Anti cd3 antibody clone okt3

Prior to co-culture experiments, activation of isolated naïve CD8+ T cells was performed. For this purpose, a 24-well plate was coated by incubation for 2 h at 37°C with 200 µl of 1.5 µg/ml anti-CD3 antibody (clone: OKT3, Biolegend) in PBS. Before T cells were seeded, wells were washed 2-times with PBS. Then, 1.3 – 1.8x10⁶ naïve CD8+ T cells were seeded in 1 ml TCM supplemented with 1.5 µg/ml anti-CD28 antibody (clone: CD28.2, Biolegend) and 60 ng/ml IL-2 (Biolegend). CD8+ T cells were cultured for 3 days at 37°C, 5% CO₂ and 86% humidity.

Seven days after editing, T cells were labelled with 5 µM of Cell Trace Violet dye (ThermoFisher Scientific) according to manufacturer’s instructions. Labelled cells were seeded on a 96-well plate pre-coated with 1 µg/ml of anti-CD3 antibody (clone OKT3, BioLegend) and cultured in X-Vivo 15 medium supplemented with human AB serum (5%). After 3 days, cellular proliferation was assessed by dye dilution by FACS analysis. Samples stained at day 0 were used as a negative control.

Primary CD4+ T cells were activated for 4 to 5 d on T25 flasks coated with 5 μg anti-CD3 antibody (clone OKT3, Biolegend) in the presence of 2 μg/mL soluble anti-CD28 antibody (clone CD28.2, Biolegend). Cells were infected with HIV-1 by spinoculation (1,200 × g for 2 h at RT) at a multiplicity of infection of 0.1. For cell-to-cell spread assays HIV-1–infected T cells (donors) were analyzed for GFP expression by flow cytometry 48 h postinfection (p.i.), and the number of GFP+ cells was measured using Precision Count Beads (Biolegend). Uninfected T cells (targets) were labeled with 2.5 μM Cell Proliferation Dye eFluor 450 (Thermo Fisher Scientific) according to manufacturer’s instructions. HIV-1–infected GFP+ donor cells were normalized to achieve equivalent numbers of infected cells (10%) to account for variability between different PBMC donors in initial infection rates and then mixed with dye-labeled targets at a 1:4 ratio, incubated at 37 °C for 24 h, and analyzed by flow cytometry. For longer-spreading infection assays, donor cells were analyzed for GFP expression by flow cytometry at 24 h p.i., infection levels were adjusted to 2% with uninfected CD4+ T cells, and cells were incubated at 37 °C and analyzed by flow cytometry at various time points.

Human PBMCs were obtained as above, and CD4⁺ T cells enriched using anti-human-CD4 microbeads (Miltenyi Biotec) before sorting naive CD4⁺ CD45RA⁺ CD25⁻T cells by flow cytometry using an Influx II cell sorter (BD Bioscience, San Diego, CA, USA). Naive T cells were co-cultured with allogenic moDC in StemXvivo (R&D Systems) serum-free medium in roundbottom plates containing interleukin-2 (10 ng ml⁻¹, Biolegend) and anti-CD3 antibody (clone OKT3, 0.2 µg ml⁻¹, Biolegend). In all, 5 × 10³ DCs were plated per 1 × 10⁵ T cells in the presence of either 100 µg ml⁻¹ anti-TGFβ (1D11), 20 µg ml⁻¹ anti-integrin β8 (clone 37e1B5,38 100 µg ml⁻¹ isotype control IgG (MOPC-21), or 5 ng ml⁻¹ active TGFβ (Peprotech).

Buffy coats from anonymized donors were purchased from the Karolinska University Hospital according to the national ethical regulations in Sweden. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation, monocytes depleted by plastic adherence and platelets removed by low-speed centrifugations (200 g). CD4+CD25- T cells were obtained from PBMCs by negative isolation with magnetic beads (human CD4 T cell isolation kit, and additional depletion of CD25+ cells with CD25 microbeads) according to the manufacturer’s instructions (Miltenyi Biotec). T cells were cultured at 5% CO₂/37 °C in serum-free X-Vivo 15 medium (Lonza) supplemented with 1% Glutamax (Life Technologies). T cells were stimulated with 5 μg/ml plate-bound anti-CD3 antibody (clone OKT3) and 1 μg/ml soluble anti-CD28 antibody (clone CD28.2; both Biolegend, LEAF grade) or with 0.5 μM ionomycin and 10 ng/ml PMA (both Sigma-Aldrich)