Commensal epitopes drive differentiation of colonic Tregs

Abstract

The gut microbiome is the largest source of intrinsic non-self-antigens that are continuously sensed by the immune system but typically do not elicit lymphocyte responses. CD4⁺ T cells are critical to sustain uninterrupted tolerance to microbial antigens and to prevent intestinal inflammation. However, clinical interventions targeting commensal bacteria-specific CD4⁺ T cells are rare, because only a very limited number of commensal-derived epitopes have been identified. Here, we used a new approach to study epitopes and identify T cell receptors expressed by CD4⁺Foxp3⁺ (T_reg) cells specific for commensal-derived antigens. Using this approach, we found that antigens from Akkermansia muciniphila reprogram naïve CD4⁺ T cells to the T_reg lineage, expand preexisting microbe specific T_regs, and limit wasting disease in the CD4⁺ T cell transfer model of colitis. These data suggest that the administration of specific commensal epitopes may help to widen the repertoire of specific T_regs that control intestinal inflammation.

Fig. 1. Activation of CD4⁺ T cell hybridomas produced using new BWNur77^GFP thymoma.

(A) Expression of GFP in NFAT^GFP (left) and Nur77^GFP (right) reporters in response to aCD3 (1 μg/ml) in two representative hybridomas. MFI, mean fluorescence intensity. (B) Up-regulation of NFAT^GFP or Nur77^GFP reporters and IL-2 secretion by aCD3 activated hybridomas. Reporter and IL-2 expression was measured by mRNA (left) and protein level (right). Each symbol marks individual hybridoma (n = 5 of each kind). The experiment was repeated three times, and representative results are shown. The statistic was calculated for t = 16 hours. (C) Expression of Nur77^GFP reporter by representative hybridoma activated by titrated aCD3 mAb. Nur77^GFP (C) and IL-2 (D) expression by hybridoma from CD4⁺TCR⁺Foxp3⁺ T_regs. GFP and IL-2 expression were measured by FACS/RT-qPCR or by HT-2 assay/RT-qPCR, respectively, from five randomly selected hybridomas. (E) Nur77^GFP expression in hybridomas declines gradually following antigen withdrawal. Hybridomas were stimulated with plate-bound aCD3 for 16 hours and then moved to uncoated wells. GFP expression was measured by FACS (left) or RT-qPCR (right) at indicated time points. Means ± SD are shown, and each symbol represents an individual hybridoma. RT-qPCR data were normalized to β-actin. Paired t test; *P < 0.05, **P < 0.01, ***P < 0.001. For statistical analysis, data points in (C) were compared to unstimulated (aCD3 = 0 ng/ml) samples, and for (D), to samples of t = 0 hours.

Fig. 2. An increase in Nur77^GFP reporter follows discrete TCR activation by bacterial antigens that do not induce detectable IL-2.

(A and B) A total of 100 hybridomas were individually cocultured with DCs pulsed with bacterial protein lysate or aCD3 mAb, and IL-2 production (A) or Nur77^GFP up-regulation (B) was measured 16 hours later. The experiment was repeated twice. (C) Nur77^GFP exclusively reports TCR activation of CD4⁺ hybridoma to antigens presented in the context of class II MHC (A^b). Antigens presented by A^b-negative DCs or DCs with antibody-blocked A^b do not up-regulate Nur77^GFP. Lack of MHCI (β2m^k/o) but not MHCII does not interfere with GFP up-regulation. aCD3 was used as positive control, and DCs not pulsed with fecal bacteria protein lysate served as a negative control. (D) Expression of Nur77^GFP reporter in response to fecal bacteria lysate in hybridomas prepared from TCR^mini, GF TCR^mini, and CNS1^k/oTCR^mini CD4⁺ cells. Fifty hybridomas of each kind were tested and MFIs of GFP were assessed by FACS. One experiment of at least three is shown. Paired t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. CD4⁺ hybridomas recognize specific microbial antigens from selected ASF bacteria.

(A) Features of TCRs expressed on specific hybridomas and specific microbial epitopes that activated these hybridomas. The table shows sequences of antigenic peptides and corresponding TCR sequences found on selected reactive hybridomas prepared from indicated CD4⁺ populations. Frequencies of TCRs in the colonic CD4⁺ cell repertoire in TCR^mini and TCR^miniCNS1^k/o mice are shown. (B) Representative responses of hybridomas from colonic CD4⁺Foxp3⁻ and CD4⁺Foxp3⁺ cells to commensal’s antigens [activated by bacterial lysate or specific peptide (3D.28)]. Nonstimulatory peptide and aCD3 mAb served as negative and positive controls, respectively. (C) Summary of hybridomas’ responses to identified reactive peptides (TCR^mini: Foxp3⁻, n = 14 and Foxp3⁺, n = 11; TCR^miniCNS1^k/o: Foxp3⁻, n = 21, Foxp3⁺, n = 10). Some hybridomas showed in (A) are marked on the graph. Each symbol represents an individual hybridoma. (D) Relative occurrence of hybridomas responding to antigens from three ASF strains (percentages show hybridomas responding to specific lysate). (E) Immunization of TCR^mini mice with E. plexicaudatum ASF492–derived 3D.28 peptide (n = 15 mice) [but not 9A.9 peptide (n = 12 mice) or control (ctr) mice with cholera toxin only (n = 15 mice)] induces colonic T_regs [(total CD4 numbers: ctr, 290.9 ± 42.5; 9A.9, 296.6 ± 23.7; 3D.28, 312.1 ± 28.5 (×10³)]. (F) 3D.28 elicits colonic T_regs with high levels of CD73 and CD39, enhancing these cells suppressor function. (E and F) Each symbol represents an individual mouse. The experiment was repeated three times. Paired t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. Epitopes from A. muciniphila activate hybridomas derived from colonic CD4⁺ T cells.

