We recently defined TIM-1 as an inclusive marker for IL-10 expressing regulatory B cells (Bregs). Moreover, Α-TIM-1 mAb prolongs allograft survival (GS) through induction of TIM-1+ Bregs, which in turn, promote Th2 deviation and Foxp3+Tregs. We recently noted that TIM-4, a TIM-1 ligand, identifies B cells enriched for IFNΓ (“Be1” B cells). Based on these findings, we hypothesized that TIM-4 blockade should also prolong GS and that this may involve B cells. Α-TIM-4 (RMT4-53; 0.25 mg d -1, 0, 5) treatment alone, resulted in long-term GS in 100% of BALB/c recipients of B6 islets (MST >100d; p<0.01). Moreover, Α-TIM-4 increases Th2 cytokines (IL-4: 2.1X; IL-10: 2.2X; p<0.05) and Foxp3+Tregs (1.4X; p< 0.05), while reducing both Th1 cytokines (IFNΓ: 1.7X; p<0.05) and CD4 proliferation (from 26% to 14%). However, when recipients were B cell deficient (JHD) or depleted (Α-CD20), Α-TIM-4 no longer induced these changes and long-term GS did not occur (MST 30d; p<0.05). Thus, as with Α-TIM-1, regulation of T cell response and prolonged GS mediated by Α-TIM-4 is also B cell dependent. Α-TIM-4 could promote GS by targeting pro-inflammatory (TIM-4+) Be1 cells. This is supported by our finding that adoptive transfer of WT, but not TIM-4-/- B cells, into B-deficient ΜMT recipients of BALB/c islets, reconstitutes long-term GS mediated by Α-TIM-4 (MST>50d vs. 25 d respectively). While Α-TIM-4 does not deplete TIM-4+ B cells, it inhibits their expression of IFNΓ by 40%, which may promote the reduced Th1 response. Next we addressed whether Bregs were also required for Α-TIM-4 induced transplant tolerance. Mutant TIM-1LOF (loss of function = mucin domain deletion) mice were reported to exhibit a defect in Bregs and develop spontaneous autoimmunity. Unlike WT B cells (MST >50d; above), reconstituting Α-TIM-4 treated ΜMT recipients with TIM-1LOF B cells has no effect (MST ∼16d; p<0.05). The requirement for Bregs led us to ask whether interfering with TIM-4:TIM-1 signals with Α-TIM-4, might augment Bregs. Unlike Α-TIM-1, Α-TIM-4 does not increase the number TIM-1+ B cells. However, Α-TIM-4 increases the % of TIM-1+ B cells expressing IL-10 (2.7X, p<0.01), resulting in a significant increase in Bregs. Taken together, our data reveal that targeting TIM-4 enhances IL-10 expressing Bregs and also reduces inflammatory Be1 B cells to promote allograft tolerance.

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