Identification of a novel splice variant transcript of HMSD encoding the mHA. (A) Summary of genome mapping around chromosome 18q21.33 showing relative positions of HMSD. Two identical cDNA clones were homologous to exons 1 and 3 plus exon 4 but lacked exon 2. This novel allelic splice variant of HMSD was designated HMSD-v (panel C). Search for potential SNPs responsible for the alternative splicing revealed 2 potential SNPs at IVS1+56 and IVS2+5 (arrowheads). Cen indicates centromere, Tel, telomere. (B) The correlation between sequence polymorphisms of the 2 SNPs and susceptibility of B-LCLs to CTL-2A12. Detection of allelic polymorphisms in B-LCLs was conducted by RT-PCR. Primers were set in exon 1 and the 5′ part of exon 4 of HMSD (horizontal arrows in panel A). Due to the lack of exon 2, the mHA⁺ allele produced a smaller PCR product. Genotyping of the 2 SNPs mentioned above and cytolysis of B-LCLs by CTL-2A12 are summarized below the results of electrophoresis. The correlation between the genotyping results of SNPs at IVS2+5, CTL-2A12 cytolysis, and the bands of electrophoresis produced by mHA⁺ and mHA⁻ allele showed complete concordance. (C) Schematic representation of HMSD and HMSD-v and mapping of the region encoding the CTL-2A12 mHA epitope by minigenes. The HMSD-v cDNA was divided into 3 minigenes, and mammalian expression plasmids containing individual minigenes were constructed. 293T/B*4403 cells were transfected with individual plasmids and cocultured with CTL-2A12. Supernatants were then harvested and assayed for IFN-γ production by ELISA. Release of IFN-γ is expressed in arbitrary units (AUs) corresponding to optical density at 630 nm.