OR2 Variable expression of HLA-C impacts T versus B cell crossmatch outcomes

Abstract

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibodies (DSA) and determine eligibility for transplantation and desensitization regimens. We measured HLA class I and HLA-C surface expression on T and B cells from various sources and examined the impact of HLA-C expression on FCXM reactivity in the presence of HLA-C DSA. HLA class I and HLA-C expression was examined on cells isolated from 18 donors using monoclonal antibodies (clone G46.2 and mAb DT9). T and B cells were differentiated using CD3 and CD19 specific monoclonal antibodies. Cells were acquired on a BD FACSCanto II; HLA expression was determined using median channel fluorescence (MCF) values and FCXM reactivity was assessed as a ratio of test serum to negative control serum. The mean HLA-C expression was significantly higher on T (11693 MCF) than B cells (5480 MCF, p < 0.05, Figure 1) while class I expression was lower on T (10167 MCF) than B cells (23602 MCF, p < 0.001) when comparing cells isolated from the same donors. This difference in HLA-C expression was mirrored in FCXM tests using sera containing HLA-C specific DSA. Mean reactivity with T cells as targets yielded consistently higher ratios than tests using B cells as targets (p < 0.001). Seven of 10 FCXM tests yielded positive T and negative B results. HLA-C expression is known to vary among HLA-C alleles (Apps et al., Science 2013); however, this study shows variability also exists between lymphocyte lineages. HLA-C is expressed higher on T than B cells and this difference impacts crossmatch reactivity. These findings should be considered when interpreting FCXM results.Download : Download high-res image (108KB)Download : Download full-size image