Clone cd28

Mouse naive CD4 T cells were seeded on plate-bound anti-CD3 (2 µg/mL, clone 17A2; BioXCell) and anti-CD28 (2 µg/mL, clone PV1; BioXCell). Human naive CD4 T cells were seeded on plate-bound anti-CD3 (5 μg/mL, clone OKT-3; BioXCell) and anti-CD28 (2 µg/mL, clone CD28.2; BioLegend). Cells (3×10⁶ cells/mL) were transfected for 6 hours with 80 µg/L cyclic dinucleotides (unless specified otherwise) using Opti-MEM Glutamax (Gibco) and Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. Alternatively, 1.5×10⁶ cells/mL CD4 T cells were treated with indicated doses of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) for 4 hours in mouse T-cell culture medium.
2′3′-cGAMP (cyclic (G(2′,5′)pA(3′,5′)p)), referred to as cGAMP; 2′3′-cGAMP control (2′5′-GpAp), referred to as control; as well as DMXAA (Murine STING ligand, Xanthenone Analog) were purchased from InvivoGen.
When indicated, mTOR inhibition was achieved by adding 10 nM of rapamycin (Rapa; Calbiochem, Merck) during the 6-hour transfection step.

Cryopreserved PBMCs from healthy women and men were thawed and CD3⁺ T cells were isolated from single-cell suspension using the Human Pan T Cell Isolation Kit (Miltenyi) according to the manufacturer’s protocol and labelled with CFSE (CellTrace™ CFSE Cell Proliferation Kit, ThermoFisher) according to the manufacturer’s protocol. CD3⁺ T cells were seeded at a density of 25,000 cells per well in an anti-CD3 (0.5 µg/ml, clone OKT3, BioLegend) coated 96-well plate. Cells were supplemented with soluble anti-CD28 (5 µg/ml, clone CD28.2, BioLegend) and anti-CD99 (5 µg/ml, clone HCD99, BioLegend) or respective isotype control (5 µg/ml, clone MOPC-173, BioLegend). Proliferation was tracked by cluster formation in the IncuCyte^® for 7 days.

T cells (1 × 10⁶ cells/mL in DPBS) were labeled with 5 μM of CellTrace Violet (Life Technologies) and incubated at 37 °C for 20 min. Medium containing 2% FBS was added (five times the original volume of the staining medium) and centrifuged once to remove free dye. The cell pellet was resuspended in 10% FBS-AIM-V medium (Life Technologies) at 1 × 10⁶ cells/mL, and 100 μL aliquots of the cells were added into wells precoated with 100 μL of anti-CD3 antibody (0.5 μg/mL, clone UCHT-1, Biolegend) and fusion antibodies to OX40 (0.25, 0.5, 1.0 and 2.0 μg/mL); soluble anti-CD28 (1 μg/mL; clone CD28.2, Biolegend) was added to the wells containing T cells. T cells in culture medium alone served as the non-stimulated cell control. The cells were then kept at 37 °C in a CO₂ incubator for 5 days. They were then harvested and the reduction of CellTrace Violet (indicating cell proliferation) was assessed via flow cytometry (BD FACSymphony A1, BD biosciences).

The proliferative rate of PBMCs was measured by MTT (Sigma Aldrich, St Louis, USA) assay. 7 × 10⁵ PBMCs were stimulated by anti-CD3 (0.157 μg/mL, Biolegend, clone OKT3, cat. 317,302) and anti-CD28 (0.085 μg/mL, Biolegend, clone CD28.2, cat. 302,901) antibodies (Figures 2sa, 2sb) and cultured in the absence or presence of 7 × 10⁴ cell line (Figures 3sa, 3sb) (E:T ratio, 10:1). Cells were treated with different concentration of drugs (piroxicam: 0, 3, 14, and 30 µM; and dexamethasone: 0, 0.1, 0.5, 1 µM) for 72 h in 48-well plates. MTT assay was used to evaluate cell proliferation. First, 20 µl of MTT dye (0.5 mg/ml) was added to the wells and the plate was incubated at 37° for 3 h. After incubation time, MTT-containing media were removed and 100 µl dimethyl sulfoxide (DMSO, Merck, Germany) was immediately added to all wells to dissolve formazan crystals. The absorbance was measured at 570 nm using ELISA reader (StatFax 2100, USA). To calculate the protective effect of piroxicam and dexamethasone on PBMCs, the following formula (Eq. (1)) was utilized [Eq. (1)]. $$\%Protection=\frac{ODTcc-ODTp}{ODCcc-ODCp}$$
ODTcc = Absorbance of PBMCs co-cultured with cell line in the treated condition, ODTp = Absorbance of PBMCs in the treated condition, ODCcc = Absorbance of PBMCs co-cultured with cell line in non-treated condition, ODCp = Absorbance of PBMCs in non-treated condition.

Purified CD4+ T cells were serially diluted in Costar plates coated with anti-CD3 (2.5μg/ml, Clone OKT3) and anti-CD28 (1μg/mL, Clone CD28.2, BioLegend 302902) monoclonal antibodies. Five serial 3-fold dilutions were performed at a starting concentration of 1x10⁶ cells/well (first dilution in a 24-well plate and following dilutions in a 96-well plate), with 6 replicates per dilution. After two days of stimulation, 50,000 or 10,000 MOLT-4/CCR5+ cells (NIH AIDS Reagent Program, 4984) were added to cell culture 24- or 96- well, respectively (day 0). Cell cultures were split twice weekly and half of cell culture supernatants (500μl or 100μl) were collected at days 7, 14 and 21 for quantification of soluble HIV-p24 protein. Supernatants were lysed and kept at -80°C until use. p24 protein was quantified by ELISA as previously described [85 (link)]. The number of wells positive for soluble p24 protein was determined, and the maximum likelihood method was applied to determine infectious units per million of cells (IUPM) (http://silicianolab.johnshopkins.edu/) [86 (link)].

PBMCs from freshly drawn heparinized blood originating from healthy volunteers were isolated by density gradient centrifugation using Ficoll Paque (GE). PBMCs (1 × 10⁵/well) were incubated for 96 h in the presence of DMSO or Cpd A (1 μM) together with anti-CD3 (3 ng/ml, clone OKT-3, BioXcell), anti-CD28 (30 ng/ml, clone CD28.2, BioLegend), TGF-β1 (0.2 ng/ml, Biolegend), IL-2 (15 ng/ml, Biolegend), anti-IFN-γ antibody (5 μg/ml, Biolegend), and anti-IL-4 (5 μg/ml, Biolegend). Cells were prepared for CD4/CD25 surface staining, followed by intracellular FoxP3 staining.