*Purpose: When antibodies bind to MHC antigens on endothelial cells a cascade of responses is initiated including exocytosis of adhesion molecules followed by attachment and activation of platelets and macrophages. The initiating mediators of antibody-mediated rejection are not fully defined.

*Methods: We have modeled these events by passively transferring donor specific antibodies (DSA) to renal allograft recipients. The influence of T cells was eliminated by using Rag deficient (Rag-/-) mice as recipients. Specifically, B10.A (H-2^a) kidneys were transplanted to C57Bl/6 (H-2^b) Rag-/- mice. After 7 days, when postoperative inflammation subsided, a mixture of IgG1, 2a and 2b monoclonal antibodies to H-2^a (or isotype controls) were transferred. Immunohistology, ELISA and NanoString analyses were performed on allografts 1 hour after antibody treatment.

*Results: By 1 hour, C4d deposited on peritubular and glomerular capillaries in a strong, diffuse pattern. Extensive aggregates of P-selectin positive platelets co-localized with C4d. Capillaries also contained platelet-monocyte conjugates. The platelets released large amounts of platelet factor 4 (CXCL4) and serotonin in the graft (10-25 ng/mg tissue). To further characterize the macrophage infiltrates mRNA arrays were performed by NanoString on kidney allografts. Passive transfer of DSA upregulated expression of genes associated with macrophages. Cd68 and Itgam (Mac1) were 3- and 4-fold higher at 1 hour following transfer of DSA and remained higher at 5 hours compared to isotype controls. However, the most highly upregulated gene was Gzmk (granzyme K). Gzmk was upregulated about 35-fold at 1 hour. Gzmk has recently been reported to be expressed by classically activated human macrophages and in separate experiments we confirmed that Gzmk is expressed by macrophages isolated from mouse renal allografts. To determine the effects of platelets on the expression of Gzmk, a group of mice was pretreated with platelet depleting antibody 1 hour before transfer of DSA. Depletion of platelets decreased Gzmk expression by half.

*Conclusions: Our finding that DSA induces GzmK expression is germane to the early responses to transplants because Gzmk has been reported to upregulate MCP-1, ICAM-1 and VCAM-1 expression in endothelial cells.

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