A PCR-SSP method to specifically select HLA-A*0201 individuals for immunotherapeutic studies

HLA-A*0201 is an important restriction element for peptide presentation to T cells in disease and cancer. Mutation studies and analyses using cytotoxi c T lymphocytes have shown the functional relevance of sub type-specific di fferences in HLA-A2 molecules for peptide binding and T-cell receptor recog nition. Therefore, many immunotherapeutic studies need to accurately select HLA-A*0201-positive individuals. We designed an easy, robust approach base d on the polymerase chain reaction using sequence-specific primers (PCR-SSP ) to specifically distinguish A*0201-positive individuals from other HLA-A2 subtypes described to date. The first step includes reactions that give in formation whether the sample donor is HLA-A2 and, if so, whether the indivi dual is homozygous or heterozygous for HLA-A2. Further, it is determined wh ether the sample has an HLA-A*020S or an HLA=A*0201 sequence at the corresp onding position in exon 4. Samples that may contain an HLA-A*0201 allele ac cording to the results of this first step are subtyped in a second step nes ted PCR. Here the strategy is focussed on the discrimination of HLA-A*0201 from the other subtypes by considering divergent nucleotide positions in tw o ways. One SSP combination amplifies the HLA-A*0201 sequence while a corre sponding SSP combination specifically amplifies the subtype or group of sub types differing from HLA-A*0201 at this position. Thus, at relevant polymor phic nucleotide positions the HLA-A*0201 sequence is both directly and indi rectly confirmed. This strategy strongly enhances the reliability of the su btyping and allows better verification of HLA-A*0201-positive patient selec tion for clinical studies.