THE GRANZYME B AND IFN-γ ELISPOT ASSAY AS ALTERNATIVES FOR THE CTLPF AFTER RENAL TRANSPLANTATION

O317* Aims: The IFN-γ Elispot assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the number of IFN-γ producing cells do not exclusively represent CTL activity as non-cytolytic cells and NK-cells can also secrete IFN-γ. The functional activity of CTLs is perhaps a more relevant surrogate marker of CTLs. Granzyme B (GrB) is present in the granules of CTLs and is involved in the lethal hit of donor target cells. Therefore, we wondered whether the GrB Elispot assay is a better marker for determining the number of CTLs than the IFN-γ Elispot assay. Methods: Peripheral blood mononuclear cells (PBMC) from 19 patients between 1 and 5 years after kidney transplantation were stimulated with donor cells or completely HLA mismatched third-party cells. We determined the CTL precursor frequency (CTLpf) by using a limiting dilution assay (LDA), and simultaneously measured the number of IFN-γ and GrB producing cells by Elispot assay. Results: In all 3 different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9/106 PBMC (range: 0-79) vs. 60/106 PBMC (0-303), p=0.0002; Elispot for IFN-γ: 10/106 PBMC (0-430) vs. 90 (20-470), p=0.0001; Elispot for GrB: 60/106 PBMC (0-590) vs. 205/106 PBMC (0-730), p=0.05. When we compared the CTLpf after third-party stimulation with the corresponding Elispot results, we found a positive correlation between the CTLpf and the number of IFN-γ producing cells (rs=0.47, p=0.05). No correlation was found between the CTLpf and the number of GrB producing cells (rs=0.23, p=0.35). However, when we compared the donor-specific CTLpf with the corresponding Elispot results, no correlation with both the Elispot for IFN-γ (rs=0.03, p=0.95) and GrB (rs=0.24, p=0.34) was found. Conclusions: After third-party stimulation, the IFN-γ Elispot assay is superior to the GrB Elispot as surrogate marker for CTL activity. Apparently, activated T-cells produce IFN-γ leading to cytolytic activity of CTL. In addition, CTL’s do not only kill via GrB, but also via FasL/Fas and TNF-α, resulting in a better association between the CTLpf and IFN-γ Elispot than the GrB Elispot assay. After donor stimulation, no correlation between de CTLpf and the IFN-γ and GrB Elispot was found. Apparently, other factors than IFN-γ play a regulatory role in donor-specific cytolytic activity. The advantage of determining CTLpf in a LDA is that this test measures the actual endpoint of cytolytic activity, independent of the pathway of killing. Therefore, we feel that the CTLpf remains the golden standard to determine donor-specific cytolytic activity after clinical organ transplantation.