Summary Oral immunotherapy has had limited success in establishing tolerance in food allergy, reflecting failure to elicit an effective regulatory T (Treg) cell response. We show that disease-susceptible mice (Il4raF709) with enhanced IL-4 receptor (IL-4R) signaling exhibited STAT6-dependent impaired generation and function of mucosal allergen-specific Treg cells. This failure was associated with the acquisition by Treg cells of T helper 2 (Th2) cell-like phenotype, also found in peripheral blood allergen-specific Treg cells of food allergic children. Selective augmentation of IL-4R signaling in Treg cells induced their reprogramming into Th2-like cells and disease susceptibility, whereas Treg cell lineage-specific deletion of Il4 and Il13 was protective. IL-4R signaling impaired the capacity of Treg cells to suppress mast cell activation and expansion, which in turn drove Treg cell Th2 cell reprogramming. Interruption of Treg cell Th2 cell reprogramming may thus provide novel therapeutic strategies in food allergy.

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Notch receptors direct the differentiation of T helper (TH) cell subsets, but their influence on regulatory T (Treg) cell responses is obscure. We here report that lineage-specific deletion of components of the Notch pathway enhanced Treg cell-mediated suppression of TH1 responses, and protected against their TH1 skewing and apoptosis. Expression in Treg cells of gain of function transgene encoding Notch1 intracellular domain resulted in lymphoproliferation, exacerbated TH1 responses and autoimmunity. Cell-intrinsic canonical Notch signaling impaired Treg cell fitness, promoted the acquisition by Treg cells of a TH1 cell-like phenotype, whereas Rictor-dependent non-canonical Notch signaling activated the AKT-Foxo1 axis and impaired Foxp3 epigenetic stability. These findings establish a critical role for Notch signaling in controlling peripheral Treg cell functions.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2 þ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8 þ T-cell proliferation and IFNg production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8 þ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival.

SUMMARY Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll like receptors (TLR). We report that Treg cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper-17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer’s patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA-responses.

“Natural” regulatory T (nTreg) cells that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” Treg (iTreg) cells are non-redundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg cell-produced IL-10 and iTreg cell fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naïve CD4+ T cells carrying a non-functional Foxp3 allele. At the onset of weight loss, mice were treated with both iTreg and nTreg cells where one marked subset was selectively IL-10-deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTreg cells were present but IL-10 deficient, iTreg cell-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ~85% of the transferred iTreg cells lost Foxp3 expression (ex-iTreg) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTreg and ex-iTreg cells, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between Treg subsets that maintains mucosal tolerance. The niche supporting stable iTreg cells is limited and readily saturated, which promotes a large population of ex-iTreg cells with pathogenic potential during immunotherapy.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d -2-hydroxyglutarate ( d -2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell–intrinsic effects of d -2HG are well understood, but its tumor cell–nonautonomous roles remain poorly explored. We compared the oncometabolite d -2HG with its enantiomer, l -2HG, and found that tumor-derived d -2HG was taken up by CD8 + T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d -2HG. d -2HG and inhibition of LDH drive a metabolic program and immune CD8 + T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.