Replacements at Structural or Functional Dimorphisms 103, 109 and 167 Distinguish HLA Class I Serologically Defined Antigens

Amino acid variations in the HLA molecule may serve as part of serologic epitopes (residues determining epitopes: DEP) detected by allo-antibodies. In current clinical histocompatibility practices, the presence of anti-HLA donor-specific antibodies (DSA) is assessed by solid phase (SP) tests with single antigen bead (SAB) panels. The antigenic risk is assessed based on the mean fluorescence intensity (MFI) values corresponding to each SAB via virtual crossmatch (VXM). To improve the accuracy of the VXM, new Associated Antigens were proposed based on DEPs; however, the antigenicity of some DEPs remained uncertain. In the current study, highly complex reactive sera were selected, and the complexity was reduced by adsorption with magnetic beads or lymphocytes followed by acid/neutralisation processes. As a proof of concept, a patient's serum was enriched with SAB coated with HLA-A*01:01. The eluate was tested using SP-SAB and showed reactivity with the antigens HLA-A1, -A9 (excluding A2403), -A80, -B12 and -B76. This allowed for validation of the antibody enrichment process and identification of the serological equivalency of DEPs 167G and 167S shared by these antigens. We identified broadly reactive sera being positive with the SAB HLA-B*35:12 (103V) and negative with the other SAB HLA-B35 (103L). Allo-antibodies were enriched by the adsorption/elution procedure with lymphocytes expressing HLA-B*35:12 and with SABs coated with HLA-B*57:01. The SAB assay using one of the eluates showed an almost identical cross-reactivity pattern: positive with all HLA-B SABs bearing 103V and no reactivity with SABs bearing 103L, suggesting that the enriched antibodies reacted with epitope(s) shared by HLA-B*35:12 and HLA-B*57:01. Additional reactivity was detected with the SABs HLA-A32, -A74 and -Cw3. This allowed for identification of putative epitope(s) containing residues 103V and 109L. Another serum with positive reactivity to the SAB HLA-B*35:02 showed reactivity to almost all HLA-A SABs including -A*02:10 and was negative with other -A*02 and -A*69:01 SABs, suggesting that the involvement of 107G and 109F may define novel epitopes. These studies allowed us to propose 13 novel HLA-B Associated Antigens; DEPs 103 and 109 in HLA class I were fully included in HLA Allele To Serotype (HATS) software update that allowed for a more detailed serologic characterisation of all common HLA alleles in the world.