Cd28 clone cd28.2

The following conjugated Abs were used for cell surface staining: CD3-Pacific Blue (clone UCHT1), CD4-Alexa Fluor 700 (clone RPA-T4), CD8-APC-H7 (clone SK1), CD160-Alexa Fluor 647 (clone BY55), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)-PE-CF594 (clone 7D3), 2B4-FITC (clone 2–69) (BD Biosciences, San Jose, CA), and programmed cell death protein-1 (PD-1)-PE (clone J105) (Thermo Fisher Scientific). Conjugated Abs for intracellular staining included the following: cytotoxic T lymphocyte antigen-4 (CTLA-4)-PE-Cy5 (clone BNI3), granzyme B-PE-CF594 (clone GB11), interferon-gamma (IFN-γ)-PE-Cy7 (clone B27), interleukin-2 (IL-2)-APC (clone MQ1-17H12), tumor necrosis factor-alpha (TNF-α)-Alexa Fluor 488 (clone MAb11) (BD Biosciences, San Diego, CA), and perforin-PE (clone B-D48) (Abcam, Cambridge, UK). All conjugated Abs were titrated and multicolor panels for flow cytometry assays were approached as previously reported [38 (link)]. The following purified (No azide/Low endotoxin) Abs were used in cultured cells: CD28 (clone CD28.2) and CD49d (clone 9F10) (BD Biosciences).

PBMCs from two different HIV-1 seronegative individuals (not included in other analysis) were thawed and T cells were activated by stimulation with CD3 (clone OKT3; eBioscience, 150 ng/mL) and CD28 (clone CD28.2; BD Biosciences, 150 ng/mL) antibodies in RPMI media supplemented with 20% FBS and recombinant human IL-2 (NIH AIDS Reagent Program, Division of AIDS, NIAID, National Institutes of Health, 30 U/mL). Activation was allowed to proceed for 72 h at 37 °C 5% CO₂. CD4+ T cells were then enriched by removal of CD8+ T cells using magnetic CD8 microbeads (Miltenyi Biotec, Germany). Cells were then infected with full-length HIV-1 infectious molecular clone virus derived from an infected individual CH058 [26 (link)] by spinoculation at 1200×g for 2 h at room temperature [42 (link)]. Infected cells were incubated in RPMI media supplemented with 20% FBS and 30 U/mL IL-2 for 24 h. Cells were then harvested and washed with PBS for scRNA-seq.

Human memory CD4+CD45RO+ and memory CD8+CD45RO+ T cells were purified from PBMC of healthy buffy coat donors. PBMC were first isolated from buffy coats (Gulf Coast Regional Blood Center, Houston, TX) by using Ficoll-Paque PLUS (GE Healthcare). Memory CD4 and memory CD8 T cells were then negatively selected from PBMC with magnetic bead-based EasySep kits (Stemcell Technologies). Purities of memory CD4+CD45RO+ T cells were usually >90% and memory CD8+CD45RO+ T cells >80% as determined by flow cytometry. Natural killer cells were purified from PBMC with negative selection kits (Stemcell Technologies), and CD3-CD56+ purity was >90%. Cells were cultured at 37°C + 5%CO₂ in complete RPMI-1640 medium, supplemented with 10% FBS, 0.1 mM MEM nonessential amino acids, 2mM sodium pyruvate, 2mM HEPES, 1× antibiotic-antimycotic and 2mM L-glutamine.
Most experiments involved no stimulation (UT) or stimulation of 1-5×10⁵ memory CD4 and memory CD8 T cells, or NK cells (200 μl/well of 96-well flat-bottom plates, or 1 ml/well of 24-well plates, as indicated) for 24 hrs with 1 ml/ml coated CD3 (clone UCHT1, BD Biosciences) or soluble CD3 (clone CD3-2, Mabtech) + 1 μg/ml CD28 (clone CD28.2, BD Biosciences) mabs. For some experiments, cells were stimulated with 100 ng/ml recombinant IL2 (Biolegend) or soluble CD3 mab alone for 24 hrs with or without brefeldin (GolgiPlug, BD Biosciences).