Soluble anti cd28 antibody

Tregs from young and old mice were cocultured with DC and Teff in 1:4:2 and 1:4:4 (DC/Teff/Treg) ratios in the presence of anti-CD3 antibody for 72 h. In a separate experiment, Treg from young and old mice were cocultured with Teff from young and old mice at 1:1 ratio in the presence of plate-bound anti-CD3 (1 μg mL⁻¹) and soluble anti-CD28 antibody (1 μg mL⁻¹, BD). After 72 h, conditioned media were collected, and IL-10 concentration was measured by using alpha-ELISA (Perkin Elmer, Waltham, MA, USA) as per manufacturer’s instructions.

FACS sorted CD8⁺ T cells were stimulated with 2 μg/mL immobilized anti-CD3ε (553058: BD Biosciences) and 2 μg/mL soluble anti-CD28 antibody (553295: BD Biosciences) during the first two days. Cells were then maintained in the medium supplemented with 20 U/ml rIL-2 (11271164001: Roche) for additional days.

Splenic CD4 T cells (1 × 10⁶) isolated from 10-week-old prediabetic female NOD mice were stimulated with plate-bound anti-CD3 antibody (3 μg/ml, clone: 145-2C11, BD Biosciences, USA) and soluble anti-CD28 antibody (3 μg/ml, clone: 37.51, BD Biosciences) in the presence of recombinant mouse IL-12 (10 ng/mL, TONBO Biosciences), recombinant mouse IL-2 (5 ng/mL, Invitrogen, USA), and anti-IL-4 antibody (10 μg/ml, clone: 11B11, BD Biosciences, USA) in 12-well culture plates. After 5 days, cells were harvested and washed, and 1 × 10⁶ differentiated Th1 cells were treated with plate-bound anti-CD3 antibody (1 μg/ml) and soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of recombinant sCD137 (240 μg/mL) for 24 h. In some wells, recombinant mouse IL-2 (25 U/ml) was added. After 24 h, cells were washed and rested in fresh TCM without IL-2 for 48 h; 0.5 × 10⁵ rested living cells were restimulated with plate-bound anti-CD3 antibody (0.5 μg/ml) and soluble anti-CD28 antibody (0.5 μg/ml) for 24 h. Supernatant was collected, and IL-2 concentration was measured by ELISA.

Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors’ and MS patients’ blood collected in EDTA-coated tubes using Ficoll density gradient as routinely performed [25 (link), 26 (link)]. CD8⁺ T lymphocytes were first isolated using CD8 Microbeads (Miltenyi Biotec), while the negative fraction devoid of CD8⁺ T lymphocytes was used to isolate CD4⁺ T lymphocytes using CD4 Microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Purity was > 95% for both T cell subsets; a representative flow cytometry analysis of isolated CD4 and CD8 T lymphocytes is shown in Additional file 1: Fig. S1. CD4⁺ and CD8⁺ T lymphocytes cultured in complete Iscove medium (containing FBS 10%, sodium pyruvate 1 mM, L-glutamine 2 mM, MEM nonessential amino acids 1%, β-mercapto-ethanol 1 µM, and antibiotics) were activated for 5 days on plate-bound anti-CD3 (OKT3 clone, eBioscience-Life technology, 2.5 μg/mL for CD4⁺ T cells and 5 μg/mL for CD8⁺ T cells) in the presence of soluble anti-CD28 antibody (BD Biosciences, 1 µg/mL). After five days, activated CD4⁺ or CD8⁺ T lymphocytes were harvested for co-culture experiments.

To measure IFNγ in CD8⁺ T cells, ICS was restricted to detection of IFNγ (a cytokine produced by CD8⁺ cells upon activation (23 (link)). Cells from spleens, lymph nodes, or TILs isolated from 4T1 tumor-bearing BALB/c mice were isolated and single cell suspensions prepared as described. Cells were then seeded into U-bottom 96-well plates precoated with 5 μg/ml anti-CD3 antibody, in RPMI media containing 10% FBS and soluble anti-CD28 antibody (Clone: 37.51. BD Biosciences, 2 μg/ml). Cells were incubated at 37°C overnight in 5% CO₂. Brefeldin A (10 μg/ml) was added to retain secretion of IFNγ in the Golgi apparatus. After 3 h, cells were stained with extracellular markers including anti-mouse PerCP-Cy5.5 CD8a (clone 53-6.7) MAb and anti-mouse PE-Cy7 CD5 (clone 53-6.7) MAb, (Biolegend, San Diego, CA)(1 μg/ml in 50 μl FACS buffer) on ice and incubated in the dark for 30 min. The samples were washed twice and fixed in 2% paraformaldehyde (50 μl). To detect CD8⁺ T cell activation, samples were stained for 60 min with anti-mouse PE-IFN-γ clone (XMG1.2) (1 μg/ml in intracellular staining permeabilization wash buffer) (Biolegend, San Diego, CA). Samples were then washed and harvested in FACS buffer for flow cytometry. Flow cytometric data were analyzed using Flowjo software (BD Bioscience).

Sorted CD4⁺ T cells were labeled with CFSE (ThermoFisher Scientific) and were cultured in custom ordered Dulbecco’s Modified Eagle Medium (D-MEM, KOHJIN BIO) supplemented with 10% heat inactivated FBS (Hyclone). 2.0 × 10⁵ cells were stimulated in 96-well flat-bottomed plate, which was pre-coated with 2 μg ml⁻¹ anti-CD3e antibody (553068, BD Bioscience) and 2 μg ml^-1 soluble anti-CD28 antibody (553295, BD Bioscience), for two days, and were maintained in D-MEM supplemented with 10 ng ml^-1 recombinant mouse IL-2 (402-ML, R&D system) for 2 to 4 days.