Human γδ T cells responded to LA-loaded CD1b/CD1c-transfected C1R cells, but not to LA-loaded CD1a/CD1d-transfected C1R cells. Purified γδ T cells and γδ T-enriched cells were prepared as described in Materials and Methods. CD1-transfected C1Rs are irradiated (4,000 rads) before use and preincubated with LA for the groups plus LA. (A) 3H-TdR-incorporation assay using purified human γδ T cells. *P < 0.05, when compared the arrow-marked group (CD1b-C1R+γδ T cell+LA) with those including CD1b-C1R+ γδ T cell, CD1b-C1R+γδ T cell+LA+anti-CD1b mAb, CD1b-C1R+γδ T cell+LA+anti-TCRγδ mAb, or CD1b-C1R+ γδ T cell+LA+anti-LA pAb; **P < 0.05 when compared the arrow-marked group (CD1c-C1R+ γδ T cell+LA) with those including CD1c-C1R+ γδ T cell, CD1c-C1R+ γδ T cell+LA+anti-CD1c mAb, CD1c-C1R+γδ T cell+LA+anti-TCRγδ mAb or CD1c-C1R+ γδ T cell+LA+anti-LA pAb. (B) CFSE labeled γδ T cell-enriched PBMCs were co-cultured with CD1a-, CD1b-, CD1c- and CD1d-transfected C1R cells preincubated with LA, with or without purified blocking Abs. The reduction of CFSE fluorescence was detected after 72 h by gating on TCRγδ+ populations. Histogram overlaps indicate the group of γδ T cell+LA+CD1-transfected C1Rs without any antibody (black filled histograms) and the groups with anti-CD1a/-CD1d (left panel), -CD1b (middle panel), -CD1c (right panel), -TCRγδ or -LA (middle & right panels) antibodies (grey line histograms), respectively. All experiments were performed at least three times with comparable results. CD1a/b/c/d-C1R: CD1a/b/c/d-transfected C1R cell lines.