Alloantigenic recognition of artificial glycosyl phosphatidylinositol-anchored HLA-A2.1

Alloantigen presentation by GPI-reanchored variants of the human class I MHC molecule HLA-A2.1 was studied in human cellular systems. To this end, we generated chimeric coding sequences for two GPI-modified HLA-A2.1 heavy chain derivatives. In these chimeras, the coding sequence for the HLA-A2.1 heavy chain was fused in-frame to alternative overlapping sequences from the 3'-end of human DAF containing the GPI-modification signal sequence. The encoded polypeptides HLA-A2.1:DAF-S and HLA-A2.1:DAF-L differed by 53 amino acids of additional DAF sequence in the latter. Both were detected on stably transfected C1R cell surfaces by HLA-A2.1-specific mAb, and their GPI-modification was confirmed by PI-PLC enzymatic cleavage. Immunoprecipitation analysis of surface-biotinylated C1R transfectants revealed heterodimeric association for both HLA-A2.1:DAF-L and HLA-A2.1:DAF-S heavy chains with beta 2m. Alloantigenic stimulation by, and cytotoxic recognition of, both HLA-A2.1:DAF-S/C1R and HLA-A2.1/CIR cells was observed; however, HLA-A2.1:DAF-L/C1R cells could not serve as allostimulators or allotargets. These findings establish that polymorphic human class I MHC molecules can function, when artificially GPI-reanchored, as alloantigenic targets. Moreover, the data suggest that the sequence bridging the HLA-A2 extracellular domain and the membrane can influence alloantigenic presentation.