*Purpose: Extensive transcript analysis has been performed on grafts at the time of rejection, but gene expression preceding overt rejection is less well studied.

*Methods: We transplanted allogeneic A/J (H-2a) hearts or control isogeneic C57BL/6 (H-2b) hearts to B6 recipients and euthanized pairs of mice at 1, 4, 6 and 8 days. Grafts were analyzed by immunohistology and NanoString gene expression panels.

*Results: Immunohistology demonstrated limited macrophage and T cell (CD4 and CD8) infiltrates at 4 days that evolved into moderate interstitial infiltrates at 6 days and then extensive infiltrates with myocardial injury at 8 days. As expected, the 25 genes that were most upregulated (>20 fold) in allografts compared to isografts at day 6 were mostly T cell and NK cell related, including Cd3, Cd8, Cd6, Cd5, granzyme B, perforin, Cxcl9, Cxcl10, Ccl5, Ifng, Il2r, Klrc1, Klrk1 and Klra2. Two additional genes in this group were the negative costimulatory receptor PD-1 (Pdcd1) and its ligand PD-L1 (Cd274) that were upregulated 50- and 30-fold, respectively. These transcript markers correlate closely with the immunohistology that demonstrated interstitial infiltrates of PD-1 positive cells. Of these 25 T cell and NK cell associated transcripts, only 9 were upregulated at day 4 in the early stages of rejection. These included Cxcl9, Cxcl10, Klra2, Ccl5, Klrk1, Gzmb, and Prf1. Remarkably PD1 and PD-L1 were also upregulated (10 and 30-fold, respectively) in allografts vs isografts at day 4. In contrast, Ctla4 a current target of therapeutic intervention was not upregulated at day 4 and was upregulated about 10-fold at day 6 when rejection is advanced.

*Conclusions: These data indicate that potent negative regulatory systems are upregulated early in the rejection process and may provide potential therapeutic targets.

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