Pharmacological Inhibition Of Semi-Direct Alloantigen Presentation and The Generation Of Cross-Dressed Antigen Presenting Cells: A Novel approach To Limit Graft Versus Host Disease Following Allogeneic Hematopoetic Stem Cell Transplantation

Background Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant (allo-HSCT) is caused by CD4+ and CD8+ donor T cells directed against mismatched recipient antigens, presented in the context of donor MHC-II (indirect pathway) and recipient MHC-I (direct pathway). Recently, the presence of ' cross-dressed ' CD11c+ antigen presenting cells (APCs) expressing both donor and recipient type MHC-I molecules has been demonstrated in animal organ and HSCT transplant models supporting 'semi-direct' pathway of allo-activation (Wang et al, Blood. 2011).These APCs can efficiently present allo-antigens to both CD4+ and CD8+ T cells and activate immune responses that could lead to allograft rejection or GVHD. Exchange of membrane fragments and associated proteins between cells, termed trogocytosis, generates cross-dressed APCs.We sought to test whether cross-dressed APCs facilitate antigen presentation to donor T cells and initiate GVHD following allo-HSCT. Further, we tested an array of drugs as inhibitors of trogocytosis, to interrupt the semi-direct pathway of allo-antigen presentation. Methods In vivo experiments used a B6(H2Kb) ˆ B10.BR(H2Kk) murine transplant model. Spleens of transplanted mice were analyzed on days 10, 15, 20 post-transplant for presence of cross dressed CD11c+cells, and their expression of CD80, CD86 and MHC-II by flow cytometry. Cross dressed donor CD11c+ FACS sorted cells from recipient spleens were co-cultured with CFSE labeled donor type T-cells for 6 days, and T-cell proliferation was measured as dilution of CFSE by flow cytometry. In vitro experiments used primary MLR consisting of CFSE labeled B6 bone marrow cells co-cultured with PKH26 (membrane dye) labeled B10.BR splenocytes. B6 antigen presenting cells were analyzed by flow cytometry for the presence of CFSE+PKH26+ double positive cells generated by trogocytosis. Pharmacological inhibitors of cytoskeleton function were added to the primary MLR and their effect on trogocytosis as well as T cell proliferation was assessed. Results Cross-dressed donor CD11c+ APCs were generated in vivo following allo-HSCT ([Figure 1][1]). Recipient spleens showed that 50%, 28.6% (p=0.01) and 12% (p=0.02) of donor type CD11c+ cells were cross dressed on days 10, 15 and 20 respectively post transplant (n=5). These cross dressed APCs expressed higher levels of co-stimulatory molecules CD80 (p<0.001) and CD86 (p<0.001), and MHC-II compared to non-cross-dressed donor CD11c+ cells ([Figure 2][2]). Sorted cross dressed CD11c+ cells from recipient mice were able to induce in vitro proliferation of co-geneic CD8 T-cells, while their non-crossdressed counterparts did not. We demonstrated that cross-dressed CD11c+ cells were generated in vitro , by exchange of plasma membrane fragments and could be inhibited in vitro by low doses of paclitaxel and VIP antagonist ([Figure 3][3]), while preserving cell viability. Further more, bone marrow treated with 0.05uM of paclitaxel, caused significantly decreased T cell proliferation in primary MLR compared to non drug treated bone marrow. ![Figure 1][4] Figure 1 Cross-dressed donor CD11c+ APCs were generated following allo-HSCT . Left and middle panels show expression of H2Kb and H2Kk in non-transplanted mice. Right panel shows cross-dressed APC on day 15 post allo-HSCT. ![Figure 2][4] Figure 2 Cross-dressed APCs (red) have increased expression of co-stimulatory molecules CD80 and CD86 compared to non-cross dressed donor CD11c+ APC (blue). ![Figure 3][4] Figure 3 Paclitaxel inhibited trogocytosis in MLR. The frequency of cross-dressed CD11c+ APC on day 6 of MLR was assessed by flow cytometry. Discussion The high frequencies of cross-dressed donor CD11c+ APCs following allo-HSCT suggests that semi-direct allo-antigen presentation may play a key role in the initiation of GVHD, while the decreasing trend could reflect replacement of host cells by donor hematopoetic cells. Reducing the generation of cross-dressed APCs by pharmacological inhibition of trogocytosis is a novel approach to reduce GVHD post allo-HSCT, targeting the semi-direct pathway of allo-antigen presentaion. Our data shows that very low doses of paclitaxel, a microtubule inhibitor and VIPHyb, an antagonist of Vasoactive Intestinal Peptide signaling, can reduce semi-direct presentaion of allo-antigen to Tcells and reduce alloreactivity without direct cytotoxic effect. Disclosures: No relevant conflicts of interest to declare. [1]: #F1 [2]: #F2 [3]: #F3 [4]: pending:yes