Cd28 49d co stimulatory antibodies

Detection of antigen specific CD4 T cells detection was quantified by activation induced marker (AIM) and intracellular cytokine staining (ICS). PBMCs/LN cells were stimulated with overlapping peptide pools of HIV consensus C and HIV-1 C.1086 Env gp140C protein (NIH AIDS Reagent Program) in AIM/R10 media in the presence of 0.2 μg CD28/49d co-stimulatory antibodies (BD) per test. As a positive control, cells were stimulated with 1 X Cell Stimulation Cocktail (PMA and ionomycin) (eBioscience, USA). Unstimulated controls were treated with volume-controlled DMSO (Sigma-Aldrich). Tubes were incubated in 5% CO₂ at 37 °C overnight for AIM assay. For ICS assay, after 1 hr of stimulations, protein transport inhibitors 2 μl/mL GolgiPlug (Brefeldin A) and 1.3 μl/mL GolgiStop (Monensin) (BD, Biosciences, USA) were added to the tubes for 8 hr at 37 °C, 5% CO₂. Following stimulation, the cells were stained for AIM and ICS surface markers (see Table 1). Cells were then fixed with cytofix/cytoperm for 10 min at 4 °C, permeabilized with 1 X Perm wash buffer (BD, Biosciences, USA), and stained for intracellular markers (see Table 1) for 45 min. Cells were then washed and acquired the same day on a BD FACS Symphony.

The polyfunctionality of CD4 T cells was assessed using intracellular cytokine staining (ICS). Brain and spleen cells were stimulated with 1X Cell Stimulation Cocktail (PMA and ionomycin) (eBioscience, USA) along with R10 media in the presence of 0.2mg CD28/49d co-stimulatory antibodies (BD) per test. Unstimulated controls were treated with volume-controlled DMSO (Sigma-Aldrich). Cells were incubated in 5% CO₂ at 37°C and after 1 hour of stimulation, protein transport inhibitors 2ml/mL GolgiPlug (Brefeldin A) and 1.3ml/mL GolgiStop (Monensin) (BD, Biosciences, USA) was added to tubes and further incubated for 3 hours at 37°C, 5% CO₂. Following stimulation, cells were stained for ICS surface markers CD3, CD4, CD8, and CD95. Subsequently, the cells were fixed using cytofix/cytoperm for 10 min at 4°C, then permeabilized with 1X Perm wash buffer (BD, Biosciences, USA), and stained with intracellular markers TNFα, IFNγ, and IL-2 for 45 min. Finally, cells were washed and acquired the same day using a BD FACSymphony flow cytometer.