Anti cd28 clone cd28

Peripheral blood mononuclear cells (PBMCs) were isolated through density gradient (Lymphoprep, STEMCELL Technologies Inc., Vancouver, Canada) by centrifugation at 400 x g for 30 minutes and cultured in 96-well V bottom plates (Costar, Corning, NY) with a density of 4 x 10⁶ cells/mL in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, 100 μg/mL of streptomycin and 2 mM L-glutamine (complete medium; all from Gibco, Carlsbad, CA). PBMCs were stimulated with 10 μg/mL of each peptide (Table 1), in presence of 1 μg/mL of both anti-CD28 (clone: CD28.2, eBioscience) and anti-CD49d (clone: 9F10, eBioscience). Cells stimulated only with anti-CD28 and anti-CD49d antibodies were used as the negative control. The PBMCs were stimulated with 1 μg/mL of staphylococcal enterotoxin B (SEB) from Staphylococcus aureus (Sigma-Aldrich) or with 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL of ionomycin (both from Sigma-Aldrich) was also included in the analysis. All stimulated cells were incubated for 12 h at 37˚C in 5% CO₂, in the presence of 10 μg/mL of brefeldin A and monensin (both from Thermo Fisher), as well as anti-human CD107a (clone H4A3, BD).

Anti-CD3ε (clone OKT-3; eBioscience, #16–0037), anti-CD28 (clone CD28.2, eBioscience, #16–0289), anti-pPAK1/2 (1:1000, Cell Signaling Technologies (CST #2601 T)), anti-pZap70 (1:1000, CST #2701P), anti-pLAT (1:1000, CST#3584S), anti-pSrc family (1:1000, CST#2101), anti-pSLP-76 (1:1000, Abcam (#ab75829)), anti-pVav1 (1:1000, #ab47282), anti-CD11/CD18-FITC (1 μg/ml, #ab13219), anti-beta-actin (1:5000, #ab6276), anti-CD69-PE (1:100, Serotec), anti-CD25-APC (1:100, Biolegend, #302,610), anti-TfR-biotin (clone OKT-9; eBioscience, #13–0719-82), anti-TfR (clone M-A712, 10 μg/ml, BD Biosciences #555,534), anti-CD18-PE (1:50, BD Biosciences #555,924) and anti-CD29-PE (1:75, clone HUTS-21; BD Biosciences, #556,049).

In vitro P. falciparum antigen-specific CD4⁺ T cell proliferation and cytokine expression were measured using P. falciparum-infected red blood cells (iRBC) as the antigen. For this, P. falciparum cultures (strain 3D7) were maintained in 0⁺ erythrocytes in RPMI-1640 medium supplemented with 0.5% AlbuMAX II (Invitrogen) at 37°C according to standard methods [72 (link)]. Schizont and late trophozoite stage parasites were isolated magnetically using the Miltenyi LD column. The percentage of infected erythrocytes after magnetic isolation was typically 80–90%. The iRBC were stored in PBS at -70°C until use in cell culture at two iRBC per T cell/PBMC. Uninfected red blood cells (uRBC), namely, the 0⁺ erythrocytes from the same donors that were used for the parasite cultures, served as negative controls at two uRBC per T cell/PBMC. CMV pp65 protein (Miltenyi, 1 μl/ml) and tetanus toxoid (10 μg/ml) served as alternative antigens. For polyclonal stimulation in proliferation assays, 1 μg/ml soluble anti-CD3 (clone UCHT1, eBioscience) and 1 μg/ml anti-CD28 (clone CD28.2., eBioscience) were used. Alternatively, anti-CD3/28-coated beads (Dynal T cell expander) were used at a ratio of five cells/bead. PHA (Sigma-Aldrich at 5 μg/ml) or PMA/Ionomycin (50ng/500ng) served as the positive controls for intracellular cytokine staining.

For intracellular staining, 2 x 10⁶ PBMNCs were re-suspended in 1ml of complete medium and cultured in 15ml, 120x17mm, polypropylene tubes (SARSTEDT, Nümbrecht, Germany), in the absence (control) or in the presence of either TT (10μg/ml), PPD (15μg/ml) or C. Alb (10μg/ml). Anti-CD28 (clone CD28.2, eBioscience) was added to all tubes (1μg/ml). After 90 minutes, brefaldin A (Golgi Plug, BD Biosciences) was added (1/1000 dilution). After 6h, cells were washed and stained for surface markers first, and then they were permeabilized and stained for intracellular markers. Data were acquired on a Canto II flow cytometer and analyzed using FlowJo (7.6.5) (Treestar) software. For analysis of antigen-specific CD4⁺ T cells, between 1.5–1.8 x 10⁶ events in the singlet gate were acquired. Responses were considered positive if the frequency of the events in antigen-stimulated cultures was ≥ 0.01%, and the frequency of background events was ≤ 30% of the frequency of events in the antigen-stimulated cultures.