Supernatants of γδ T cells enhanced the Foxp3 T cell response.

TCRαβ and CD3⁺ T cells were purified from B6 or TCR-δ^-/- mice immunized with the human interphotoreceptor retinoid-binding protein (IRBP) peptide IRBP_1-20, as described previously; non-activated and activated γδ T cells were prepared by isolation from naïve and immunized mice, respectively (materials and methods). Two-day culture supernatants were collected from γδ T cells (at a density of 1 x 10⁶/well of 24-well plate). They were tested by addition to TCR-δ^-/- responder T cells at a volume of 1:5. A) Addition of γδ T cell supernatant enhanced, whereas direct addition of γδ T cells into the culture inhibited the Foxp3 T cell response. In a 24-well plate, 1 x 10⁶/well TCR-δ^-/- responder T cells were cultured in IL-2-containing medium in the absence or presence of activated γδ T cells (2%, right panel) or γδ T cell supernatant (1:5 vol:vol, middle panel). Foxp3⁺ T cells were evaluated as detailed before. B) Supernatants of cultured γδ, but not dendritic cells and αβ T cells, enhanced the Foxp3 T cell response in vitro. Supernatants collected from γδ T cells, BMDCs, and αβ T cells (at a density of 1 x 10⁶/well of 24-well plate) were added to TCR-δ^-/- responder T cells at a volume of 1:5. Foxp3⁺ T cells were evaluated as detailed before. C) The culture supernatants of both activated and non-activated γδ T cells had an enhancing effect on Foxp3 T cell response in culture. Supernatants collected from non-activated γδ T cells from naïve mice and activated γδ T cells from immunized mice [15,18,29] were added to TCR-δ^-/- responder T cells at volume of 1:5. Foxp3⁺ T cells were evaluated as detailed before. D) The enhancing effect of the γδ T cell supernatants was abolished after treatment by adenosine-degrading enzyme (ADA). γδ supernatants, with or without a prior treatment of ADA (1U/ml), were added to TCR-δ^-/- responder T cells at volume of 1:5,. Foxp3⁺ T cells were evaluated as detailed before.