Molecular characterization of HLA‐C incompatibilities in HLA‐ABDR‐matched unrelated bone marrow donor‐recipient pairs

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Abstract
While the influence of HLA‐AB and ‐DRB1 matching on the outcome of bone marrow transplantation (BMT) with unrelated donors is clear, the evaluation of HLA‐C has been hampered by its poor serological definition. Because the low resolution of standard HLA‐C typing could explain the significant number of positive cytotoxic T lymphocyte precursor frequency (CTLpf) tests found among HLA‐AB‐subtype, DRB1/B3/B5‐subtype matched patient/donor pairs, we have identified by sequencing the incompatibilities recognized by CD8+ CTL clones obtained from such positive CTLpf tests. In most cases the target molecules were HLA‐C antigens that had escaped detection by serology (e.g. Cw*1601, 1502 or 0702). Direct recognition of HLA‐C by a CTL clone was demonstrated by lysis of the HLA class I‐negative 721,221 cell line transfected with Cw*160l cDNA. Because of the functional importance of Cw polymorphism, a PCR‐SSO oligotyping procedure was set up allowing the resolution of 29 Cw alleles. Oligotyping of a panel of 382 individuals (including 101 patients and their 272 potential unrelated donors, 5 related donors and 4 platelet donors) allowed to determine HLA‐C and HLA A‐B‐Cw‐DRB1 allelic frequencies, as well as a number of A‐Cw, B‐Cw, and DRB1‐Cw associations. Two new HLA‐Cw alleles (Cw*02023 and Cw*0707) were identified by DNA sequencing of PCR‐amplified exon 2‐intron 2‐exon 3 amplicons. Furthermore, we determined the degree of HLA‐C compatibility in 287 matched pairs that could be formed from 73 patients and their 184 potential unrelated donors compatible for HLA‐AB by serology and for HLA‐DRB1/ B3/B5 by oligotyping. Cw mismatches were identified in 42.1% of these pairs, and AB‐subtype oligotyping showed that 30% of these Cw‐incompatible pairs were also mismatched for A or B‐locus subtype. The degree of HLA‐C incompatibility was strongly influenced by the linkage with B alleles and by the ABDR haplotypes. Cw alleles linked with B*4403, B*5101, B18, and B62 haplotypes were frequently mismatched. Apparently high resolution DNA typing for HLA‐AB does not result in full matching at locus C. Since HLA‐C polymorphism is recognized by alloreactive CTLs, such incompatibilities might be as relevant as AB‐subtype mismatches in clinical transplantation.

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