Th17/1-Biased Inflammatory Environment Involved in the Response of Epithelial Cells to Antigen Stimuli in Nasal Polyps

Several studies showed that IL-17A was significantly increased in nasal polyps (NPs). However, the source and characteristics of IL-17A-producing cells in NPs were not fully understood. We isolated mononuclear cells from NPs and uncinate tissues and analyzed them using flow cytometry. The results indicated that IL-17A was increased in NP tissues compared to uncinate tissues. The main IL-17A-expressing cells were CD3+ T cells in NP tissues, including Th17 cells, Tc17 cells, and γδT17 cells. Not similar to those in uncinate tissues, the majority of Th17 cells highly coexpressed IFN-γ in NP tissues, such as Th17/1 cells, which highly expressed CXCR3, CCR6, RORγt, and T-bet. Furthermore, Th17/1-biased environment increased the response of nasal epithelial cells to bacterial and viral stimuli, implying that Th17/1 cells play a greater role in the pathological development of NPs than Th17 or Th1 cells.

Type 17 immunity promotes the exhaustion of CD8+ T cells in cancer

BackgroundMultiple types of immune cells producing IL-17 are found in the tumor microenvironment. However, their roles in tumor progression and exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) remain unclear.MethodsTo determine the role of type 17 immunity in tumor, we investigated the growth of B16F10 melanoma and the exhaustion of CD8+ TILs in Il17a−/− mice, Il17aCreR26DTA mice, RORγt inhibitor-treated mice, or their respective control mice. Adoptive transfer of tumor-specific IL-17-producing T cells was performed in B16F10-bearing congenic mice. Anti-CD4 or anti-Ly6G antibodies were used to deplete CD4+ T cells or CD11b+Gr-1hi myeloid cells in vivo, respectively. Correlation between type 17 immunity and T cell exhaustion in human cancer was evaluated by interrogating TCGA dataset.ResultsDepletion of CD4+ T cells promotes the exhaustion of CD8+ T cells with a concomitant increase in IL-17-producing CD8+ T (Tc17) cells in the tumor. Unlike IFN-γ-producing CD8+ T (Tc1) cells, tumor-infiltrating Tc17 cells exhibit CD103+KLRG1−IL-7Rαhi tissue resident memory-like phenotypes and are poorly cytolytic. Adoptive transfer of IL-17-producing tumor-specific T cells increases, while depletion of IL-17-producing cells decreases, the frequency of PD-1hiTim3+TOX+ terminally exhausted CD8+ T cells in the tumor. Blockade of IL-17 or RORγt pathway inhibits exhaustion of CD8+ T cells and also delays tumor growth in vivo. Consistent with these results, human TCGA analyses reveal a strong positive correlation between type 17 and CD8+ T cell exhaustion signature gene sets in multiple cancers.ConclusionIL-17-producing cells promote terminal exhaustion of CD8+ T cells and tumor progression in vivo, which can be reversed by blockade of IL-17 or RORγt pathway. These findings unveil a novel role for IL-17-producing cells as tumor-promoting cells facilitating CD8+ T cell exhaustion, and propose type 17 immunity as a promising target for cancer immunotherapy.

IFN-γ- and IL-17-producing CD8+ T (Tc17-1) cells in combination with poly-ICLC and peptide vaccine exhibit antiglioma activity

