A Polymorphic Oncofetal Antigen Recognized by CD8+ CTL from Two Patients Experiencing Regression of Metastatic Renal Cell Carcinoma after Allogeneic HCT.

Abstract

Regression of metatstatic renal cell carcinoma (RCC) has been observed in 8–57% of patients undergoing nonmyeloablative allogeneic hematopoietic cell transplant (HCT) from HLA-matched donors. Tumor regression in this setting typically occurs after complete donor T cell engraftment is established, and is correlated with the development of GVHD. We previously isolated from two patients experiencing regression of metastatic RCC after nonmyeloablative allogeneic HCT CD8+ CTL clones that recognized a RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201, and localized the gene encoding this antigen to chromosome 19q. To identify the gene that encodes this antigen, a cDNA expression library made from a HLA-A*0201+ minor H antigen-positive tumor cell line was screened using a transfection assay in COS-7 cells. After two rounds of screening, a 1.4 kb cDNA was identified that stimulated HLA-A*0201-dependent cytokine release from minor H antigen-specific CTL. Sequencing showed that this cDNA was derived from the MGC13170 gene on chromosome 19q, within the region of significant linkage. Sequencing of MGC13170 alleles in minor H antigen-positive and minor H antigen-negative cells revealed six single nucleotide polymorphisms (SNPs), inherited as a haplotype and all previously identified, that determined susceptibility or resistance to lysis by minor H antigen-specific CTL. Analysis of 5′ and 3′ truncation constructs derived from the MGC13170 cDNA identified an interval encoding a 48-residue open reading frame that contained the epitope and spanned one of the six SNPs (dbSNP rs3745526) that had been shown to correlate with CTL recognition. Further analysis of minigenes spanning this nonsynonymous T↔A SNP identified an interval containing T at the polymorphic position and encoding a predicted 11-residue peptide that stimulated maximal HLA-A2-dependent cytokine release from minor H antigen-specific CTL. The T↔A SNP is predicted to create a Ser↔Thr polymorphism in the encoded peptide. A synthetic 11-mer peptide containing Ser at the polymorphic residue, but not the homologous peptide containing Thr at this position, was recognized by minor H antigen-specific CTL when pulsed onto minor H antigen-negative HLA-A*0201+ cells, thus demonstrating that the T allele of MGC13170 encodes the minor H antigenic peptide. The function of MGC13170 is unknown, but a previous study (Zhou et al., Ai Zheng 21:341–345, 2002) suggested a role in resistance of tumor cells to chemotherapeutic agents. The range of tissues in which the epitope-encoding MGC13170 sequence is expressed was evaluated in silico by probing the translated human EST database with the predicted sequence of the 48-residue polypeptide encoded by the epitope-containing ORF. Of the several hundred ESTs identified that encoded the Ser- or Thr-containing peptide sequence, 72% were derived from tumor cells or cell lines (including RCC), 5% from embryonic or fetal tissues, 9% from embryonic stem cells, and 11% from normal tissues. Thus, MGC13170 encodes a novel HLA-A*0201-restricted minor H antigen that is expressed in a wide variety of neoplastic, embryonic, and fetal cells. Further studies of MGC13170 as a possible target for antitumor immune responses after allogeneic HCT are underway.