Background. T cells specific for minor histocompatibility antigens (mHAgs) play a major role in both graft-versus-host disease and the graft-versus-tumor effect after HLA-identical bone marrow transplantation (BMT). However, characterization of individual T-cell responses to mHAgs is difficult and has generally involved extensive screening of T-cell clones isolated from bulk T-cell cultures generated from BMT recipients. In this report, we describe a new approach that permits both direct visualization of CD8(+) T-cell responses to mHAgs and cloning of T cells reacting with mHAgs presented by individual HLA alleles of interest.
Methods and Results. Panels of Epstein-Barr virus. transformed B-cell lines (B-LCL) expressing retrovirally transduced HLA cDNA were used as stimulator cells in an enzyme-linked immunospot (ELISPOT) assay to identify CD8(+) T cells reacting with mHAgs in cultures generated from postBMT recipient peripheral blood. T cells specific for mHAgs presented by selected HLA alleles could then be captured and cloned using an interferon-gamma secretion assay and magnetic bead selection. A majority of T-cell clones thus isolated exhibited cytolytic activity against the same HLA-transfected B-cell lines used for the ELISPOT assay.
Conclusion. The ELISPOT assay was useful for identification of the HLA alleles presenting mHAgs recognized by individual T-cell lines. This approach for isolating mHAgs-specific CD8(+) T-cell clones should assist in characterizing responses restricted by HLA alleles of interest, which are common in a certain ethnic group.