TOLEROGENIC ENDOTHELIAL CELLS INHIBIT THE DIRECT RECOGNITION PATHWAY

Abstract

P928 Aims: Allograft rejection is mediated by T cells with direct and indirect allorecognition capacity. The direct pathway involves T cells which interact directly with professional (dendritic cells, macrophages) or nonprofessional (endothelial cells) Antigen Presenting Cells (APC) of the donor. The indirect pathway involves T cells which recognize dominant allopeptides derived from the processing of necrotic or apoptotic cells of the graft by host APC. Both in the periphery and within the graft, the frequency of T cells with direct allorecognition capacity is 10,000 times higher than that of allopeptide reactive T cells at all times. We have explored the hypothesis that tolerogenic endothelial inhibit the direct recognition pathway. Methods: Human endothelial cells (EC) were conditioned by use of IFN-γ and used for T cell priming. Conditioned EC were incubated with allospecific CD4+CD25+CD45RO+ FOXP3+ T regulatory cells (TR) or CD8+CD28- T suppressor cells. The expression on EC of CD31, CD40, CD54, CD62E CD106 CD58, HLA class I, HLA class II, ILT3, and ILT4, prior and following exposure to TS or TR was measured using flow cytometry and RT-PCR. T suppressor cells were tested in proliferation assays for their capacity to inhibit TH reactivity. Results: The ILT3high, ILT4high phenotype is characteristic of tolerogenic DC which induce regulatory T cells inhibiting the direct allorecognition pathway. However, after the departure of donor APC from the graft, rejection can be triggered by activated endothelial cells (EC). We have explored the hypothesis that EC may be involved in the maintenance of quiescence. Our studies have shown that alloantigen specific CD8+CD28- T cells with suppressor activity are FOXP3 positive and interact with human dendritic cells as well as EC. The interaction of TS with EC results in the induction of the inhibitory receptor ILT4, down-regulation of costimulatory and adhesion molecules and tolerization of EC. In turn, tolerized ILT4high EC elicit the differentiation of CD8+CD28- FOXP3+ T suppressor cells. Similarly alloantigen specific TR elicited the transcriptional activation of ILT3 and ILT4 in EC. Alloantigen specific CD8+CD28- FOXP3+ T cells, which trigger the transcriptional activation of ILT4 in EC, were found in the circulation of 21 heart allograft recipients in quiescence. Patients with a history of early acute rejection episodes (N=7) and/or chronic rejection (N=3) showed no TS or TR. The assay which we developed facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppression, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Conclusions: Endothelial cells are an important target of antigen specific T effector cells not only in transplantation but also in numerous autoimmune diseases. Agents which inhibit EC activation and/or render them tolerogenic are likely to have a beneficial effect in treatment of rejection, autoimmunity and possibly atherosclerosis.