BackgroundWhile adoptive transfer of T-cells has been a major medical breakthrough for patients with B cell malignancies, the development of safe and effective T-cell-based immunotherapy for central nervous system (CNS) tumors, such as glioblastoma (GBM), still needs to overcome multiple challenges, including effective homing and persistence of T-cells. Based on previous observations that interleukin (IL)-17-producing T-cells can traffic to the CNS in autoimmune conditions, we evaluated CD8+ T-cells that produce IL-17 and interferon-γ (IFN-γ) (Tc17-1) cells in a preclinical GBM model.MethodsWe differentiated Pmel-1 CD8+ T-cells into Tc17-1 cells and compared their phenotypic and functional characteristics with those of IFN-γ-producing CD8+ T (Tc1) and IL-17-producing CD8+ T (Tc17) cells. We also evaluated the therapeutic efficacy, persistence, and tumor-homing of Tc17-1 cells in comparison to Tc1 cells using a mouse GL261 glioma model.ResultsIn vitro, Tc17-1 cells demonstrated profiles of both Tc1 and Tc17 cells, including production of both IFN-γ and IL-17, although Tc17-1 cells demonstrated lesser degrees of antigen-specific cytotoxic activity compared with Tc1 cells. In mice-bearing intracranial GL261-Quad tumor and treated with temozolomide, Tc1 cells, but not Tc17-1, showed a significant prolongation of survival. However, when the T-cell transfer was combined with poly-ICLC and Pmel-1 peptide vaccine, both Tc1 and Tc17-1 cells exhibited significantly prolonged survival associated with upregulation of very late activation antigen−4 on Tc17-1 cells in vivo. Glioma cells that recurred following the therapy lost the susceptibility to Pmel-1-derived cytotoxic T-cells, indicating that immuno-editing was a mechanism of the acquired resistance.ConclusionsTc17-1 cells were equally effective as Tc1 cells when combined with poly-ICLC and peptide vaccine treatment.

Identification of RORg+T Cells as Key Players in Thyroid Autoimmunity From Checkpoint Immunotherapy

Purpose: Immune checkpoint inhibitors (ICI) are powerful new cancer therapies that leverage the body’s own immune system to attack cancer cells. Unfortunately, their use may be limited by the development of immune-related adverse events (IrAE) in up to 60% of patients. Thyroiditis is a common IrAE, with shared and distinct features from spontaneous thyroid autoimmunity, i.e. Hashimoto thyroiditis (HT). The cause of IrAE remains unknown, however, recent data suggest that toxicity can be uncoupled from anti-tumor effects. Methods: We developed a novel mouse model to study mechanisms of IrAE, in which ICI (anti-PD-1 and/or anti-CTLA-4) treatment leads to multi-organ immune infiltrates, including thyroiditis. To understand immune changes occurring with ICI-autoimmunity, we first evaluated changes in the frequency and activation status of different immune cells in our mice using immunohistochemistry (IHC) and flow cytometry. Then we confirmed these findings in peripheral blood and thyroid fine needle aspiration (FNA) specimens from patients with ICI-thyroiditis, HT, or no IrAE, using flow cytometry and single cell RNA sequencing (scRNAseq) techniques. Results: In our mouse model, ICI treatment of autoimmune-prone non-obese diabetic mice induces multi-organ autoimmunity. Modeling ICI-IrAE observed in humans, our mice developed increased immune infiltrates in multiple tissues (e.g. thyroid, colon, liver, lung), autoantibodies, and acceleration of underlying autoimmune risk (i.e. diabetes). Increased frequency of autoimmune disease was seen with combination (anti-PD-1 + anti-CTLA-4) vs. single agent ICI. We found increased IL-17A+ T cells in secondary lymphoid tissues of ICI-treated mice, a cytokine produced by RORγ + Th17 and Tc17 cells and associated with autoimmunity. IHC studies on thyroid infiltrates showed accumulation of CD4+ and CD8+ T cells and macrophages in ICI-treated vs. isotype control mice. This finding was confirmed by flow cytometry analyses of thyroid-infiltrating leukocytes in ICI-thyroiditis mice, which showed significantly increased T cells, specifically RORγ + T cells, and rare B220+ B, CD11b+ myeloid, or NKp46+ NK cells. In patients with ICI-thyroiditis, thyroid FNA showed that thyroid immune infiltrates were predominately T cells. scRNAseq studies in patients with ICI-thyroiditis showed enrichment of Th17 and Tc17 (RORγ + IL23R+ CD161+) T cells, compared to ICI-treated patients without IrAE. Conclusion: We have identified a role for RORγ + Th17 and Tc17 cells in thyroid autoimmunity from ICI using a newly developed mouse model of ICI-associated IrAE and translational studies in patients with ICI-thyroiditis. Th17 and Tc17 cells have previously been associated with spontaneous autoimmune disease, including HT, but have not yet been characterized in IrAE. These cells provide a potential therapeutic target for prevention of endocrine IrAE from ICI.

