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 IFNγ/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 IFNγ. 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 IFNγ/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 IFNγ 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 IFNγ T cell response and T cell vaccine response. In addition, a lower IFNγ+IL-2+ T-cell response after vaccine was observed in the group of patients treated with TNFα 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.

PD-1-expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPV-associated head and neck cancer.

Head and neck cancers positive for human papillomavirus (HPV) have a more favorable clinical outcome than HPV-negative cancers, but it is unknown why this is the case. We hypothesized that prognosis was affected by intrinsic features of HPV-infected tumor cells or differences in host immune response. In this study, we focused on a comparison of regulatory Foxp3(+) T cells and programmed death-1 (PD-1)(+) T cells in the microenvironment of tumors that were positive or negative for HPV, in two groups that were matched for various clinical and biologic parameters. HPV-positive head and neck cancers were more heavily infiltrated by regulatory T cells and PD-1(+) T cells and the levels of PD-1(+) cells were positively correlated with a favorable clinical outcome. In explaining this paradoxical result, we showed that these PD-1(+) T cells expressed activation markers and were functional after blockade of the PD-1-PD-L1 axis in vitro. Approximately 50% of PD-1(+) tumor-infiltrating T cells lacked Tim-3 expression and may indeed represent activated T cells. In mice, administration of a cancer vaccine increased PD-1 on T cells with concomitant tumor regression. In this setting, PD-1 blockade synergized with vaccine in eliciting antitumor efficacy. Our findings prompt a need to revisit the significance of PD-1-infiltrating T cells in cancer, where we suggest that PD-1 detection may reflect a previous immune response against tumors that might be reactivated by PD-1/PD-L1 blockade.

Adjuvant properties of Cytosine-phosphate-guanosine oligodeoxynucleotide in combination with various polycations in an ovalbumin-vaccine model.

Oligonucleotides containing CpG motifs (cytosine-phosphate-guanosine oligodeoxynucleotide [CpG ODN]) display strong immunostimulatory effects, and polycations have been previously reported as cellular delivery system. In the present study, we investigated the adjuvant properties of combinations of a CpG ODN with various polycations (poly-arginine, poly-lysine, poly-histidine, or chitosan) in an ovalbumin vaccination model. We showed that, when combined to CpG ODN, poly-arginine and poly-histidine, but not poly-lysine or chitosan, enhanced efficiently both the IgG antibody production and the number of splenocytes secreting interferon-gamma after stimulation with a CD8+ T cell-restricted peptide. Interestingly, CpG ODN-poly-arginine, which was the most efficient, compared favorably to the complete Freund's adjuvant and aluminium salts and induced no local toxicity, making this combination a very attractive adjuvant for vaccines.