Fig. 1.

γδ T-APCs induce CD8⁺ T cell proliferation and activation. γδ T cells stimulated by zoledronate and IL-2 for 10 days were used as γδ T-APCs. A, B γδ T-APCs or DCs were, respectively, pretreated for 2 h with 10 μg/ml MAGEA3 peptide (γδ T-MAGEA3 and DC-MAGEA3) and then washed extensively. After that, peptide non-pulsed γδ T-APCs (γδ T), γδ T-MAGEA3 and DC-MAGEA3 were then co-cultured with CFSE-labeled CD8⁺ T cells for 10 days at an APC/responder cell ratio of 1:10. Proliferating CFSE⁻ CD8⁺ T cells were measured by flow cytometry. C, D γδ T-APCs or DCs were pretreated for 2 h with 10 μg/ml MAGEA3 peptide and then washed extensively. After that, peptide non-pulsed γδ T-APCs, γδ T-MAGEA3 and DC-MAGEA3 were then co-cultured with CD8⁺ T cells for 5 days at an APC/responder cell ratio of 1:10. CD69 expression levels on CD8⁺ T cells were measured by flow cytometry. E–J γδ T-APCs or DCs were pretreated for 2 h with 10 μg/ml MAGEA3 peptide and then washed extensively. After that, peptide non-pulsed γδ T-APCs, γδ T-MAGEA3 and DC-MAGEA3 were then co-cultured with CD8⁺ T cells for 7 days at an APC/responder cell ratio of 1:10. After that, the cells in each group received the same stimulation as the previous one for 4 h followed by further analysis. E, F CD107a expression levels on CD8⁺ T cells were measured by flow cytometry. G, H Perforin expression levels in CD8⁺ T cells were measured by flow cytometry. I, J IFN-γ expression levels in CD8⁺ T cells were measured by flow cytometry. K CD8⁺ T cells were treated with MAGEA3 peptide or co-cultured, respectively, with peptide non-pulsed γδ T-APCs, irrelevant peptide-pulsed γδ T-APCs (γδ T-IP), γδ T-MAGEA3 or DC-MAGEA3 for 10 days at an APC/responder cell ratio of 1:10. Specificity of the generated MAGEA3-specific T cells was assessed by IFN-γ ELISpot. Cells were plated in triplicates at 10,000 cells per well of anti-IFN-γ capture antibody-coated plates and IFN-γ spot-forming units/well was shown in histogram. L, M γδ T-APCs or DCs were pretreated with indicated concentrations of MAGEA3 peptide or 10 μg/ml irrelevant peptide (IP) for 2 h, then washed extensively and co-cultured with CD8⁺ T cells for 10 days at an APC/responder cell ratio of 1:10. MAGEA3-specific responder cells were quantified by MAGEA3-tetramer staining using flow cytometry. Representative data were shown from 2 to 4 independent experiments. All the values were presented as mean ± SD. ***p < 0.001 versus Control or 0 μg/ml of the corresponding group (M). NS, not significant