U bottom tissue culture plate

CD4⁺ T cells were enriched from lung and BAL cells using a negative selection CD4 enrichment kit per manufacturers’ instructions (StemCell). They were then stained with antibodies against CD4, GITR and CD25 alongside Topro3 as a viability dye. Pools of 100,000 live CD4⁺ GITR⁺ CD25⁺ cells were then sorted to >95% purity on a BD Aria III under Bsl2 conditions. Naïve CD4⁺ T cells were isolated from the spleens of uninfected mice using a mouse naïve CD4⁺ T cell isolation kit according to manufacturers’ instructions (StemCell). They were then stained with Tag-it Violet (TV) proliferation and cell tracking dye (Biolegend). 10,000 labelled naïve CD4⁺ T cells were then co-cultured with increasing ratios of CD4⁺ GITR⁺ CD25⁺ up to 1:1 in the presence of 30 U/ml rIL-2 and Mouse activator CD3/28 Dynabeads (ThermoFisher) in 96-well U bottom tissue culture plates (Corning). The cells were then incubated for 5 days in the dark at 37°C. Cells were then washed with PBS and stained with far-red fixable viability dye (ThermoFisher) and resuspended in 200 μl of FACS buffer. Each well was then run at a constant speed and time on a flow cytometer. The number of viable, proliferating effector cells (viability dye negative, TV diluted) in each well was then calculated. Percent proliferating effector cells was calculated versus naïve CD4⁺ T cells stimulated in the absence of Tregs.

Primary human monocytes (CD14⁺) obtained from healthy human donors were purchased and differentiated into dendritic cells as described above. Differentiated MoDC were seeded at 10⁶ cells in 100 uL MoDC media in 96-well U-bottom tissue culture plates (Corning, Inc., Corning, NY, USA). Single-cell suspensions were then stimulated with 10⁴ particles of heat-killed Bifidobacterium breve (Bb15700), Akkermansia muciniphila (AmBAA-835), Fusobacterium nucleatum (Fn23726), Bacteroides fragilis (Bf25285), Faecalibacterium prausnitzii (Fp27766), Enterococcus hirae (Eh8043), Bacteroides fragilis (Bf43858), Fusobacterium nucleatum (Fn25586), or Escherichia coli K-12 (K12), or 1 ug/mL high molecular weight (HMW) polyinosinic:polycytidylic acid (Poly I:C) in 100 uL MoDC media overnight (16–18 h) at 37 ℃, 5% CO₂, without agitation [Escherichia coli K-12 from Sigma-Aldrich, St. Louis, MO, USA; all other strains from ATCC; HMW Poly I:C from InvivoGen, San Diego, CA, USA]. PBS (ThermoFisher Scientific, Waltham, MA, USA) served as a negative control. N = 6 independent donors.

Venous blood was collected in EDTA vacutainers (BD Diagnostics-Preanalytical Systems), the plasma was removed, and cells were processed within 24 h. 2 x 10⁵ PBMCs in 100 μl/well were cultured in RPMI-1640 containing 10% human AB serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES in 96-well U-bottom tissue culture plates (Corning Life Sciences) and stimulated with 2% (v/v) phytohaemagglutinin (PHA, M-form, Invitrogen), 10 ng/ml staphylococcal enterotoxin B (SEB, Sigma-Aldrich) or two concentrations (6,250 ng/ml and 24 ng/ml total protein) of two independently prepared PAO1-L lysates at 37°C, 5% CO₂ for 6 days. On Day 6 PBMCs were re-stimulated with 15 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) overnight, and supernatants harvested for cytokine analysis on Day 7.