C1-B are potent inducers of Tregs. (A and B) Frequency, activation, and proliferation of FOXP3⁺ Tregs in the LNs of 10 d immunized ACB and BQ mice (n = 5 mice/group). (C–E) Frequency and activation of LAG3⁺CD49b⁺ Tr1 cells in the LNs of 10 d immunized ACB and BQ. (F and G) Flow cytometry plots depicting the frequency of endogenous COL2-reactive Vβ8.3 proliferating Tregs (FOXP3⁺Ki67⁺) after in vitro culture of LN cells derived from ACB, heterozygous and homozygous ACB.Col2^R360Q mice (n = 5 mice/group). (H) Frequency of CD44⁺ Tregs gated on F. (I) Experimental setup for contact-dependent and independent Treg induction experiment. (J and K) Flow cytometry plots depicting the frequency of proliferating Tregs in direct contact (pink), contactless (gray) culture alone or with natural C1-B and B cells purified from BQ mice in the presence or absence of COL2 (n = 18 mice, each symbol represents three mice pooled). Error bars represent mean ± SEM. Statistics in A, B, D, and E were determined by two-tailed Mann–Whitney U test. Significance in G, H, and K was determined by two-way ANOVA followed by Sidak’s test. ****P < 0.0001.