MULTIPLE MINOR HISTOCOMPATIBILITY ANTIGEN-SPECIFIC CYTOTOXIC T-LYMPHOCYTE CLONES CAN BE GENERATED DURING GRAFT-REJECTION AFTER HLA-IDENTICAL BONE-MARROW TRANSPLANTATION

Graft rejection after T cell-depleted HLA-genotypically identical bone marrow transplantation (BMT) is probably mediated by mil antigen-spec ific cytotoxic T lymphocytes (CTL). We have analyzed peripheral blood mononuclear cells (PBMC) from a female bone marrow graft recipient, co llected during graft rejection after a sex mismatched HLA-identical BM T. A CTL line was generated by stimulating recipient PBMC collected du ring graft rejection with donor PBMC and donor EBV-transformed lymphob lastoid cell lines. From this CTL line a large number of clones of dif ferent specificity and phenotype was established by limiting dilution. These clones exhibited several mil antigen specificities, restricted by HLA-B7, -B27 or -DR2 as shown by differential recognition of family members and unrelated individuals sharing potential restriction eleme nts. The CD3(+)CD4(+) and CD3(+)CD8(+) bulk culture was cloned, result ing in 50 HLA-B7 restricted CD3(+)CD4(-)CD8(+) CTL clones, three HLA-B 27 restricted CD3(+)CD4(-)CD8(+) CTL clones, one HLA-DR2 restricted CD 3(+)CD4(+)CD8(-) CTL clone and two additional HLA class II restricted CD3(+)CD4(+)CD8(-) CTL clones with a different specificity. One repres entative clone of each specificity was selected for further analysis. The CTL line and the HLA-B7 restricted CD8(+) CTL clone, but not the H LA class II restricted CD4(+) CTL clone, inhibited the growth of donor hematopoietic progenitor cells (HPC). In conclusion, these results sh ow that graft rejection after HLA-identical BMT may be mediated by mul tiple CTL clones that specifically recognize one mil antigen peptide p resented by different HLA molecules or different mil antigens expresse d on donor cells and that CD8(+) CTL, but not CD4(+) CTL inhibited don or HPC growth.