*Purpose: De novo anti-HLA-II DSAs (dnDSA) are associated with antibody-mediated rejection. However, some patients with anti-HLA-II DSAs will not develop rejection, suggesting that the development of antibodies is not sufficient to trigger the process. We hypothesized that a determinant of rejection is the expression of the associated HLA antigen in the graft. More specifically, we hypothesized that HLA-II antigen expression on allo-endothelial cells will vary between individuals and between subclasses (DR, DP and DQ) within the same individual.

*Methods: To examine the distribution of expression of HLA-II antigens on endothelial cells in humans, we generated endothelial-colony forming cells (ECFCs) from PBMC samples biobanked from patients and healthy volunteers (n=31). ECFCs were first activated during a 4-day course with IFN-γ, followed by a 10-day time-course. HLA-I and II (DR, DP and DQ subclasses) expression was measured by flow cytometry for both ECFCs and PBMCs from the same individual. The relationship between the level of expression of HLA-II and specific alleles was analyzed by a multivariate model.

*Results: On ECFCs, we found a high variability for HLA-DQ expression between individuals ranging from 0% to 86% for minimal and maximal values respectively (standard deviation of HLA-DQ positive cells 28%). HLA-DR and DP expression were more similar (standard deviation of 3% and 8% respectively). In the cohort, the percentage of ECFCs expressing HLA-DQ correlated with the percentage found on CD14+ cells. Analysis of the HLA typing revealed that patients who had the DQ5 and DQ6 alleles had substantially more HLA-DQ antigen expression (13±6 vs. 37±11% positive cells and 10±5% vs. 39±11% for DQ5 and DQ6 respectively; p=0.03 and 0.01 respectively). On the opposite, DQ7-patients had substantially less endothelial surface expression of the antigen (42±10 vs. 7±5%, p=0.03).

*Conclusions: These data suggest an underappreciated heterogeneity in HLA expression on endothelial cells, which could be predictable by HLA typing. These results are clinically relevant since most anti-HLA antibodies are directed against HLA-DQ. Our observations could explain why some patients do not develop antibody-mediated rejection despite the development of anti-HLA DSAs.

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