Under physiological conditions, prompt clearance of dying cells provides protection from the release of pro-inflammatory contents, induces T cell tolerance to self-antigens and prevents autoimmunity. Furthermore, experimental data suggests that exposure to apoptotic bodies, adoptively transferred prior to transplantation, may promote tolerogenic DCs and allo-specific tolerance. Here, we investigated the effects of blockade of phagocytosis at the time of transplant.

In a fully mismatched model, administration of gadolinium (Gad; 100mcg on D-2, D5) to cardiac allograft recipients completely abrogates the graft-prolonging effects of CTLA4-Ig (MST 8d vs 30d, p<0.001). This was associated with a 3-fold and 5-fold increase in graft-infiltrating CD4 and CD8 effector memory cells, respectively. After blockade of phagocytosis, a 2-fold increase in IFNg production by graft infiltrating CD4 T cells was seen. As expected, the % of AnnexinV+7AAD+ (dying/dead) cells was increased in recipient spleens treated with Gad (25.2+/-0.9 vs 15.66+/-1.9%, p<0.05). Upon ex vivo restimulation, splenocytes from Gad-treated mice showed a general increase in pro-inflammatory cytokine production: IFNg (2.5x), IL4 (2x), and IL17 (1.5x), but no differences in IL10 production. To determine whether these cytokines were produced by or in response to dying apoptotic cells, we depleted the splenocyte fraction of AnnexinV+ cells by magnetic isolation before re-stimulation. Surprisingly, we still found a 2-fold increase in IFNg and IL4 and a 3-fold increase in IL17 production, but a 2-fold reduction in IL10 production from splenocytes taken from Gad-treated recipients. Taken together, these data indicate that blockade of phagocytosis at the time of transplant promotes a pro-inflammatory milieu largely by promoting heightened allo-specific responses from live cells, rather than simply through non-specific cytokine release from dying cells. Thus, strategies to enhance uptake and elimination of these dying cells, particularly at the time of transplant when ischemia-reperfusion injury promotes cell death, may be of therapeutic benefit.

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