Anti cd28 37

For polyclonal CD4+ T cell stimulation, single cell suspensions from pooled infection site-draining secondary lymphoid organs (mediastinal, cervical, inguinal, and mesenteric) or lungs were sorted using the L3T4 Positive CD4+ T Cell Selection kit and LS Columns from MACS Miltenyi according to the manufacturer’s instructions. After sorting, cells were washed once in MACS buffer, counted using a Muse Cell Analyzer (Millipore Sigma), and plated at a density of 1 x 10⁶ cells per well of a 96-well round-bottom plate. Plates were either uncoated or coated with 1 μg/mL each of anti-CD3 (145-2C11, BioLegend) and anti-CD28 (37.51, BioLegend). Cells were cultured in cRPMI for 72 hours at 37°C at 5% CO₂.
For whole lung cell stimulation with 2W1S peptide or anti-CD3, single cell suspensions from lungs were prepared and plated at a density of 2 x 10⁵ cells per well in a 96 well round-bottom plate. For anti-CD3 stimulation, plates were pre-coated with 1 μg/mL anti-CD3 (145-2C11, BioLegend) and cells were resuspended in CD4+ T cell media (cRPMI + 1 mM Sodium Pyruvate, 1 mM L-Glutamine, 50 μM MEM non-essential amino acids (NEAA), 5 mM HEPES, and 50 μM β-mercaptoethanol, 10 ng/mL mouse recombinant IL-2). For peptide stimulation, cells were resuspended in CD4+ T cell media containing 100 μg/mL 2W1S peptide and 1 μg/mL anti-CD28 (37.51, BioLegend).

T cells were seeded in primary T cell medium, i.e. RPMI1640 supplemented with 10% FCS (PAA Laboratories), 50 µM β-mercaptoethanol, 50 µg/ml each of penicillin and streptomycin, 1% non-essential amino acids and 1 mM sodium pyruvate (all from Life technologies). Bafilomycin A1 was obtained from Enzo Life Science. For Treg cell stimulation, 2 µg/mL anti-CD3 (145-2C11, BioLegend), 4 µg/mL anti-CD28 (37.51, Biolegend) and 10 ng/ml murine IL-2 (402-ML, R&D) were used. For in vitro Treg cell induction, 2 x 10⁵ FACS-sorted naive T cells (CD4⁺, CD62L⁺ and CD25^-) were cultured in 200 µl primary T cell medium for 5 days in the presence of 1 µg/mL plate-bound anti-CD3 (145-2C11, BioLegend), 2 µg/mL anti-CD28 (37.51, Biolegend), 10µg/ml anti-IL-4 (11B11, self-purified), 10µg/ml anti-IFNγ (XMG1.2, self-purified), 10 ng/ml murine IL-2 (402-ML, R&D), and 5 ng/mL TGF-β (R&D Systems) in a flat-bottom 96-well plate.