The granzyme B and interferon-gamma enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplantation.
BACKGROUND
The interferon (IFN)-gamma 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-gamma ELISPOT assay. METHOD.: 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-gamma- 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 60/10(6) PBMC (P=0.0002); number of IFN-gamma pc: 10 versus 90/10(6) PBMC (P=0.0001); number of GrB pc: 60 versus 205/10(6) 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-gamma pc (r(s)=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (r(s)=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-gamma (r(s)=0.10, P=0.69) or GrB (r(s)=-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.
van Besouw
Nicole M
NM
Department of Internal Medicine-Transplantation, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. n.vanbesouw@erasmusmc.nl
Zuijderwijk
Joke M
JM
de Kuiper
Petronella
P
Ijzermans
Jan N M
JN
Weimar
Willem
W
van der Mast
Barbara J
BJ
D015415:Biomarkers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D053804:Granzymes; D006801:Humans; D007371:Interferon-gamma / Q000097:blood*; D016030:Kidney Transplantation / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D012697:Serine Endopeptidases / Q000097:blood*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D015415:Biomarkers; D007371:Interferon-gamma; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases