The granzyme B interferon-γ enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplatation

Background. The Interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the functional activity of CTL might be a more relevant surrogate marker of CTL. Therefore, the authors wondered whether the granzyme B (GrB) ELISPOT assay is a better marker for determining the number of CTL than the IFN-γ ELISPOT assay. Methods. Peripheral blood mononuclear cells (PBMC) from 19 kidney transplant patients were stimulated with donor cells or third-party cells. The authors determined the CTL precursor frequency (CTLpf) and simultaneously measured the number of IFN-γ- and GrB-producing cells (pc) by ELISPOT assay. Results. In all three different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9 versus 60/106 PBMC (P=0.0002); number of IFN-γ pc: 10 versus 90/106 PBMC (P=0.0001); number of GrB pc: 60 versus 205/106 PBMC (P=0.05). When the authors compared the CTLpf after third-party stimulation with the corresponding ELISPOT results, they found a positive correlation between the CTLpf and the number of IFN-γ pc (rs=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (rs=0.23, P=0.36). However, when they compared the donor-specific CTLpf with the corresponding ELISPOT results, no correlation with the ELISPOT for IFN-γ (rs=0.10, P=0.69) or GrB (rs= -0.24, P=0.34) was found. Conclusions. The authors feel that the CTLpf, as a measure of the actual endpoint of cytolytic activity and independent of the pathway of killing, remains the "gold standard" for determining donor-specific cytolytic activity after clinical organ transplantation. Copyright

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