Graft rejection is a significant complication in cord blood transplantation (CBT), but little is known about the mechanism of rejection. In the present study, to investigate the potential role of T lymphocytes in graft rejection, we isolated a cytotoxic T lymphocyte (CTL) clone from a patient who developed graft rejection after CBT. A female patient received CBT from an unrelated male donor using a reduced-intensity preparative regimen consisting of 125 mg/m2 fludarabine and 180 mg/m2 melphalan. The infused total nuclear cell dose was 2.9 × 107 /kg. The patient was diagnosed as having graft rejection on day 28 based on the marrow hypoplasia, with complete loss of donor chimerism. The patient achieved primary engraftment after a second CBT. DNA typing of the HLA-A and B loci demonstrated that the recipient was A*1101/A*2402 and B*4404/B*5603, the first CBT donor was A*1101/A*2402 and B*1501/B*5603, and the second CBT donor was A*2402/A*3303 and B*4403/B*5101. We obtained the peripheral blood mononuclear cells (PBMCs) just after the development of graft rejection following CBT, cultured them in interleukin-2-containing media without stimulator cells for 14 days, and isolated two T lymphocyte clones by limiting dilution. One of the two clones, designated N19D8, lysed Epstein Barr virus-transformed lymphoblastoid cells (B-LCL) from the donor, but failed to lyse B-LCL from the patient. Thus, we further investigated the N19D8 clone because it may have been involved in the immunological graft rejection. Flow cytometry and sex chromosome fluorescent in situ hybridization revealed that the N19D8 clone was CD3+CD4−CD8+ T lymphocyte and originated from the patient. In a cytotoxicity assay for a panel of B-LCL derived from unrelated individuals, N19D8 CTL lysed all of five B-LCL lines from unrelated individuals that shared HLA-B*1501, which is the mismatch antigen in the first CBT, but it failed to lyse B-LCL from nine unrelated individuals without B*1501. To determine if the lack of recognition by N19D8 CTL was solely due to the absence of HLA-B*1501 gene expression, the patient’s B-LCL were transfected with HLA-B*1501 cDNA and then employed as targets in cytotoxicity assays. The B*1501-transfected recipient’s B-LCL were lysed almost as effectively as the first CBT donor’s B-LCL. Furthermore, COS cells transfected with HLA-B*1501 cDNA alone stimulated interferon-γ production of N19D8 CTL. Thus, we concluded that the N19D8 CTL clone recognizes the mismatched HLA-B*1501 molecule as an alloantigen, but not a minor histocompatibility antigen presented by the HLA-B*1501 molecule such as an unidentified male-specific H-Y antigen. We next determined if the N19D8 clone was developed before transplantation, using nested PCR assays specific for the CTL clone’s uniquely rearranged T cell receptor Vβ17 chain gene. PCR products were produced by amplification of DNA from pre-transplant as well as post-transplant PBMC. Additionally, the presence of microchimerism of B*15-positive cells in the pre-transplant PBMCs was confirmed by PCR assay specific for the HLA-B*15. These data demonstrated that the N19D8 CTL clone developed in the patient before the first CBT. The present study demonstrated a potential role of pre-transplant CTLs in graft rejection following CBT. Further studies on mismatched HLA-specific CTLs should help determine the optimal strategy for overcoming graft rejection in CBT.

Author notes

Disclosure: No relevant conflicts of interest to declare.