper transcription factor activating transcription factor-like (Batf) contributes to transcriptional programming of multiple effector T cells and is required for T helper 17 (Th17) and T follicular helper (Tfh) cell development. Here, we examine mechanisms by which Batf initiates gene transcription in developing effector CD4 T cells. We find that, in addition to its pioneering function, Batf controls developmentally regulated recruitment of the architectural factor Ctcf to promote chromatin looping that is associated with lineage-specific gene transcription. The chromatin-organizing actions of Batf are largely dependent on Ets1, which appears to be indispensable for the Batf-dependent recruitment of Ctcf. Moreover, most of the Batf-dependent sites to which Ctcf is recruited lie outside of activating protein-1-interferon regulatory factor (Ap-1-Irf) composite elements (AICEs), indicating that direct involvement of Batf-Irf complexes is not required. These results identify a cooperative role for Batf, Ets1, and Ctcf in chromatin reorganization that underpins the transcriptional programming of effector T cells.