Self and microbiota-derived epitopes induce CD4+ T cell anergy and conversion into CD4+Foxp3+ regulatory cells

The physiological role of T cell anergy induction as a key mechanism supporting self-tolerance remains undefined, and natural antigens that induce anergy are largely unknown. In this report, we used TCR sequencing to show that the recruitment of CD4+CD44+Foxp3−CD73+FR4+ anergic (Tan) cells expands the CD4+Foxp3+ (Tregs) repertoire. Next, we report that blockade in peripherally-induced Tregs (pTregs) formation due to mutation in CNS1 region of Foxp3 or chronic exposure to a selecting self-peptide result in an accumulation of Tan cells. Finally, we show that microbial antigens from Akkermansia muciniphila commensal bacteria can induce anergy and drive conversion of naive CD4+CD44-Foxp3− T (Tn) cells to the Treg lineage. Overall, data presented here suggest that Tan induction helps the Treg repertoire to become optimally balanced to provide tolerance toward ubiquitous and microbiome-derived epitopes, improving host ability to avert systemic autoimmunity and intestinal inflammation.


Bacterial strains
A. muciniphila (ATCC BAA-835) was received from Dr. B. Chassaing (INSERM, France) and was grown in an anaerobe BHI broth (Fisher) in an anaerobic chamber (Coy Lab Products). A. muciniphila was grown overnight, washed twice in PBS and 100 l (2x10 8 CFU) per mouse was gavaged to 6-8 weeks old mice. E. coli BL21 DE3 strain (Invitrogen) was propagated overnight in LB, washed twice in PBS and 2x10 8 CFU was gavaged per a mouse.

Real-time PCR
RNA was prepared from FACS-sorted CD4 + cells using Qiagen kit. DNA-free RNA was converted to cDNA with SuperScript III kit (Invitrogen). An equal amount of material was run in triplicate on Quant Studio 3 Real-Time PCR instrument (Fisher) using TaqMan assays (Invitrogen). A comparative CT (CT) method was used for analysis with data normalized to -actin.

Tissue preparations, FACS analysis, and cell sorting
Colon samples were prepared and stained as reported 12 . Spleens were mechanically disrupted and passed through 70 m filters (Fisher). Erythrocytes were lysed with buffered ammonium chloride and washed with HBSS before staining. FACS gating strategy is shown in Fig. S5. For the detection of intracellular cytokines, cells were stimulated for 4 hours with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of brefeldin A and monensin (each 1:1,000; BioLegend). Surface markers were stained before fixation and permeabilization (Foxp3 kit, eBioscience). T cells ex vivo proliferation was assessed by measuring dilution of eFluor670 (eBioscence) dye according to the manufacturer's instruction. Cells were analyzed on day 4 by flow cytometry. FACS analysis was done using CytoFlex (B5-R3-V5) flow cytometer (Beckman) and cells were sorted with SH800 sorter using 100 m chips with purity >98%, and viability >98% (as assessed by DAPI or trypan blue staining). In some experiments, CD4 + cells were first negatively enriched using MS columns (Miltenyi) to >90% purity before sorting on SH800 instrument. FACS data were analyzed with FlowJo v.10 (FlowJo, LLC).

Cell culture
All cells were cultured in CTM media and IL-2 was measured with HT-2 assay 12 . Inhibition assays were done as reported 38 . Briefly, FACS-purified naïve CD4 + CD44 -Foxp3cells were labeled with eFluor670 dye and activated with soluble aCD3 (100 ng/ml) in the presence of GM-CSF-elicited bone marrow-derived dendritic cells (DCs) 22 . The tittered CD4 + Foxp3 + cells were added to selected wells and cells were FACSanalyzed at day 4. In some experiments, CT-1 peptide (RQPKIWFPNRRKPWKKRPRPDDLEI (American Peptide Company), 30 nM) was added to co-cultured cells. The selected experiments involved the knocking-down ex vivo expression of Cx43 in Tregs. For that purpose, the pool of Cx43 or scrambled siRNA (Santa Cruz) was delivered with HiPerfect (Qiagen) to FACS-sorted Tregs which then were incubated overnight with 10 ng/ml of IL-2, washed and co-cultured with tittered number of Tregs in suppression assay. During co-culture Tregs and CD4 + Foxp3cells were activated by plate-bound aCD3 (10 g/ml) and aCD28 (1 g/ml). Downregulation of Cx43 by siRNA was detected with real-time PCR and by measuring transfer of calcein violet as reported 27 . Briefly, in vitro activated Tregs were loaded with calcein violet (0.5 M), mixed with activated CD4 + Foxp3cells and calcein transfer was assessed 5 hours later by FACS.

Immunization
Antigenic peptides (ABI Scientific, purity >90%) were mixed with 10 g cholera toxin (CT; Sigma) at 30 g/mouse and mice were immunized by gavage. Seven days later mice were boosted with ip injection of 15 g peptide in PBS and sacrificed on day 14. For vaccination studies, a pool of four A. municiphila-derived peptides (25 g of each per a mouse) was mixed with CT and gavages to mice in 100 l total volume per mouse. One day later FACS-purified naïve CD4 + CD44 -Foxp3were intravenously injected into mice. Animals were gavaged every other day for 3 additional times followed by i.p. peptide injections 3 times a week for 4 weeks of peptides adsorbed on alum (Alhydrogel; Brenntag) for 5 min before injection. Control mice received alum only.

Adoptive transfer
FACS-purified naïve CD4 + CD44 -Foxp3 GFPcells (1x10 6 ) were adoptively transferred into TCR k/o mice expressing wild type A b or A b covalently linked with Ep or 63K peptides 20 . Animals were monitored for signs of disease (weight loss, diarrhea) and euthanized when met humane endpoint or no later than day 120.

Statistical analysis
Data were analyzed with Origin 2017 (OriginLab). Data were presented as the mean values ± SD or survival (Kaplan-Meier) plots. Statistical significance of differences (p values) was calculated using ANOVA with Bonferroni correction when more than two groups were analyzed or with paired sample Student's t-test if two groups were analyzed. A log-rank test was applied to compare survival. Differences were considered as significant when *p<0.05, **p<0.01, ***p<0.001.
For TCR analysis, only CDR3 regions found more than five times in each population per organ and mouse were analyzed. TCR CDR3 species frequencies were obtained from 2 biological replicates each with 2 technical replicates sequenced both ways. These frequencies were used to determine the number of TCRV2 CDR3 clonotypes. Statistical significance of the results was performed using Origin Pro (Origin Lab). The analysis was performed using independent samples t-test or a paired sample Student t-test as appropriate. The results were expressed as the mean ± SEM unless stated otherwise. *p≤0.05, **p<0.01, ***p<0.001 as statistically significant. For the comparative analysis of the overlap, a mutual information index (I-index) was used as defined 43 . Diversity was quantified using the effective number of species (ENS) and presented in the form of diversity profiles (see 43 for definitions).   Fig. S2. The magnitude of TCR mini CD4 + cells ex vivo activation by many wild-type peptides or single peptide bound to A b . (A, B) Expression of activation marker CD44 + and proliferation marker CD71 + on CD4 + Foxp3cells from TCR mini , EpTCR mini or 63KTCR mini mice co-cultured for 4 days with DCs expressing A b wt, A b Ep or A b Ep63K complexes. Representative staining from one of two experiments is shown, and stimulation with aCD3 MoAb (0.1 µg/ml) was used as a control.