Granzyme B ELISPOT Assay Determines the Cytotoxic T Lymphocyte Precursor Frequency After HLA-Identical Living-Related Kidney Transplantation

Abstract

A major goal in organ transplantation is to define the optimal immunosuppressive dose. Recently, we demonstrated that the frequency of cytotoxic T lymphocytes (CTLpf) identifies patients in whom the immunosuppressive load can be safely reduced. However, in peripheral blood mononuclear cells (PBMC) from HLA-identical living-related kidney transplant patients, no donor-specific CTLpf can be measured. The determination of the functional activity of cytotoxic T lymphocytes (CTLs) could be an alternative method for the CTLpf. Granzyme B (GrB) is present in the granules of CTLs and is involved in the direct lethal hit of donor target cells. Therefore, we wondered whether the GrB ELISPOT assay is an alternative method to determine the activity of CTLs after HLA-identical living-related kidney transplantation. We measured the number of GrB producing cells (pc) against donor PBMC and third-party PBMC in PBMC from HLA-identical patients who were reduced in their immunosuppression from 100% to 50% azathioprine with 5 to 10 mg/day prednisone. We found low numbers of GrB pc before reduction of immunosuppression, as only 20% of the patients' PBMC responded to donor cells, whereas 57% of the patients' PBMC responded to donor cells after reduction of immunosuppression. After third-party stimulation, the number of GrB pc increased after tapering the immunosuppressive load ( P = .03). Our results demonstrate that the GrB ELISPOT assay might be used as an alternative for the CTLpf after HLA-identical living-related kidney transplantation.