Isolation and Proliferation Analysis of T Cell Subsets

Buffy coats were obtained from healthy volunteers, with approval by the local ethical committee (Landesaerztekammer Rheinland-Pfalz). CD8⁺ T cells, CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells were isolated as previously described.[14 (link), 44 (link), 45 (link)] For some experiments, peripheral blood mononuclear cells (PBMC) were depleted of T cells using CD3-Dynabeads (0.5 beads/cell, Invitrogen).
For proliferation assays, cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cultured in 48 well plates at 10⁶ cells/ml, stimulated with 0.5 μg/ml anti-CD3 mAb (clone OKT3) plus 1 μg/ml anti-CD28 mAb (clone 28.2, eBioscience) in presence or absence of different ratios of melanoma cells.
To analyze the effect of sGARP on proliferation and granzyme B expression of CD8⁺ T cells, T cells were stimulated with anti-CD3 mAb (0.5 μg/ml) and anti-CD28 mAb (1 μg/ml) or with melanoma cell line D05-MEL#6 (5 × 10⁴/ml) in presence or absence of sGARP (10 μg/ml) from R&D Systems (#6055-LR, <0.01 endotoxin units per 1 μg) as shown before.[9 (link)] At day 7, T cells were harvested and re-stimulated with melanoma cells or anti-CD3 mAb (0.5 μg/ml) plus irradiated (90 Gy) T cell-depleted PBMC. For some experiments, T cell proliferation was measured by additional 16h-pulse with ³H TdR (37 kBq/well) using a liquid beta-scintillation counter.