Tc17 CD8+ T cells accumulate in murine atherosclerotic lesions, but do not contribute to early atherosclerosis development

Aims CD8+ T cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces interleukin-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis. Methods and results Flow cytometry analysis of atherosclerotic lesions from apolipoprotein E-deficient mice revealed a pronounced increase in RORγt+CD8+ T cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8−/− low-density lipoprotein receptor-deficient mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of interleukin-17A production in vivo. Moreover, Tc17 cells produced lower levels of interferon-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice. Conclusion These findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart.

IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis

IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond.

Expansion of Th/Tc17 T Cells and Loss of CX3CR1+ Macrophages Play a Critical Role in the Pathogenesis of Steroid-Refractory Acute Intestinal Gvhd

Acute gut graft-versus-host disease (GVHD) is the major contributor to mortality and morbidity of allogeneic hematopoietic stem cell transplantation recipients. Steroids are the first-line therapy for acute GVHD, but approximately 30% of patients develop steroid refractory GVHD (SR-GVHD). The pathophysiology of SR-GVHD is poorly understood. In the current studies, we explored the mechanisms of SR-GVHD, using a murine model of MHC-mismatched HCT with C57BL/6 donors and BALB/c recipients. Acute GVHD can be mediated by either Th/Tc1 or Th/Tc17 T cells. We evaluated the impact of dexamethasone (DEX) treatment on acute GVHD induced by spleen and BM cells from WT, IFN-γ-/- or RORγt-/- C57BL/6 donors. WT donor T cells give rise predominantly to Th/Tc1 cells early after HCT, but Th/Tc17 cells can expand as time goes on. IFN-γ-/- donor T cells give rise to Th/Tc17 cells but not to IFN-γ-producing Th/Tc1 cells, while RORγt-/- donor T cells give rise to Th/Tc1 cells but not to Th/Tc17 cells. BALB/c recipients were treated with single dose dexamethasone (DEX) at 5 mg/Kg on day 3 after HCT. DEX treatment effectively prevented acute GVHD in recipients given WT or RORγt-/- grafts for up to 20 days after HCT. GVHD then recurred in recipients grafted with WT cells but not in recipients grafted with RORγt-/- cells. In contrast, DEX treatment had no effect on acute GVHD in recipients grafted with IFN-γ-/- cells. Recurrence of GVHD in DEX-treated recipients grafted with WT cells and the lack of DEX effect in recipients grafted with IFN-γ-/- cells was associated with expansion of Th/Tc17 cells that produced both IL-17 and IL-22. In recipients grafted with IFN-γ-/- cells, we also observed marked reduction of donor-type CX3CR1+ intestinal macrophages that play a critical role in controlling bacterial translocation from the intestinal lumen to the liver and blood. In addition, the DEX-treated recipients showed augmentation of Th/Tc17 differentiation and exacerbation of acute GVHD, When CX3CR1GFP/GFP mice that are deficient in generating CX3CR1+ intestinal macrophages were used as donors. These results indicate that DEX-treatment can cause expansion of Th/Tc17 and loss of protective donor-type CX3CR1+ intestinal macrophages, leading to SR-GVHD. Disclosures Martin: Abgenomics: Research Funding; Enlivex Therapeutics: Consultancy; Genentech: Consultancy, Other: One-time advisory board; Neovii: Other: One-time advisory board; Pfizer: Other: Data and Safety Monitoring Committee; Pharmacyclics LLC: Other: One-time advisory board; Procter and Gamble: Equity Ownership; Xenikos: Research Funding.

TCF-1 limits the formation of Tc17 cells via repression of the MAF–RORγt axis

Interleukin (IL)-17–producing CD8+ T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ–producing effector CD8+ T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 (Tcf7) regulates CD8+ T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1–driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8+ T cell subsets. IL-17–producing CD8+ T cells isolated from healthy humans were also distinct from CD8+IL-17− T cells and enriched in pathways driven by MAF and RORγt. Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.