Cd28 and cd49d antibodies

T-cell responses in PBMC were measured by flow cytometric intracellular cytokine staining (ICS) as previously described [36 (link)]. One million PBMC were incubated with overlapping 15-mer peptide pools spanning HIV-M (subtype B) Gag or Nef, and co-stimulated with CD28 and CD49d antibodies (BD Biosciences) for 1 hour, followed by incubation with Brefeldin A (Sigma-Aldrich) for 8 hours. Similarly, stimulation using human cytomegalovirus (HCMV) 15-mer peptide pools spanning IE1 and pp65 served as positive controls, while no antigen incubation served as background controls. Cells were surface stained with antibodies against CD3, CD4, CD8, CD95, CD28, and amine-reactive dye, and then fixed with 2% paraformaldehyde (PFA). BD FACS Permeabilizing Solution (BD Biosciences) was used to permeabilize the membranes before staining intracellularly for IFN-γ, TNF-α, and CD69. Samples were collected on a BDLSR-II instrument with FACsDIVA version 6.1, and analyzed with FlowJo v10 (Tree Star) by gating on singlet, live, CD3+, CD4+ or CD8+ cells. Positive responding CD4+ and CD8+ memory (CD95+) T-cells were measured by Boolean gating on CD69+/TFN-α+ and/or CD69+/IFN-γ+ cells. Presented as HIV positive responses were sums of individual HIV Gag and Nef responses, while positive HCMV responses were sums of individual HCMV IE1 and pp65 responses.

Cryopreserved PBMC were thawed and incubated overnight at 37°C, 5% CO₂. Cells were plated at 1 x 10⁶ per well and stimulated with L. major whole lysate (1μg/mL, generous gift from David Sacks) for 24 hours at 37°C, 5% CO₂. Brefeldin A (10μg/mL, Sigma) was added to all wells at 18 hours. All cells were costimulated with 1 μg/mL CD28 and CD49d antibodies (BD Biosciences). Following stimulation, cells were stained for population identification markers (CD3, CD4, CD8, CD14 and CD19) and intracellular cytokine expression (TNF-α, IFN-γ, and IL-2). T cell receptor (TCR) phenotyping antibodies were included for the αβ TCR and γδ TCR.