Human anti cd8 microbeads

Peripheral blood mononuclear cells (PBMCs) were obtained from patients chronically infected with HBV, HCV, and healthy donors visiting the outpatient clinic of the University Hospital Freiburg. All donors gave written informed consent according to the local ethic committee's of the University hospital Freiburg ruling, federal laws and the Declaration of Helsinki. PBMCs were isolated using density-gradient centrifugation (Pancoll; PAN Biotech, Aidenbach, Germany).
For some experiments, CD8⁺ T cells were isolated by autoMACS separation (Miltenyi) using anti-human CD8 Microbeads (Miltenyi). Virus-specific CD8⁺ T cells were analysed using the following APC-labelled MHC class I-tetrameric complexes: HLA-A*02/HBV core18–27 (FLPSDFFPSV), HLA-A*02/HBV pol455–463 (GLSRYVARL), HLA-A*02/HCV NS31073–1081 (CINGVCWTV), HLA-A*02/HCV NS31406-1415 (KLVALGINAV), HLA-A*02/HCV NS5B2594–2602 (ALYDVVTKL), HLA-B*27/HCV NS5B2841–2849 (ARMILMTHF), HLA-A*02/CMV pp65495-503 (NLVPMVATV). PBMCs were stained with tetramer for 15 min at 37 °C and blocked with murine IgG1 before further staining for surface markers or intracellular cytotoxic proteins.

Peripheral blood of patients was stored in anticoagulated tubes, diluted with PBS, and lightly spread over Lymphocyte Separation Medium (YEASEN). Peripheral Blood Mononuclear Cells (PBMCs) were obtained by centrifugation using density gradient centrifugation for 25 min. Anti-human CD8 Microbeads (Miltenyi) and anti-human CD14 Microbeads (Miltenyi) and MACS® MultiStand (Miltenyi) were employed for T cells and Mononuclear cells sorting, respectively. T cells were activated by CD3/CD28, and Mononuclear cells were active by M-CSF. Activated T cells and macrophages were co-cultured with sh-NC or sh-LDHA PC PDOs.

CD8⁺ T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK). After enrichment, the T cells were cultured at 37°/5% CO₂ for 48 hr in AB-RPMI medium supplemented with interleukin-7 (IL-7; 10 ng/ml; Peprotech, London, UK) to allow for the activation of autologous CLL cells to be used as a source of antigen-presenting cells. AB-RPMI consisted of RPMI-1640 (Sigma-Aldrich) supplemented with 5% human AB serum, glutamine (2 mmol/l), streptomycin (100 μg/ml), penicillin (100 U/ml), HEPES (25 mmol/l) (all sourced from Life Technologies, Paisley, UK). After 48 hr the T cells were harvested, washed and cultured in the presence of irradiated activated autologous CLL cells at a 4 : 1 ratio in AB-RPMI supplemented with Bax peptide pool (10 μg/ml; Mimotopes, Clayton, Victoria, Australia)8 (link) and IL-2 (40 U/ml; Proleukin, Novartis, Frimley, UK). After 3 days, 500 μl of AB-RPMI supplemented with IL-2 (120 U/ml) and IL-7 (30 ng/ml) was added. The cultures were re-stimulated weekly with the peptide pool and autologous activated CLL cells. On day 35 the T cells were harvested and Bax peptide immunogenicity was tested by ELISpot assay.