Comparison of a combined MHC tetramer and interferon-γ (IFN-γ) intracellular cytokine staining (ICS) assay with individual MHC-tetramer and IFN-γ ELISPOT assays to monitor cancer immunotherapy

1866 Background: Validation of ex vivo immune monitoring methods is a high priority for the clinical development of novel cancer-immunotherapy strategies. We investigated whether the combination of IFN-γ ICS and tetramer assay resulted in an assay that combines the robustness and quantitative results of MHC tetramer and the functional information derived from IFN-γ enzyme-linked immunospot (ELISPOT) assays, resulting in a more efficient way to conduct immune-monitoring assays. Methods: Eligible subjects were HLA-A*0201 with a baseline level of MART-126-35-, EBV-BMLF1- and/or CMVpp65-reactive CD8+ T cells above the tetramer assay’s low limit of detection (LLD) of 0.03%. Subjects received no intercurrent therapy. 100 ml of peripheral blood was collected twice separated by at least 15 days. Samples were cryopreserved and analyzed by iTAg MHC Tetramer IFN-γ kit from Beckman Coulter and compared to our standardized gold standard assays of MHC tetramer staining and IFN-γ ELISPOT (Comin-Anduix et al. Clin Ca Res 06). The Pearson coefficient with a 95% confident interval was computed to establish correlation between the assay results. Results: 10 subjects (2 healthy donors and 8 melanoma patients) had EBV-BMLF1, 1 had MART-126-35 and 4 had CMV-pp65-specific cells above the tetramer LLD. Following the manufacturer’s instructions, the combined tetramer-IFN-γ ICS assay reproducibly overestimated the number of antigen-specific T cells by approximately 1 log compared to the results with the tetramer assay alone (mean EBV-BMLF1 tetramer 0.86vs. 5.68 by ICS-tetramer, paired t-test p Conclusion: A modified tetramer-IFN-γ ICS assay can efficiently quantitate circulating antigen-specific T cells. Although results of the tetramer-IFN-γ ICS assay do not correlate with the functional IFN-γ ELISPOT assay, the addition of ICS to the tetramer assay provides specific functional phenothyping information on antigen-specific IFN-γ producing cells in peripheral blood.