Acute rejection of cardiac allografts is characterized by interstitial infiltrates and diagnosed by endomyocardial biopsies. In contrast, chronic rejection is distinguished by vasculopathy in large arteries and is diagnosed by angiography. Hearts transplanted from male to female C57BL/6, which differ only at H-Y, undergo acute rejection that transitions into chronic rejection. Diffuse interstitial infiltrates of T-cells and macrophages that spared the large arteries were documented at 2 weeks. By 6 weeks the interstitial infiltrates resolved, and about half of the large arteries developed macrophage infiltrates together with neo-intimal proliferation of smooth muscle cells. To test the role of CD4 T-cells in orchestrating acute and chronic pathology, male B6 hearts were transplanted into female B6 Rag1-/- mice, and 1 week later purified CD4 T–cells were transferred from female Marilyn transgenic mice, in which all CD4 T-cells are specific for H-Y. This resulted in macrophage-rich interstitial infiltrates at 2 weeks and arteriopathy at 6 weeks. To identify local mediators, we separated interstitial and arterial infiltrates by laser capture microdissection. An array of 86 genes by was screened by real time RT PCR. CD274 (PDL1) and a set of IFNg regulated genes: CXCL9 (MIG), CCL5 (RANTES) and CCL2 (MCP-1) were highly upregulated in the interstitium at 2 weeks. By 6 weeks, expression of these genes diminished together with the interstitial infiltrates. ELISA measurements of intragraft protein levels confirmed gene expression. PDL1 upregulation at 2 weeks suggested that negative co-stimulatory signaling of T cells might contribute to the resolution of acute interstitial infiltrates. Therefore, we blocked PD1:PDL1 interaction by 3 doses of 200¯o;g of purified rat monoclonal antibody to PDL1 or 200¯o;g of isotype control. Blocking PD1:PDL1 in the acute phase increased interstitial infiltrates leading to complete rejection of 2 of 4 cardiac allografts by 2 weeks. Delayed blocking of PD1:PDL1 increased interstitial infiltrates moderately, but did not alter arteriopathy. A different set of genes was upregulated in the arterial compartment: IL1R2, IL1R1, TLR6 and TRAF6 at 2 weeks and TLR6, MyD88, CCR3, CCL5, CXCL9 and CCL1 at 6 weeks. The expression of CCR3, CCL5, CXCL9 and CCL1 support the T cell dependency of the macrophage-rich arterial lesions. TLR6 and MyD88 indicate a possible role for endogenous or exogenous molecular pattern recognition in the development of arteriopathy.

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