T cell immunoglobulin and mucin domain (TIM)-4, expressed on dendritic cells and macrophages, functions both as a costimulatory molecule and a phosphatidylserine receptor, mediating phagocytosis of apoptotic cells. The relative importance of these functions in alloimmunity remains unclear. Here, we investigated whether the graft-prolonging effects of anti-TIM-4 are mediated by blockade of either T cell costimulation or phagocytosis of apoptotic cells.

In a fully mismatched model, synergistic blockade using CTLA4-Ig and anti-TIM-4 mAb led to graft acceptance (MST >100d vs 30d with CTLA4-Ig alone, p<0.001). This was associated with a marked reduction in CD4 and CD8 Teffector cells. However, a 10-fold increase in number of splenic apoptotic cells was seen on TUNEL staining in aTIM-4 treated mice. We hypothesized that this effect could be due to impairment of phagocytosis of apoptotic cells by TIM-4 expressing DCs or macrophages. Since disruption of AB clearance leads to a pro-inflammatory response, we hypothesized that blockade of TIM-4 costimulation promotes GS, whereas blockade of its phagocytic function may be detrimental. To test this, we compared GS of recipients treated with CTLA4-Ig and gadolinium (which blocks global phagocytosis), with and without aTIM-4. As expected, gadolinium markedly accelerated rejection, but the additional administration of aTIM-4 still led to a 3.5-fold increase in GS (MST 30d vs 8d, p<0.001), presumably by blocking TIM-4 costimulation. We then sought to investigate whether aTIM-4 or TIM-4 deficiency leads to impaired phagocytosis in DCs or macrophages. While DC phagocytosis was unaffected by aTIM-4, in contrast, phagocytosis by splenic and peritoneal macrophages was markedly decreased by both blockade of and the absence of TIM-4 (2- and 3-fold reduction, respectively). Finally, to confirm that interruption of DC TIM-4 costimulatory function is the predominant mechanism leading to graft acceptance, we generated CD11c-DTR chimeras to allow long-term depletion of DCs. Chimeric mice were then transplanted and treated with aTIM-4 or control IgG. Consistent with our hypothesis, we found GS achieved by TIM-4 blockade is markedly attenuated by near-complete DC depletion (MST 26d vs 20d).

In summary, TIM-4 appears to have opposing roles in alloimmunity. Its function as a costimulatory molecule promotes graft rejection, whereas its role as a PS receptor may be protective.

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