Bregs play a key role in immune tolerance by negatively regulating immune responses via IL-10. We showed that TIM-1 is an inclusive marker for IL-10+ Bregs and these cells can directly transfer allograft tolerance. Anti-TIM-1 (RMT1-10) induces IL-10+ Breg, suggesting that TIM-1 is more than a marker for Breg. Indeed, we recently showed that TIM-1 mucin domain deficient (TIM-1^Δmuc) mice developed progressive reduction in Breg and spontaneous autoimmunity. We now show that TIM-1^Δmuc (B6) recipients of BM12 heart allografts exhibit accelerated rejection (MST 27d vs. >60d) and more severe graft vasculopathy than wt recipients. Moreover, splenocytes from TIM-1^Δmuc recipients exhibit decreased IL-4 (2X), IL-5 (6X), and IFNΓ (4X), but a 5X increase in IL-17 compared to wt recipients. TIM-1 expression on TIM-1^Δmuc vs. wt B cells is higher (8% vs. 11%), IL-10 expression by TIM-1+ B cells (and total B cells) is lower (10% vs. 14%; p<0.05). Transfer of 10⁶ alloimmunized TIM-1+ but not TIM-1^– B cells into TIM-1^Δmuc recipients prolonged GS (MST from 27 to 42d; p<0.05). This suggests that the relative Breg deficiency in TIM-1^LOF mice contributes to their immune hyper-reponsiveness. Next, we assessed the role of TIM-1 mutation in Breg induction per se. Apoptotic cells (AC) are known to induce Bregs. The mucin domain of TIM-family members are known receptors for phosphatidyl serine (PtdS) on AC. To address the role of TIM-1 mucin domain on Breg induction, we treated wt and TIM-1^Δmuc mice with autologous AC (thymocytes). AC induced wt Bregs 2.5X but did not effect TIM-1^Δmuc Bregs. This could contribute to the Breg defect in TIM-1^Δmuc mice. Next addressed the response of B cells from wt vs. TIM-1^Δmuc mice to in vivo ligation with anti-TIM-1. Importantly, anti-TIM-1 (RMT1-10) binds with equivalent affinity to TIM-1 from both mice. Whereas anti-TIM-1 induces a 1.6-2X increase (to 22%) in TIM-1+IL-10+ Bregs in wt mice, this mAb had no affect on Bregs from TIM-1^Δmuc mice. (10% = control levels). Thus, TIM-1^Δmuc lack TIM-1-mediated induction of IL-10+ Bregs. So TIM-1 is important not only as a marker for Breg but also as an important functional determinant of basal and induced Breg. The TIM-1 mucin domain appears to play a key role in Breg induction by AC through PtdS binding, as well through anti-TIM-1mediated signaling, and in turn, plays an important role in immune homeostasis, allo- and autoimmunity.

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