Gut resident T-bet expressing Tr1 cells driven by dietary signals dominate the small intestinal immune landscape I

Intestinal immunity provides defense against enteric pathogens, mediates symbiotic relationships with the resident microbiota, and provides tolerance to food antigens, safeguarding critical nutrient absorption and barrier functions of this mucosal tissue. Despite the large abundance of tissue resident activated T cells, how T cell immunity contributes to these various roles remains poorly understood. Here we describe a previously uncharacterized dominant T-bet expressing Tr1 T cell population in the small intestine lamina propria present at homeostasis. Importantly, this IL-10 secreting CD4^+^T-bet^+^ T cell population appears in the small intestine at the time of weaning independently of the resident microbiota, presenting similar accumulation, functional capacity, and TCR repertoire in germ free conditions. Instead, small intestinal T-bet^+^ Tr1 T cells respond to dietary signals driving their accumulation, suppressive program, and clonal selection; and depend on cDC1s, IL-27 signaling, and T-bet, but not IL-12. Altogether, this work uncovers a previously unappreciated dominant player in homeostatic small intestinal immunity with the potential to play critical suppressive roles in oral tolerance to dietary antigens, raising important implications for the understanding of food allergy and celiac disease. Overall design: SILP CD4 T cells (Live, CD45+CD90.2+, TCRb+, CD4+) from 4 SPF and 4 GF T-bet-ZsGreen mice (adult , steady state) were sorted from samples labeled with TotalSeqC hashtags antibodies (Biolegend) using a Sony MA900 sorter. Sorted cells were pooled during sorting and 32000 cells per lane (total of 8 lanes) were loaded on a Chromium Single Cell Controller (10X Genomics) to encapsulate the cells into droplets. The gene expression (GEX), hashtag oligonucleotide (HTO) and VDJ (TCR) sequencing libraries were prepared using Chromium Single Cell 5' v2 reagent kits following the manufacturer's instructions, and sequenced on an Illumina Nextseq2000 platform (NextSeq 1000/2000 P2 reagents).