70-P : ALLOGENEIC EXPANSION OF CD4+ T CELL SUB-POPULATIONS BY HUMAN MICROVASCULAR ENDOTHELIAL CELLS IS MODIFIED BY HLA-DR ANTIBODY PRE-ACTIVATION OF THE ENDOTHELIAL CELL

Aim Antibody mediated graft rejection (ABMR) is mainly due to alloantibodies to donor HLA antigens. Microvascular ECs are targets of ABMR. The hallmark of chronic rejection, Transplant Glomerulopathy, is associated with high levels of HLA class II alloantibodies with or without complement activation. We have shown that EC expression of HLA-DR permits expansion of allogeneic pro-inflammatory CD4+IL-17+-T cells (Th17) via an IL-6/STAT-3 pathway and regulatory CD4+FoxP3+ T cells (Tregs) via CD54 expression by ECs under inflammatory conditions (Taflin et al PNAS 2011). This study examines whether activation of ECs by HLA class II Ab modifies CD4+ T subset expansion. Methods HLA-DR Ab stimulation of human microvascular ECs was carried out under signaling (not blocking) conditions before recuperating cells for proteomic studies, further EC culture or co-culture with allogeneic CD4+ T lymphocytes. F(ab’) 2 Ab fragments were tested. Cytokines were detected by ELISA. T cell proliferation was measured by CFSE labeling and expansion of T cell subsets was determined by intracellular cytokine staining. Results Activation of the allogeneic EC via HLA-DR modified tyrosine kinase activation and cytokine production by intact HLA-DR Ab or the F(ab’)2 fragment. HLA-DR mediated activation of the EC increased expansion of the Th17 population and decreased generation of Treg cells and this too was reproduced by the F(ab’)2 fragment thus excluding a requirement for Fc mediated effects to induce the functional changes in this experimental model. Conclusions These data demonstrate that HLA-DR antibodies can activate ECs and that activation has functional consequences for the EC expansion of allogeneic CD4+ T pro-inflammatory or regulatory populations. The results reveal an interaction between humoral and cellular alloresponses and lead to the suggestion that one mechanism of HLA-DR alloantibody driven allograft damage could be due to modified EC allogenicity and increased local pro-inflammatory Th17 expansion.