Purification and Activation of αβ and γδ T Cells

αβ T cells were purified from B6 mice immunized with the human interphotoreceptor retinoid-binding protein (IRBP) peptide IRBP_1-20, as described previously [4 (link),7 (link),8 (link)], while γδ T cells were purified from immunized and control (naïve) B6 or A2AR^-/- mice. Nylon wool-enriched splenic T cells from naïve or immunized mice were incubated sequentially for 10 min at 4°C with FITC-conjugated anti-mouse γδ TCR or αβ TCR Abs and 15 min at 4°C with anti-FITC Microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany); the cells were then separated into bound and non-bound fractions on an autoMACS^TM separator column (Miltenyi Biotec GmbH). The purity of the isolated cells was >95%, as determined by flow cytometric analysis using PE-conjugated Abs against αβ or γδ T cells. Resting γδ T cells were prepared either by isolation from naïve mice or by incubating activated γδ T cells in cytokine-free medium for 5–7 d, at which time they show down-regulation of CD69 expression [3 (link)]. Highly activated γδ T cells were prepared by incubating resting γδ T cells for 2 d with Abs against the γδ TCR (GL3) and CD28 (both 2 μg/ml, both from Bio-Legend, San Diego, CA), or cytokine combination (IL-1,IL-7 and IL-23).