Thymus-derived regulatory T cells contribute to tolerance to commensal microbiota

Abstract

Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4(+) Foxp3(+) regulatory T (Treg) cells generated in the thymus or extrathymically by induction of naive CD4(+) Foxp3(-) T cells. Previous studies suggested that the T-cell receptor repertoires of thymic Treg cells and induced Treg cells are biased towards self and non-self antigens, respectively, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Treg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage and favour tolerogenic presentation of antigens to naive CD4(+) T cells, suggesting that intestinal homeostasis depends on microbiota-specific induced Treg cells. Here, to identify the origin and antigen-specificity of intestinal Treg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4(+) Foxp3(+) and CD4(+) Foxp3(-) T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived Treg cells constitute most Treg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic Treg cells, and not induced Treg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.

Fig. 1. TCR repertoires of intestinal Tregs are similar to the TCR repertoire of CD4⁺Foxp3⁺thymocytes

a, The frequencies of fifteen dominant TCRs selected for each population from indicated organs in all organs (based on single cell TCR sequencing, see Table S1 for the number of TCR CDR3 sequences analysed). Color shades reflect the relative frequency with which a given TCR was found in each organ. b, The frequencies of dominant TCRs from colonic Tregs in the population of CD4⁺Foxp3⁺ thymocytes. c, The hierarchical diagrams depict similarity indices (MII) for TCR repertoires from CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ populations ((b and c are based on HT sequencing, see Table S1 for the number of TCR CDR3 sequences analysed).

Fig. 2. In TCRβFoxp3^GFP transgenic mice the majority of colonic CD4⁺Foxp3⁺ T cells share TCRs with CD4⁺Foxp3⁺ thymocytes

a, Dominant TCRs from colonic Tregs and their frequencies on CD4⁺Foxp3⁺ thymocytes b, The hierarchical dendrogram depicts MII indices between TCR repertoires from CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ populations from the indicated organs. For calculation of MII, the dataset from CD4⁺Foxp3⁺ thymocytes was limited as described in Methods Summary.

Fig. 3. Antibiotic-induced changes in colonic flora have profound influence on the TCR repertoire of colonic tTregs

a, The effect of antibiotic treatment on the proportion of Tregs in indicated organs. Three mice per group were analyzed. b, Fifty dominant TCRs (Table S4) of colonic Tregs from untreated (red bars) or antibiotic-treated (black bars) mice and their frequencies in analyzed repertoires. TCRs not found on CD4⁺Foxp3⁺ thymocytes are star-marked. c, Diversity index (Renyi Entropy Function) of Tregs from indicated organs of untreated and antibiotic-treated mice. REF close to “0” corresponds to diversity of low abundant TCRs and close to “2” for high abundant TCRs. d, MII indices for TCR repertoires of CD4⁺Foxp3⁻ and Treg populations from antibiotic-treated mice.

Fig. 4. TCRs from colonic tTregs recognize microbial antigens

a. The response (± s.e.m from three experiments) of cloned colonic Treg hybridomas, which responded to cecal lysate from untreated TCR^miniFoxp3^GFP mice (Fig. S6), to re-stimulation with cecal lysate from untreated or antibiotic-treated mice. b, The abundance of Treg TCRs from hybridomas that responded to cecal lysates from untreated mice (shown in a) on CD4⁺Foxp3⁺ thymocytes. Hybridomas marked by asterisk also responded to cecal lysate from antibiotic-treated mice. c, Phylogenetic distance of bacterial strains tested here. d. The response of hybridomas highlighted in panel 4b to indicated bacterial sonicates.