Research ArticleIMMUNOLOGY

Commensal epitopes drive differentiation of colonic Tregs

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Science Advances  17 Apr 2020:
Vol. 6, no. 16, eaaz3186
DOI: 10.1126/sciadv.aaz3186
  • Fig. 1 Activation of CD4+ T cell hybridomas produced using new BWNur77GFP thymoma.

    (A) Expression of GFP in NFATGFP (left) and Nur77GFP (right) reporters in response to aCD3 (1 μg/ml) in two representative hybridomas. MFI, mean fluorescence intensity. (B) Up-regulation of NFATGFP or Nur77GFP 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 Nur77GFP reporter by representative hybridoma activated by titrated aCD3 mAb. Nur77GFP (C) and IL-2 (D) expression by hybridoma from CD4+TCR+Foxp3+ Tregs. 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) Nur77GFP 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 Nur77GFP 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 Nur77GFP up-regulation (B) was measured 16 hours later. The experiment was repeated twice. (C) Nur77GFP exclusively reports TCR activation of CD4+ hybridoma to antigens presented in the context of class II MHC (Ab). Antigens presented by Ab-negative DCs or DCs with antibody-blocked Ab do not up-regulate Nur77GFP. Lack of MHCI (β2mk/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 Nur77GFP reporter in response to fecal bacteria lysate in hybridomas prepared from TCRmini, GF TCRmini, and CNS1k/oTCRmini 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 TCRmini and TCRminiCNS1k/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 (TCRmini: Foxp3, n = 14 and Foxp3+, n = 11; TCRminiCNS1k/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 TCRmini 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 Tregs [(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 Tregs 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 TCRmini and TCRminiCNS1k/o mice are also shown. (B) Representative up-regulation of Nur77GFP 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 Tregs in TCRmini mice after immunization with A. muciniphila–derived 2C.1 peptide. (E) Frequencies and total number of colonic Tregs in TCRmini 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) Tregs 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 Tregs in vivo expansion.

    (A) 16S ribosomal RNA analysis of microbiomes from TCRminiCNS1k/o and TCRmini 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 TCRminiFoxp3GFP mice with A. muciniphila expands colonic Tregs. (C) A. muciniphila induces pTregs and expands tTregs. Naïve CD4+CD44lowFoxp3 cells from congenic TCRmini mice were adoptively transferred into CNS1k/oTCRmini 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) TCRminiCNS1k/o mice were colonized with A. muciniphila, and 8 weeks later, their colonic tTregs were analyzed. Graphs show percentage (left) and numbers (right) of CD4+Foxp3+ cells. (E) tTregs 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 pTregs 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 pTregs 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.

Supplementary Materials

  • Supplementary Materials

    Commensal epitopes drive differentiation of colonic Tregs

    Michal P. Kuczma, Edyta A. Szurek, Anna Cebula, Benoit Chassaing, Yu-Jin Jung, Sang-Moo Kang, James G. Fox, Bärbel Stecher, Leszek Ignatowicz

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