Clinical validation of IFNγ/IL-10 and IFNγ/IL-2 FluoroSpot assays for the detection of Tr1 T cells and influenza vaccine monitoring in humans

The type of T cell polarization and simultaneous production of multiple cytokines have been correlated with vaccine efficacy. ELISpot is a T cell detection technique optimized for the measurement of a secreted cytokine at the single cell level. The FluoroSpot assay differs from ELISpot by the use of multiple fluorescent-labeled anticytokine detection antibodies, allowing optimal measurement of multiple cytokines. In the present study, we show that an IFN gamma/IL-10 FluoroSpot assay is more sensitive than flow cytometry to detect Tr1 regulatory T cells, an immunosuppressive T cell population characterized by the production of IL-10 and IFN gamma. As many tolerogenic vaccines are designed to induce these Tr1 cells, this FluoroSpot test could represent a standard method for the detection of these cells in the future. The use of an IFN gamma/IL-2 FluoroSpot assay during influenza vaccine monitoring showed that the influenza-specific IL-2-producing T-cell response was the dominant response both before and after vaccine administration. This study therefore questions the rationale of using the single-color IFN gamma ELISpot as the standard technique to monitor vaccine-specific T-cell response. Using this same test, a trend was also observed between baseline levels of IFN gamma T-cell response and T-cell vaccine response. In addition, a lower IFN gamma+ IL-2(+) T-cell response after vaccine was observed in the group of patients treated with TNF alpha inhibitors (P = 0.08). This study therefore supports the use of the FluoroSpot assay due to its robustness, versatility, and the complementary information that it provides compared with ELISpot or flow cytometry to monitor vaccine-specific T-cell responses.