Cd28 cd49d antibodies

HLA-B57+ primary CD4 T cells were either treated with LRA, stimulated with anti-CD3/CD28 or kept in culture with no stimulation for 48 hours. The cells were thoroughly washed and magnetic anti-CD3/CD28 beads were removed. For each condition, 2 million of CD4 T cells were infected with 20 ug HIV Gag p24 equivalent of NL4-3-Δenv-GFP virus pseudotyped with Vesicular Stomatitis Virus glycoprotein (VSV-g) in the presence of 5ug/mL polybrene (Sigma) by spinfection for 1.5 h at 2000x g as in [37 (link)]. At 24, 48 and 72 h post-infection, CD4 T cells were plated with epitope-specific CD8 T cells at a ratio of 1:4 (CD4:CD8) and an aliquot of CD4 T cells was also harvested to measure HIV-1 infection through GFP and HIV-1 p24 intracellular expression, as well as cellular activation and HLA class I surface expression by flow cytometry. After 30 min of co-culture, CD107a-Pe-Cy7 (BD Biosciences) and CD28/CD49d antibodies (BD Biosciences) were added to each well to measure CTL degranulation. After 6 h cells were harvested for surface staining.
The avidity of the CTL clones was assessed by performing a concentration-course titration. Target HLA-B57 CD4 T cells were incubated with increasing concentration of the cognate peptides (0.000002–2 ug/mL) before co-culture with the corresponding CD8 T cells as described above.

PBMC were thawed and washed twice in CM containing benzonase (Novagen, 25KU, 1/5000 V/V). Viability was assessed using trypan blue exclusion after a resting period of 2–6 hours at 37°C in complete medium. Cells were stimulated for 12h to 16h in complete medium containing Brefeldin A (Sigma-Aldrich, 5ug/ml) and CD28/CD49d antibodies (Pharmingen, 1ug/ml) with CMVpp65 peptide set (JPT Peptide Technologies, 2ug/ml), Staphylococcal Enterotoxin B (Sigma-Aldrich, 0.8ug/ml) or nothing as a negative control. Intracellular cytokine staining was performed using a standard staining protocol [10 (link)]. Acquisition of cells was performed on BD FACS Canto2 (NIMR-MMRC, Mbeya) or BD Fortessa (IHI-BRTC, Bagamoyo). Instruments were calibrated before each run using BD Cytometer Setup and Tracking Beads according to manufacturer’s recommendations. Data were analysed using FlowJo 9.X (Tree Star). T cell responses were considered positive, if the frequency of IFNγ⁺ cells was above 0.05% of the parent population and >2-fold the background frequency in the unstimulated control. GraphPad Prism (San Diego, CA, USA), version 4.03, was used for statistical analysis. Anonymized patient characteristics and the respective flow cytometry data statistics are provided as S1 Table.

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient and stored in liquid nitrogen until processed, as previously described (26 (link)). Cryopreserved PBMC were thawed and washed in pre-warmed RPMI 1640 supplemented with 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Corning cellgro, Manassas, VA) (complete RPMI). PBMC were resuspended in complete RPMI containing 0.1 μg/ml CD28/CD49d antibodies (BD Biosciences, San Jose, CA, United States) at a cell concentration of 2 to 3 × 10⁶/ml. After the addition of CD28/CD49d antibodies, 1-ml aliquots of the cell suspension were transferred in 12 × 75 mm polystyrene FACS tubes and either stimulated with 10 μg/ml PPD (Staten Serum Institute, Copenhagen, Denmark) or left unstimulated. A mixture of 25 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, United States) and 0.5 μM ionomycin calcium salt (Enzo Life Sciences, Farmingdale, NY, United States) served as positive control for T cell stimulation. Tubes were incubated for 2 or 6 h at 37°C in a 5% CO₂ humidified atmosphere.