Dynabeads cd8

Splenocytes were depleted for CD4 or CD8 T cells using CD8 Dynabeads^™ (Thermofisher, #11145D) or CD4 Microbeads (Miltenyi Biotec, #130–117-043), according to manufacturer’s instructions. The remaining sample was enriched for CD4 T cells (via CD8 T cell depletion) or enriched for CD8 T cells (via CD4 T cell depletion). Splenocytes were maintained in 4°C for 36 hours before processing for CD8 T cell enrichment. For CD8 depletion, the Dynabeads^™ were washed in isolation buffer and placed in the magnet. At the same time, cells were prepared at a concentration of 1 × 10⁷ cells per mL in isolation buffer. The prewashed beads were added, and the solution was incubated for 30 minutes at 4°C with gentle tilting. After, the tubes were placed in the magnet for 2 minutes and the supernatant was transferred to a new tube for further analysis. The beads and associated cells were discarded. For CD4 depletion via Microbeads, the cells were prepared in the appropriate buffer and the microbeads were added. The solution was incubated for 10 minutes at 4°C. The cell suspension was added to the LD column and the flow through, representing the CD8 enriched (CD4 depleted) population, was collected for further analysis.

Buffy coats were purchased from Sanquin. Human PBMCs (peripheral blood mononuclear cells) were isolated by density gradient centrifugation using Ficoll (1.078 g/mL, Fisher Scientific #11743219). CD8 T lymphocytes were positively selected by CD8 Dynabeads (Thermo Fisher Scientific) following manufacturer’s instructions. Anti-CD3 and anti-CD28 antibodies (eBioscience, 5 mg per well in 24-well plate format) were pre-coated for CD8 activation. After 48h of in vitro activation, T cells were transduced with retrovirus encoding 1D3 TCR. Transduction efficiency was assessed by FACS analysis and tumor cell-T cell ratios for co-culture were corrected for 1D3 TCR transduction efficiency. Multiple T cell donors (male and female origin) were used throughout this manuscript and can vary in killing efficiency between experiments.

TZM-bl cells were cultured in DMEM (Invitrogen) containing 10% FCS, MEM non-essential amino acids (0.1mM, Invitrogen), penicillin and streptavidin (pen/strep), both 100U/ml. Raji DC-SIGN cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 100U/ml pen/ strep. The peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (Sanquin) of three healthy CCR5 wild-type homozygous donors using ficoll-hypaque density centrifugation. The cells were pooled and kept at −150°C until required. After thawing PBMCs were cultured in RPMI supplemented with 10% FCS, 100U/ml pen/strep and 100U/ml IL2. 3μg/ml phytohemaglutinin was used to activate the cells and CD4⁺ T-lymphocytes were enriched by removing the CD8⁺ cells using CD8 dynabeads (Life Technologies) according to manufacturer’s protocol. Monocytes were isolated from buffy coats by ficoll-hypaque density centrifugation followed by selection for CD14⁺ cells via MACS (Miltenyi Biotec). Monocytes were cultured in RPMI containing 10% FCS supplemented with 500U/ml IL4 and 800U/ml GM-CSF (Schering-Plough) for 6 days which differentiated them into iDCs [19 (link)].

Raji DC-SIGN cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 100U/ml penicillin and 100U/ml streptomycin (100U/ml Pen/ Strep). Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (purchased Sanquin) obtained from healthy volunteers using ficoll-hypaque density centrifugation. PBMCs of 3 CCR5 wild-type homozygous donors were pooled and cultured in RPMI 1640 containing 10% FCS, 100U/ml Pen/ Strep, 100U/ml recombinant IL-2 (Chiron), and 2μg/ml phytohemagglutinin (PHA, Remel) was used to activate the cells. After 5 days of culture PBMCs were enriched for CD4⁺ T-cells by depleting the CD8⁺ T-cell population using CD8 dynabeads (Life Technologies) according to manufacturer’s protocol. Monocytes were isolated from PBMC as previously described [49 (link)]. Monocytes cultured in IMDM (Gibco) containing 5% FCS, 86μg/ml gentamycin (Duchefa), 500U/ml GM-CSF (Schering-Plough) and 10U/ml IL-4 (Miltenyi Biotec) for 6 days differentiated into immature DCs (iDCs). The CD4⁺ T-cell isolation MACS kit (Miltenyi Biotec, 130-091-155) was used according to manufacturer’s protocol to isolate CD4⁺ T-cells from the PBL fraction. Subsequently, the CD4⁺CD45RA⁺CD45RO^- naïve T-cells were isolated from the CD4⁺ T-cells using anti-CD45RO-PE (DAKO, R084301) and anti-PE beads (Miltenyi-Biotec, 130-048-801), described in detail [49 (link)].

The SupT1 T cell line (10⁵ cells) was infected with 293T-produced virus (equivalent of 2.5 ng CA-p24). To perform infections on CD4⁺ T lymphocytes, we first isolated peripheral blood mononuclear cells (PBMCs) from buffy coats (Sanquin) of healthy volunteers using Ficoll–Hypaque density centrifugation [30 (link)]. Cells of three donors (homozygous wild-type CCR5) were pooled and cultured in RPMI containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 100U/ml recombinant IL-2 and the first 3 days also with 2 µg/ml phytohemagglutinin (PHA) to activate the T lymphocytes. At day 5 the CD4⁺ T lymphocytes were isolated by depleting the CD8⁺ population using CD8 Dynabeads (Invitrogen) according to manufacturer’s protocol. The CD4 T lymphocytes (4 × 10⁵) were infected at 37 °C with equal amounts of 293T-produced virus (equivalent of 5 ng CA-p24). Cells were maintained with 100 units/ml recombinant interleukin-2 following infection.