Clone okt3

For testosterone treatment, we used RPMI 1640 Medium, without L-glutamine, without phenol red (Capricorn) supplemented with 1% GlutaMAX™ Supplement (Gibco), 1% Pen Strep (Gibco) and 5% charcoal stripped FBS Standard (PAN Biotech). For charcoal stripping of FBS, 2 g dextran-coated charcoal (DCC, Merck) were added to 100 ml FBS and incubated on a shaker overnight at 4 °C followed by centrifugation and filtration. Cryopreserved PBMCs from male healthy individuals were thawed and seeded at a density of 2 × 10⁵ cells per well in an anti-CD3 (10 µg/ml, clone OKT3, BioLegend) coated 96-well plate and supplemented with soluble anti-CD28 (2.5 µg/ml, clone CD28.2, BioLegend) or left unstimulated. Subsequently, testosterone (3, 30 and 300 ng/ml, Sigma Aldrich) or 5α-dihydrotestosterone (0.3, 3 and 30 ng/ml, SigmaAldrich) was added. Samples were incubated for 48 h at 37 °C and 5% CO₂, stained with antibodies listed in Table S4 and analyzed by flow cytometry after 2, 24 and 48 h.

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.

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated using Ficoll-Paque Plus (GE Healthcare) from anonymized healthy blood donor buffy coats which were purchased from Karolinska University Hospital. “Untouched” CD4+CD25– T cells were isolated from PBMCs using the CD4+ T cell Isolation Kit, human (Miltenyi Biotec) including additional depletion of CD25+ cells with CD25-specific MACS beads (8 μl per 10⁷ cells) as described earlier (22 (link)). Purity of the isolated T cells was accessed with flow cytometry and defined as CD3+ CD4+ CD25– CD8– T cells. T cells were stimulated with antibodies against CD3 (0.2 μg/ml, clone OKT3, BioLegend, LEAF grade), CD28 (2 μg/ml, clone 15E8, Miltenyi Biotec, functional grade), and goat anti-mouse Ig as a cross-linker (2 μg/ml, Southern Biotech) mimicking TCR and co-stimulation. The cells were stimulated for either 15 min or 1 h for proteomics studies or alternatively 3 h for mRNA studies. Jurkat T cells (clone E6.1) were stimulated with either above-described TCR and co-stimulation or “P/I stimulation” with Phorbol 12-myristate 13-acetate (PMA; 10 ng/ml; Sigma Aldrich) and ionomycin (375 ng/ml; Sigma Aldrich) for 1 h for imaging studies.

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)].