Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class І MHC within 24 hrs after graft reperfusion in mice. Our recent studies indicate that prolonged cold ischemic graft storage provokes intense inflammation within hours after allograft reperfusion and promotes endogenous memory CD8 T cell rejection of the allograft. The role of graft TLR4 signaling in early post-transplant inflammation and downstream alloimmune responses to allografts subjected to prolonged cold ischemic storage was tested.

Wild type (WT) BALBc (H-2d) or BALB/c.TLR4−/− hearts were subjected to 8 hrs of cold ischemic storage in University of Wisconsin solution and then transplanted to C57BL/6 (H-2b) mice. T cell, macrophage, and neutrophil infiltration into allografts on day 2 post-transplant were assessed by flow cytometry and immunohistochemistry.

Marked decreases in neutrophil, but not CD4 and CD8 T cell and macrophage, infiltration were observed in TLR4-/- vs. WT BALB/c allografts on day 2 post-transplant. This decrease was associated with marked decreases in intragraft mRNA expression of the neutrophil chemoattractants CXCL1 and CXCL2 on day 2. The absence of allograft TLR4 signaling did not affect intragraft mRNA expression of CXCL10, TNF-α and IL-1β. On day 7, TLR4-deficient allografts had virtually no infiltrating CD4 T cells, macrophages or neutrophils and decreased CD8 T cell infiltration that was accompanied by decreases in mRNA expression of perforin, granzyme B and FasL. Treatment with peri-transplant anti-CD154 mAb extended the survival of WT allografts from day 8 to day 38 whereas survival of TLR4-/- allografts was extended from day 12 to 50-60. This extension in allograft survival was due to the absence of TLR4 signaling on bone marrow-derived, and not on parenchymal, cells in the allograft.

These data indicate that the absence of TLR4 signaling in allografts subjected to prolonged cold ischemic storage inhibits the early post-reperfusion inflammation and inflammation at times post-transplant when wild-type allografts are undergoing acute rejection. The results suggest that therapeutic targeting of TLR4 will reduce the negative impact of early inflammatory events on allograft outcome and contribute to graft survival-promoting strategies.

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