Microbiota-activated CD103+ DCs stemming from microbiota adaptation specifically drive γδT17 proliferation and activation

Chris Fleming, Yihua Cai, Xuan Sun, Venkatakrishna R. Jala, Feng Xue, Samantha Morrissey, Yu-ling Wei, Yueh-hsiu Chien, Huang-ge Zhang, Bodduluri Haribabu, Jian Huang, Jun Yan

IL-17-producing γδT cells (γδT17) promote autoinflammatory diseases and cancers. Yet, γδT17 peripheral regulation has not been thoroughly explored especially in the context of microbiota-host interaction. The potent antigen-presenting CD103(+) dendritic cell (DC) is a key immune player in close contact with both γδT17 cells and microbiota. This study presents a novel cellular network among microbiota, CD103(+) DCs, and γδT17 cells. Immunophenotyping of IL-17r(-/-) mice and IL-17r(-/-) IRF8(-/-) mice were performed by ex vivo immunostaining and flow cytometric analysis. We observed striking microbiome differences in the oral cavity and gut of IL-17r(-/-) mice by sequencing 16S rRNA gene (v1-v3 region) and analyzed using QIIME 1.9.0 software platform. Principal coordinate analysis of unweighted UniFrac distance matrix showed differential clustering for WT and IL-17r(-/-) mice. We found drastic homeostatic expansion of γδT17 in all major tissues, most prominently in cervical lymph nodes (cLNs) with monoclonal expansion of Vγ6 γδT17 in IL-17r(-/-) mice. Ki-67 staining and in vitro CFSE assays showed cellular proliferation due to cell-to-cell contact stimulation with microbiota-activated CD103(+) DCs. A newly developed double knockout mice model for IL-17r and CD103(+) DCs (IL-17r(-/-)IRF8(-/-)) showed a specific reduction in Vγ6 γδT17. Vγ6 γδT17 expansion is inhibited in germ-free mice and antibiotic-treated specific pathogen-free (SPF) mice. Microbiota transfer using cohousing of IL-17r(-/-) mice with wildtype mice induces γδT17 expansion in the wildtype mice with increased activated CD103(+) DCs in cLNs. However, microbiota transfer using fecal transplant through oral gavage to bypass the oral cavity showed no difference in colon or systemic γδT17 expansion. These findings reveal for the first time that γδT17 cells are regulated by microbiota dysbiosis through cell-to-cell contact with activated CD103(+) DCs leading to drastic systemic, monoclonal expansion. Microbiota dysbiosis, as indicated by drastic bacterial population changes at the phylum and genus levels especially in the oral cavity, was discovered in mice lacking IL-17r. This network could be very important in regulating both microbiota and immune players. This critical regulatory pathway for γδT17 could play a major role in IL-17-driven inflammatory diseases and needs further investigation to determine specific targets for future therapeutic intervention.