Anti human cd28 cd49d

Peripheral blood was collected in heparin coated tubes. Whole blood was either stimulated with CMV cell lysate (10 µg/mL) or control lysate (10 µg/mL, HEL-299) (both Lophius Biosciences) for six hours in the presence of anti-human CD28/CD49d (1 µg/mL, clones L293 and L25, BD Biosciences, Heidelberg, Germany). If cytokine production of antigen specific T-cells was determined, Brefeldin A (Sigma Aldrich, Munich, Germany) was added after the first two hours of incubation. Following incubation, whole blood was lysed (FACS lysing solution, BD Biosciences) and permeabilized (FACS Permeabilizing Solution 2, BD Biosciences). Cells were then stained for lineage markers (anti-human CD3, Pacific Blue, clone UCHT1, and anti-human CD4 APC, clone 13B8.2, both Beckman Coulter Krefeld, Germany; anti-CD8 APC-H7, clone SK1, BD Biosciences) and IFN-γ (anti-IFN-γ, FITC, clone 45.15, Beckman Coulter). If CD154 expression was determined on antigen-specific T-cells, whole blood was washed after six hours of incubation and then stained for the above-mentioned lineage-markers, as well as for CD154 (anti-human CD154 FITC, clone 24–31, Biolegend, San Diego, CA, USA). Whole blood was lysed (VersaLyse, Beckman Coulter) and washed twice. Samples were then measured with a flow cytometer (Navios, Beckman Coulter). Data was analyzed using Kaluza Software Version 1.5a (Beckman Coulter).

To evaluate T-cells function, cells were incubated with 5 μg/mL antigenic peptides in the presence of brefeldin A (7.5 μg/mL) (Sigma-Aldrich), anti-human CD28/CD49d as costimulatory reagents (1 μg/mL) (BD Biosciences) for 4 hours. Subsequently, cells were stained for surface markers, then fixed, and permeabilized for intracellular staining using Foxp3 staining buffer set from eBiosciences according to the manufacturer’s instructions. Permeabilized cells were then incubated with IFNγ, IL-2, TNFα (BD Biosciences), Ki67 (BioLegend) antibodies (BD Biosciences) and resuspended in PBS 2% FBS 1% PFA before acquisition. For degranulation potential, BD GolgiStop solution, anti-CD107a and anti-granzyme B (BD Biosciences) were used. T cells treated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) were used as positive controls. Acquisition was performed with a LSRII flow cytometer and data were analyzed using Flowlogic software.