Wednesday, April 2, 2025
4/2/2025
Gut Treg cells at the microbiome interface - Coexistence with the commensal microbes that populate the mammalian gastro-intestinal tract requires a balance to prevent microbial invasion while tolerating symbionts and limiting inflammatory damage. FoxP3+ T regulatory (Treg) cells play a central role in gut homeostasis. Conversely, the gut microbiota shapes local Treg pools, in particular by regulating a subpopulation of Rorg+Maf+ Treg cells which co-exists with Helios+Gata3+ Tregs more involved in tissue repair. Some colonic Tregs are of thymic origin (tTreg), others differentiate locally (pTregs), but the equivalence between Rorg+/pTreg and Helios+/tTreg is debated. In recent studies we discovered that maternal imprinting establishes setpoints of colon Treg that are stable in adults, charted transcriptional programs across colonic Tregs, and observed that many TCRs expressed by gut Tregs are reactive against microbe or food antigens. Our overarching goal here is to understand how microbes and food antigens establish these gut Treg phenotypes and TCR repertoires during the key postnatal and weaning periods, as well as during pTreg generation later in adults. 1. Early trajectory of intestinal Treg cells. Colonic Treg pools transition from early Helios+ Tregs in postnatal mice where when maternal imprinting occurs, to Rorg+ Tregs at weaning, when bacterial diversity explodes. The mechanisms behind these important transitions (successive waves, reprogramming by microbes or varied foods?) are unknown. We will combine lineage tracing, lineage ablation, abTCR analysis and single-cell transcriptomics to determine how these transitions occur, and to parse maternal, food and microbial influences. 2. TCR expressed by intestinal Tregs conditions their fate. Our preliminary data identified many microbe- or food-reactive TCRs, and show that the TCR conditions pTreg differentiation. To assess the determinants of pTreg generation, we will use a powerful new approach to CRISPR- edit TCRs in mature CD4+ Tconv cells, and track their differentiation into Teff or pTregs in response to food or microbe antigens. As this TCR editing scales and multiplexes readily, we will generate Tconv cells expressing a panel of 24 TCRs of different origin (colon Treg, Tconv) and specificity (food or microbes). We will assess the dependence of pTreg formation on particular antigen-presenting cells, and use scRNAseq to relate TCR and Treg programs, leveraging the unique opportunity to compare pTregs driven by food and microbial antigens in the same host. 3. Do microbe- induced and -reactive Tregs affect microbes? Microbes induce and manipulate Tregs, but a reciprocal effect of microbe-specific Tregs on microbial physiology is mostly unknown. To this end, we will infuse microbe-specific T cells and measure the consequences on competitive fitness of target microbes (fluorescent strains present at traceable but low frequencies), their coating by IgA, and their phenotype (bacterial RNAseq). Overall, this program should illuminate how microbes set the phenotype and genetic regulatory network of colon Treg cells at key junctures, and how these Treg talk back and mold the microbiota.
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