γδ T cells enhance the autoreactive T cell response by competing with the binding of adenosine.

<p>A) Activated γδ T cells express an increase of >1000-times the A2AR but only minimally increased A2BR. Activated γδ T cells and naïve γδ T cells were isolated from immunized from naïve B6 mice, respectively. The expression of A2AR/A2BR was determined by real-time PCR assay. Real-time RT-PCR analysis of A2AR and A2B transcripts among total RNA isolated from αβ and γδ T cells isolated from naïve and IRBP_1-20-immunized B6 mice before and after in vitro activation. qPCR was performed with Gapdh as the internal reference. Results were represented as 2^-ΔCt. **, p < 0.01. B) Competitive binding test revealed a hierarchical order of adenosine capture by immune cells as γδ T cells >> αβ T cells >> DCs. αβ responder T cells were isolated from IRBP_1-20-immunized TCR-δ^-/- mice. DCs were from 5 d GM-CSF-cultured bone marrow cells. In a 24-well plate the T cells were seeded at 1 x 10⁶/well and DCs at 5 x 10⁵/well. After incubation with1,000 nM H³-labeled adenosine for 1 h, the cells were harvested, and cell-bound H³-adenosine was counted by liquid scintillation. ** p < 0.01. C) Comparison of adenosine binding of γδ T cells isolated from naïve and immunized mice γδ T cells from immunized or naïve B6 mice, respectively. 1 x 10⁵/well cells were cultured in 96-well plate for 48h. 0–12,000 nM H³-labeled adenosine for 1 h, then the cells were harvested and cell-bound H³-adenosine counted by liquid scintillation. D) A2AR^+/+γδ T cells, but not A2AR^-/- γδ T cells, possess an enhancing effect, which converted the inhibitory effect to enhancing on the αβ T cell response of an A2AR agonist. αβ and γδ T cells (1 x 10⁵) were isolated from IRBP_1-20-immunized TCR-δ^-/- mice. The responder αβ T cells were labeled with CFSE and incubated for 5 d with immunizing peptide in the presence of APCs alone (A) or in the presence of the A2AR^+/+ or A2AR^-/- γδ T cells (B), in the absence or presence of an A2AR agonist, and with or without an addition of A2AR^+/+γδ T cells. The results shown are representative of three experiments.