Fan Pan

Abstract Innate lymphoid cells (ILCs) and CD4 + T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4 + T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4 + T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4 + T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4 + T cells and ILCs to maintain intestinal homeostasis.

Myeloid-derived suppressor cells (MDSC) play a key immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). In this study, we characterized CD14+HLA-DR(-/lo) cells sorted from the tumors, draining lymph nodes, and peripheral blood of HNSCC patients. CD14+HLA-DR(-/lo) cells were phenotyped as CD11b+, CD33+, CD34+, arginase-I+, and ROS+. In all 3 compartments, they suppressed autologous, antigen-independent T cell proliferation in a differential manner. The abundance of MDSC correlated with stage, but did not correlate with previous treatment with radiation or subsites of HNSCC. Interestingly, MDSC from all 3 compartments showed high phosphorylated STAT3 levels that correlated with arginase-I expression levels and activity. Stattic, a STAT3-specific inhibitor, and STAT3-targeted siRNA abrogated MDSC’s suppressive function. Inhibition of STAT3 signaling also resulted in decreased arginase-I activity. Analysis of the human arginase-I promoter region showed multiple STAT3-binding elements, and ChIP demonstrated that phosphorylated STAT3 binds to multiple sites in the arginase-I promoter. Finally, rescue of arginase-I activity after STAT3 blockade restored MDSC’s suppressive function. Taken together, these results demonstrate that the suppressive function of arginase-I in both infiltrating and circulating MDSC is a downstream target of activated STAT3.

BACKGROUND: The PD-1/PD-L1 checkpoint is a central mediator of immunosuppression in the tumor immune microenvironment (TME) and is primarily associated with IFN-g signaling. To characterize other factors regulating PD-L1 expression on tumor and/or immune cells, we investigated TME-resident cytokines and the role of transcription factors in constitutive and cytokine-induced PD-L1 expression. METHODS: Thirty-four cultured human tumor lines [18 melanomas (MEL), 12 renal cell carcinomas (RCC), 3 squamous cell carcinomas of the head and neck (SCCHN), and 1 non-small-cell lung carcinoma (NSCLC)] and peripheral blood monocytes (Monos) were treated with cytokines that we detected in the PD-L1+ TME by gene expression profiling, including IFN-g, IL-1a, IL-10, IL-27 and IL-32g. PD-L1 cell surface protein expression was detected by flow cytometry, and mRNA by quantitative real-time PCR. Total and phosphorylated STAT1, STAT3, and p65 proteins were detected by Western blotting, and the genes encoding these proteins were knocked down with siRNAs. Additionally, the proximal promoter region of PDL1 (CD274) was sequenced in 33 cultured tumors. RESULTS: PD-L1 was constitutively expressed on 1/17 cultured MELs, 8/11 RCCs, 3/3 SCCHNs, and on Monos. Brief IFN-g exposure rapidly induced PD-L1 on all tumor cell lines and Monos regardless of constitutive PD-L1 expression. PD-L1 mRNA levels were associated with protein expression, which was diminished by exposure to transcriptional inhibitors. siRNA knockdown of STAT1 but not STAT3 reduced IFN-g- and IL-27-induced PD-L1 protein expression on tumor cells. In contrast, STAT3 knockdown in Monos reduced IL-10-induced PD-L1 protein expression, and p65 knockdown in tumor cells reduced IL-1a-induced PD-L1 expression. Notably, constitutive PD-L1 expression was not affected by knocking down STAT1, STAT3, or p65. Differential effects of IFN-g, IL-1a, and IL-27 on individual tumor cell lines were not due to PDL1 promoter polymorphisms. CONCLUSIONS: Multiple cytokines found in an immune-reactive TME may induce PD-L1 expression on tumor and/or immune cells through distinct signaling mechanisms. Factors driving constitutive PD-L1 expression were not identified in this study. Understanding complex mechanisms underlying PD-L1 display in the TME may allow treatment approaches mitigating expression of this immunosuppressive ligand, to enhance the impact of PD-1 blockade.