Dynabeads cd3

Tonsillar B cells were isolated using negative isolation with CD3 Dynabeads (Thermo Fisher, MA, USA). CD3-depleted cells were split in two populations and incubated with antibodies for 10 min. CD44^- GC B cells were obtained through negative selection by combining Biotin Binder Dynabeads (Thermo Fisher, 50 μl beads per 10x10⁶ cells) with biotinylated anti-CD44 (BD Pharmingen) (1:50). A naïve/memory joint population was isolated using Biotin Binder Dynabeads (Thermo Fisher) with biotinylated anti-CD38 (eBioscience) (2.35 μg/mL). Cells and Dynabeads were incubated in the dark at 4°C with rotation. IgD-depleted memory B cells were obtained by negative selection by incubating CD19⁺ B cells with Pan Mouse IgG Dynabeads (Thermo Fisher) coated with mouse anti-human IgD Abs (BD) for 30 min at 4°C, followed by removal of beads.

PBMCs were isolated from healthy donors by density gradient centrifugation using Biocoll separating solution (Biochrom GmbH, Berlin, Germany) after informed consent. CD3⁺ cells were subsequently depleted by CD3 Dynabeads (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. CD3-depleted PBMCs were seeded at a cell density of 1.0 × 10⁶ in 25 ml culture flasks in RPMI 1640 medium (Biochrom) supplemented with 10% fetal bovine serum (Biochrom), 2 mM L-glutamine (Biochrom), 100 U/ml penicillin (Biochrom), and 100 μg/ml streptomycin (Biochrom) in the presence of 5% CO₂ in a humidified atmosphere at 37 °C. All experiments involving human tissues were approved by the ethics committee at the Medical Faculty of the Eberhard Karls University and the University Hospital Tuebingen (349/2013BO) and informed consent was obtained from healthy donors in accordance with the Helsinki Declaration of 1975 (revised in 2008).

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.