Multiple minor histocompatibility antigen-specific cytotoxic T lymphocyte clones can be generated during graft rejection after HLA-identical bone marrow transplantation

Abstract

Graft rejection after T cell-depleted HLA-genotypically identical bone marrow transplantation (BMT) is probably mediated by mH antigen-specific cytotoxic T lymphocytes (CTL). We have analyzed peripheral blood mononuclear cells (PBMC) from a female bone marrow graft recipient, collected during graft rejection after a sex mismatched HLA-identical BMT. A CTL line was generated by stimulating recipient PBMC collected during graft rejection with donor PBMC and donor EBV-transformed lymphoblastoid cell lines. From this CTL line a large number of clones of different specificity and phenotype was established by limiting dilution. These clones exhibited several mH antigen specificities, restricted by HLA-B7, -B27 or -DR2 as shown by differential recognition of family members and unrelated individuals sharing potential restriction elements. The CD3+CD4+ and CD3+CD8+ bulk culture was cloned, resulting in 50 HLA-B7 restricted CD3+CD4-CD8+CTL clones, three HLA-B27 restricted CD3+CD4-CD8+CTL clones, one HLA-DR2 restricted CD3+CD4+CD8-CTL clone and two additional HLA class II restricted CD3+CD4+CD8-CTL clones with a different specificity. One representative clone of each specificity was selected for further analysis. The CTL line and the HLA-B7 restricted CD8+CTL clone, but not the HLA class II restricted CD4+ CTL clone, inhibited the growth of donor hematopoietic progenitor cells (HPC). In conclusion, these results show that graft rejection after HLA-identical BMT may be mediated by multiple CTL clones that specifically recognize one mH antigen peptide presented by different HLA molecules or different mH antigens expressed on donor cells and that CTL, but not CD4+ CTL inhibited donor HPC growth.