Project Description
Els Goulmy, Co-chair
Paul Martin, Co-chair
The goal of the Minor Histocompatibility Antigen Component of the 13th IHWG is to provide investigators with the opportunity to map genetic loci that encode human minor histocompatibility antigens (mHA) recognized by cytotoxic T lymphocyte (CTL) clones. In preliminary work to support this goal, we have obtained a panel of 183 well characterized Epstein-Barr virus-transformed B lymphoblastoid cell lines (EBV-LCL) from 12 extended CEPH families, each containing 2 parental probands, 2-4 grandparents, and 8-15 offspring of the parents. In previous work by CEPH, these cell lines have been allele typed for thousands of genetic markers. The cell lines have been expanded and cryopreserved for distribution, and identity of each has been validated by testing a panel of known genetic markers. In further preliminary work, we have prepared vaccinia vector constructs encoding HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-B*0702 and HLA-B4402, so that these molecules can be expressed in EBV-LCL used as targets in CTL assays with clones that recognize mHA.
New preliminary work by Dr. Edus H. Warren has demonstrated the feasibility of using vaccinia vectors to express HLA restriction molecules for presentation of mHA in CTL assays. For this purpose, he used a CTL clone that recognizes HA-8, an HLA-A2-restricted minor histocompatibility antigen. The HA-8 peptide is encoded by KIAA0020, a gene located in the distal region of chromosome 9p. Cell line from 3 CEPH families were tested. All members of family 1413 were HLA-A2-positive, and all members of family 1332 were HLA-A2-negative. Family 1362 contained both HLA-A2-positive and HLA-A2-negative individuals. HLA-A2-negative EBV-LCL transfected with a vector construct encoding HLA-A2 expressed HLA-A2 at levels comparable to that of HLA-A2-positive EBV-LCL, as determined by cell surface staining with an HLA-A2-specific monoclonal antibody (Fig. 1). All cell lines were tested in cytotoxicity assays after transfection with either wild-type vector or HLA-A2 vector. In addition, all cell lines were tested for the presence of HA-8 by analysis of a restriction fragment length polymorphism in an amplified DNA segment to detect the KIAA0020 allele that confers susceptibility to the HA-8-specific CTL clone.
Figure 1. Expression of HLA-A2 after infection of an HLA-A2-negative EBV-LCL with a vaccinia vector containing an HLA-A2 cDNA insert. The grey line depicts control staining with a FITC-conjugated antibody specific for murine IgG. The black line shows staining with an HLA-A2-specific monoclonal anti-body.
Cytotoxicity was uniformly detected when the HA-8-specific CTL clone was tested against wild-type vector-transfected HA-8-positive cell lines from HLA-A2-positive family members but not against wild-type vector-transfected HA-8-negative cell lines from HLA-A2-positive family members (Fig. 2). No cytotoxicity was detected against wild-type vector-transfected HA-8-positive or negative cell lines from HLA-A2-negative family members. Cytotoxicity was uniformly detected against HLA-A2-transfected HA-8-positive cell lines from HLA-A2-negative family members but not against HLA-A2-transfected HA-8-negative cell lines from HLA-2-negative family members. Results of the cytotoxicity assay with HLA-A2-transfected cells correlated completely with the presence or absence of HA-8 as determined by genetic testing.
Figure 2. Lysis of CEPH cell lines by a CTL clone that recognizes HA-8. Solid symbols indicate HA-8-positive cell lines, and open symbols indicate HA-8-negative cell lines. Squares identify HLA-A2-positive cell lines transfected with wild-type vector. Triangles identify HLA-A2-negative cell lines transfected with wild-type vector. Circles identify HLA-A2-negative cell lines transfected with HLA-A2 vector.
Two-point linkage analysis of data from these three families demonstrated that the gene encoding HA-8 is tightly linked with a cluster of marker loci on the distal portion of chromosome 9p (maximum LOD score, 7.47). These results are consistent with previous studies demonstrating that the HA-8 peptide is encoded by KIAA0020. By analysis of informative recombinants within the three families, the gene encoding the HA-8 peptide was located to a 4.3 mb segment containing 8 known genes, including KIAA0020.
These results show that the transfection strategy proposed for this component of the IHWG can be used successfully to map genes that encode mHA among cell lines that do not express the appropriate MHC restriction molecule. With the HA-8-specific CTL clone, HLA-A2-transfected EBV-LCL from HLA-A2-negative individuals had a signal-to-noise ratio comparable to that of wild-type-transfected EBV-LCL from HLA-A2-positive individuals. Mapping of genes encoding other mHA should be feasible if similarly favorable signal-to-noise ratios can be achieved with other CTL clones. The considerable narrowing of candidate genes achieved through genetic mapping should facilitate the identification of mHA peptides through expression cloning strategies.
The CEPH family cell lines and vaccinia vectors are now available for distribution to investigators who have CTL clones that recognize human minor histocompatibility antigens. Further information can be obtained by contacting Paul Martin by e-mail at pmartin@fhcrc.org. The IHWG meeting will be held in Victoria, British Columbia from May 12-16, 2002, and the 13th International Congress on Histocompatibility and Immunogenetics will be held in Seattle from May 18-22, 2002.
