Peripheral education of the immune system by colonic commensal microbiota

Abstract

The instruction of the immune system to be tolerant of self, thereby preventing autoimmunity, is facilitated by the education of T cells in a specialized organ, the thymus, in which self-reactive cells are either eliminated or differentiated into tolerogenic Foxp3(+) regulatory T (T(reg)) cells. However, it is unknown whether T cells are also educated to be tolerant of foreign antigens, such as those from commensal bacteria, to prevent immunopathology such as inflammatory bowel disease. Here we show that encounter with commensal microbiota results in the peripheral generation of T(reg) cells rather than pathogenic effectors. We observed that colonic T(reg) cells used T-cell antigen receptors (TCRs) different from those used by T(reg) cells in other locations, implying an important role for local antigens in shaping the colonic T(reg)-cell population. Many of the local antigens seemed to be derived from commensal bacteria, on the basis of the in vitro reactivity of common colon T(reg) TCRs. These TCRs did not facilitate thymic T(reg)-cell development, implying that many colonic T(reg) cells arise instead by means of antigen-driven peripheral T(reg)-cell development. Further analysis of two of these TCRs by the creation of retroviral bone marrow chimaeras and a TCR transgenic line revealed that microbiota indigenous to our mouse colony was required for the generation of colonic T(reg) cells from otherwise naive T cells. If T cells expressing these TCRs fail to undergo T(reg)-cell development and instead become effector cells, they have the potential to induce colitis, as evidenced by adoptive transfer studies. These results suggest that the efficient peripheral generation of antigen-specific populations of T(reg) cells in response to an individual's microbiota provides important post-thymic education of the immune system to foreign antigens, thereby providing tolerance to commensal microbiota.

Figure 1. The colonic Treg TCR repertoire is unique

(a) TCR usage between colonic T cell subsets. A total of 2,892 TRAV14 TCRα sequences from colonic naive, memory, and Treg cells of five individual mice were compared (Supplementary Fig. 2). Each symbol represents a Morisita-Horn similarity comparison between two different T cell subsets within each mouse (top), or a comparison of the same T cell subset between different mice (bottom). Bars represent mean ± S.E.M. (b) Analysis of individual TCRs. The relative distribution within all T cell subsets is shown for the 20 most common individual TCRs in each colonic T cell subset. For example, a TCR with equal percentage in the Foxp3⁺ and CD44^hi subset would be shown as a half-green/half-orange bar. This analysis uses the pooled data set, which includes sequences from individual mice as well as 9,680 sequences from Expt. 1–3, each consisting of cells from 3–5 mice (Supplementary Fig. 2). Note that 1 TCR is found in both Foxp3⁺ (#15) and CD44^hi (#3) plots, and 1 in both CD44^lo (#5) and CD44^hi (#12) plots; all others appear in only one plot. (c) Anatomic distribution of colonic TCRs. Morisita-Horn indices comparing the colon data to other locations(closed symbols), or between each of the other locations (open symbols), are shown. Abbreviations: mes, mesenteric; cerv, cervical. (d) Analysis of individual TCRs. The 20 most prevalent colon TCRs for each subset in the pooled data set are shown, and their presence at other locations represented in a manner analogous to (b).

Figure 2. In vitro reactivity of colonic Treg TCRs to colonic contents and bacterial isolates

(a) Reactivity to colonic contents. Colonic Treg TCR -expressing NFAT-GFP hybridoma cells were cultured with Flt3L-induced dendritic cells in the presence of autoclaved food homogenate, or autoclaved colonic contents (CC) isolated from Rag1^−/− mice from Jackson Labs (Jax Rag1^−/−), Jax Rag1^−/− mice co-housed with mice from our colony(Co-housed Rag1^−/−), germ-free mice, and conventionally housed (Conv.) mice in our colony.(b) Reactivity to bacterial pools. Cultures of heat-killed commensal bacteria isolated from our colony (Supplementary Fig. 6) were pooled (denoted by culture conditions and a letter) and screened for their ability to stimulate colonic Treg TCR expressing hybridomas. For (a,b), see Supplementary Fig. 4 for additional TCRs. (c)Reactivity to individual isolates. Hybridomas showing reactivity against a pool of bacterial isolates were re-screened against the individual constituents (numbered). Data shown in (a-c) are the mean fold change in %GFP⁺ over the no antigen control ± S.E.M. from 2–4 experiments. (d) Specificity of colonic Treg TCRs. A panel of heat-killed Parabacteroides and Bacteroides spp. (Supplementary Fig. 7) was tested against CT2, CT6, and CT7-expressing hybridomas. Data shown are the mean ± S.E.M. from 3 experiments.

Figure 3. Colonic Treg TCRs facilitate thymic Treg cell development poorly, if at all

(a) Assessment of thymic Treg cell development from TCRαβ transduced Rag1^−/− thymocytes. The gating strategy (left), and summary of 2–4 experiments per TCR (right) is shown. See Supplementary Fig. 3 for additional TCR information, and Supplementary Fig. 8–9 for plots and analysis of clonal frequencies. Comparison of colon versus other Treg TCRs revealed p-values < 0.01. (b) Mixed retroviral bone marrow chimeras. The percentage of Foxp3⁺ cells in the CT2 or CT6 expressing CD45.2⁺ CD4⁺ population is shown in hosts with (right) or without (left) co-housing with mice from our colony. See Supplementary Figs. 10–11 for additional analyses. (c) Peripheral conversion of CT6 TCR transgenic cells. Naive CD45.2 CT6 cells and CD45.1 CD4⁺ filler cells were adoptively transferred into Tcrb^−/− hosts for 5 weeks. The percentage (left) of Foxp3⁺ CT6 cells (Vα2⁺Vβ6⁺CD45.2⁺CD45.1⁻CD4⁺) are shown. The number of CT6 cells (right) was determined by flow cytometry of the entire colonic lamina propria. Data are from 3 experiments; bars represent mean ± S.E.M. See Supplementary Fig. 13 for flow cytometric plots. (d) Helios expression in Treg cells. Representative intracellular Helios staining in CD4⁺ Foxp3⁺ cells from conventionally-housed and germ-free Foxp3^gfp mice is shown, and summarized in Supplementary Fig. 14. For all plots in this Figure, each symbol represents data from an individual host.

Figure 4. Pathogenic potential of colonic Treg TCRs

(a) Adoptive transfer of peripheral T cells transduced with CT2 or CT6 into co-housed Rag1^−/− hosts. Non-transduced (none) or naive TCR (B8) transduced T cells were used as controls. Each line represents an individual recipient. One representative experiment is shown (summary in Supplementary Fig. 15b). (b) A representative H&E section of the descending colon is shown at 4x magnification 7–10 weeks after T cell transfer.