Previously we reported an organ-specific immunomodifying therapy that provides protection from early rejection without systemic immunosuppression. The ImmunoCloak (IC) therapy is a membrane of laminin, proteoglycans, fibronectin and collagen. IC coats the surfaces of the renal vasculature and acts as a barrier between donor vascular endothelial cells (VEC) and the recipient’s immune cells. The resulting apical surface significantly delays the onset of rejection 5-fold over controls. In this study we elucidate the mechanisms of IC protection.Methods
IC was applied to the surfaces of VEC from human umbilical cords in 96 well plates. The assays were performed in triplicate using allogenic mononuclear cells (MNC) with or without IC treatment (n=5). Antigen presentation and T Cell alloresponses were measured by cytokine synthesis on the Luminex platform. Flow cytometry analysis was used to enumerate T-cell activation after 18H by quantification of CD8+, CD69+ lymphocytes. MNC proliferation was measured using a BrdU ELISA. Transmigration of MNC was tested on a VEC coated porous filter that separated 2 distinct compartments as a static model of chemotactic leukocyte transendothelial migration. MNC were added to the upper chambers and the chemoattractant SDF-1 was added to the lower chambers for a chemokine gradient.Results
IC therapy resulted in significant inhibition of cytokines: IL-1β, γ-IFN, TNF-α, IL-8, IP-10, MIP-1α and MIP-1β (p<0.05) (Table 1). The inhibition of cytokines production after stimulation with IC treated allogeneic VEC suggests that antigen presentation was prevented. The upregulation of T cell activation markers, IL-6, IL-2, MCP-1, and MIG was prevented with IC treatment. Therefore, the presence of IC on VEC blocks T cell responses. The results of flow cytometric analysis demonstrated prevention of CD69 activation after IC therapy. When MNCs are stimulated with allogeneic VEC a strong proliferative response occurred. The IC membrane inhibited these alloresponses by 99%. In transmigration studies 65% of cells were found to have migrated into the chamber containing SDF-1 within 3H. With IC applied to the VEC, the leukocyte migration into the lower chambers containing the SDF-1 was inhibited by a mean of 93%.Discussion
IC is a short-term therapy that could provide the opportunity to introduce effective adjunct therapies during the early posttransplant period. Eliminating the need for nephrotoxic immunosuppressive drugs during the early posttransplant period could help to ameliorate the severity of delayed graft function and could provide a path to utilizing more ischemically damaged renal allografts.Conclusion
The results of these studies support our hypothesis that the membrane provides protection in an organ-specific manner to prevent the cascade of events resulting in early allorecognition that normally occurs upon reperfusion.Conclusion
National Institute of Health - NIAID Grant # R44AI081372.