Recombinant human il 2

Ectopic ESCs were cultured in 24-well plates (Corning, Steuben County, NY, USA) at a density of 1 × 10⁵ cells/well. The co-culture systems were established by incubating 2 × 10⁵ monocytes with ESCs or alone, adding of estrogen (10⁻⁸ M; Sigma), 1-MT (0.05 mM; Sigma), or estrogen (10⁻⁸ M; Sigma) plus 1-MT (0.05 mM; Sigma). Meanwhile, naive CD4⁺ T cells were cultured in 24-well plates that coated with monoclonal anti-CD3 (5 μg/ml; Biolegend) and monoclonal anti-CD28 (1 μg/ml; Biolegend), in the presence of recombinant human IL-2 (50 ng/ml; Biolegend). The monocytes-derived macrophages and 2 × 10⁵ naive CD4⁺ T cells were collected to co-culture with ESCs in 1 ml medium/well for 5 days (Supplementary Figure 1).

Jurkat cells were cultured in RPMI1640 medium supplemented with 10% FBS and activated with 10 ng/mL phorbol myristate acetate (PMA; Thermo Fisher Scientific) or with Dynabeads Human T-activator CD3/CD28 (Thermo Fisher Scientific). After activation, the cells were collected, stained with a cell viability dye (Ghost V450, TONOBO), and the following allophycocyanin (APC)-conjugated antibodies: anti-human CD69 (BioLegend, clone FN50), or with isotype control (BioLegend, clone MOPC-173 and clone MOPC-21; isotype 1 and isotype 2, respectively) and analyzed using flow cytometry.
Human peripheral blood mononuclear cells (PBMC) and healthy mouse splenocytes were cultured in RPMI1640 medium supplemented with 10% FBS at a density of 1 × 10⁶ cells/mL. Cells were activated with 25 μL of CD3/CD28 beads per 1 × 10⁶ cells (Gibco, Dynabeads human or mouse T-activator) and recombinant human IL2 (BioLegend). Subsequently, the cells were collected and stained with a cell viability dye (Ghost UV780, TONOBO), blocked with TruStain FcX (BioLegend, human or anti-mouse CD16/32, clone 93), and stained with CD3 (BioLegend, anti-human, clone HIT3a or anti-mouse, clone 17A2) and CD69 (BioLegend, anti-human, clone FN50 or anti-mouse, clone H1.2F3) for flow cytometric analysis.

Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Stemcell Technologies Inc.) density gradient centrifugation. CD4⁺CD25⁺ T cells were obtained by positively selection using CD4⁺CD25⁺ T cells (Regulatory T cells) micro-magnetic beads according to the manufacturer instructions (Miltenyi Biotec). CD4⁺CD25⁻ cells were obtained by negatively selection according to the manufacturer instructions (Miltenyi Biotec). CD4⁺CD25⁺ T cells were cultured in 96-well plates that coated with monoclonal anti-human CD3 (5 μg/ml; Biolegend, clone: OKT3) and monoclonal anti-human CD28 (1 μg/ml; Biolegend, clone: CD28.2), in the presence of recombinant human IL-2 (50 ng/ml; Biolegend), while CD4⁺CD25⁻ cells were cultured in 24-well plates that coated with monoclonal anti-CD3 (5 μg/ml; Biolegend) and monoclonal anti-CD28 (1 μg/ml; Biolegend). T_reg cells were collected to culture with non-treated ESCs, 1-MT-pretreated ESCs, vector-pretreated ESCs, and MRC2-silenced ESCs or not for 48 h after T_reg cells proliferation for two weeks. After that, T_reg cells from these groups respectively cultured with paired CFSE-labeled CD4⁺CD25⁻ T cells (T_eff) for 48 h and then detected by flow cytometry.

Dsg-specific memory B cells were measured using a polyclonal stimulation assay, essentially as previously described (Pinna et al., 2009 (link)). Briefly, PBMCs were cultured at 1×10⁶ cells/mL in RPMI supplemented with penicillin/streptomycin, L-glutamine, 10% FBS, a 50 μM of beta-mercapto-ethanol (Sigma) (R10) containing R848 (1 μg/mL, Invivogen) and recombinant human-IL2 (10 ng/mL, Biolegend) for 3 days at 37°C. Total and Dsg3-specific IgG-secreting cells were quantified by ELISPOT assay, in which a 96-well ELISPOT filter plates (Millipore) were coated overnight with rDsg3 antigen (3 μg/mL, provided by the Hertl Lab), rDsg1 antigen (3 μg/mL, EuroImmun), or polyvalent goat anti-human Ig (25 μg/mL, Rockland) in PBS/1 mM CaCl₂. Plates were washed and blocked by incubation with R10 at 37°C for 2 h. PBMCs were added to the plates in a dilution series and incubated for 5 hs at 37°C. Plates were washed with PBS, followed by PBS/0.05% Tween, and incubated with biotinylated anti-human IgG antibody (Invitrogen) at room temperature for 90 min. After washing, plates were incubated with an avidin-D horseradish peroxidase conjugate (Vector laboratories) and developed using 3-amino-9-ethyl-carbazole substrate (Sigma). Plates were scanned and analyzed using an automated ELISPOT counter (CTL, Cellular Technologies).

Human peripheral blood samples were obtained from five different healthy volunteers (BWH Specimen Bank). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (StemCell Technologies). PBMCs were cultured at 1.5 × 10⁶ cells per mL in RPMI 1640 media (Lonza), supplemented with 10% human serum (GemCell) (10% HS-RPMI), recombinant human IL-2 10 ng per mL (or 100 U per ml, BioLegend) and R848 1 µg per mL (Cayman Chemicals) for 5 days, at 37 °C, 5% CO₂. PKS21221 in DMSO, or DMSO only for control, was added to the culture media on day 5, and cells were incubated for additional 12, 24, and 48 h. Cells were then stained with surface marker antibodies and analyzed by flow cytometry. The viability and apoptosis were assessed using Annexin V Apoptosis Detection Kit (BD Biosciences).