(A) Antigenic peptides and CDR3 sequences from specific TCRα chain expressed on hybridomas. Frequencies of these TCRs on colonic CD4⁺ cells in TCR^mini and TCR^miniCNS1^k/o mice are also shown. (B) Representative up-regulation of Nur77^GFP reporter by colonic CD4⁺ hybridoma in response to epitope (2C.1 or 2C.9) from A. muciniphila. Nonstimulating peptide and aCD3 served as negative and positive controls, respectively. (C) Summary of hybridoma responses to A. muciniphila epitopes. Each symbol represents an individual hybridoma. Responding hybrids from (A) are marked (Foxp3⁻, n = 5; Foxp3⁺, n = 3). (D) Representative dot plots show an expansion of colonic T_regs in TCR^mini mice after immunization with A. muciniphila–derived 2C.1 peptide. (E) Frequencies and total number of colonic T_regs in TCR^mini mice primed with cholera toxin only (ctr) (n = 10) or with toxin with 6H.3 (n = 10) or 2C.1 (n = 12) peptides [total CD4 numbers: ctr, 350.9 ± 59.2; 6H.3, 296.6 ± 57.5; 2C.1, 340.6 ± 53.3 (×10³)]. (F) T_regs elicited by 2C.1 immunization express higher levels of CD73 and CD39. Gated colonic CD4⁺Foxp3⁺ cells were tested. (E and F) Each symbol represents an individual mouse. The experiment was repeated twice. Paired t test; **P < 0.01, ***P < 0.001.

Fig. 5. A. muciniphila supports T_regs in vivo expansion.

(A) 16S ribosomal RNA analysis of microbiomes from TCR^miniCNS1^k/o and TCR^mini mice. Data were confirmed by RT-qPCR (shown below legend). Reads were first normalized to UniF340/UniR514 and then to CNS1^+/+ A. muciniphila signal. (B) Inoculation of GF TCR^miniFoxp3^GFP mice with A. muciniphila expands colonic T_regs. (C) A. muciniphila induces pT_regs and expands tT_regs. Naïve CD4⁺CD44^lowFoxp3⁻ cells from congenic TCR^mini mice were adoptively transferred into CNS1^k/oTCR^mini hosts, and half of the recipients received A. muciniphila. Graphs show frequencies (left) and total number (right) of Foxp3⁺ cells gated according to their origin. (D) TCR^miniCNS1^k/o mice were colonized with A. muciniphila, and 8 weeks later, their colonic tT_regs were analyzed. Graphs show percentage (left) and numbers (right) of CD4⁺Foxp3⁺ cells. (E) tT_regs up-regulate the CD71 proliferation marker in response to A. muciniphila. Summary and typical dot plots are shown. For (B) to (D), each symbol represents individual animals, and data were obtained in at least two independent experiments. Paired t test; *P < 0.05, **P < 0.01.

Fig. 6. A. muciniphila–induced pT_regs prevent colitis in the adoptive transfer model.

(A) Outline of the experiment. Mice were treated with a cocktail of antibiotics (Abx) and inoculated with indicated bacteria (n = 8 for each condition). At indicated time points, tail blood and feces samples were collected. i.v., intravenously. (B) Survival curves of adoptive transfer recipients precolonized with indicated bacteria (Kaplan-Meier curves and log-rank test). (C) Loss of initial body weight following adoptive transfer. Each point represents the mean percentage of initial body weight for the cohort ± SEM. (D) Frequency of pT_regs in the blood at indicated time points for CNS1^+/+ cell recipients. (E) Representative staining of analysis of colonic CD4⁺CNS1^+/+ cells in recipient mice. (F) Proportions and total number of colonic CD4⁺ cells in individual mice after colonization and adoptive transfer. (G) Histological analysis of colonic sections. Representative H&E staining (×40 magnification) and colitis scores (scores are presented as an average of three fragments of each tissue) are shown. Scale bars, 100 μm. The experiment was repeated twice with three to five mice per group. Each symbol depicts individual animal. For survival analysis, the log rank test was performed; otherwise, paired t tests were applied; *P < 0.05, **P < 0.01, ***P < 0.001. BL21-E. coli BL21DE3.