Lsr fortessa 4

Staining buffer was PBS supplemented with 0.5% FCS and 4 mM EDTA. Whole blood collected in Lithium Heparin vacutainers was lysed and fixed with BD FACS lysing solution (Becton Dickinson) and lymphocytes permeabilized with BD FACS permeabilising solution 2 (Becton Dickinson) according to the manufacturer’s instructions. Cells were then resuspended in 50 μl of staining buffer containing anti-human CD4-BV510 (Becton Dickinson, 1:200 dilution), anti-human CD8-APC-H7 (Becton Dickinson, 1:400 dilution), anti-human CD19-PE-Cy7 (Biolegend, 1:200 dilution), anti-human CD38-APC (Biolegend, 1:400 dilution), anti-human Perforin-PE (Biolegend, 1:400 dilution), anti-human Granzyme B-Pacific Blue (Biolegend, 1:400 dilution) and 1 μl of human Fc receptor blocking solution (Human TruStain FcX, Biolegend) for 30 minutes at room temperature, washed and resuspended in staining buffer before acquisition on LSR Fortessa 4 (Becton Dickinson) with Diva software. FlowJo software version 6.0 was used for gating.

Approximately 5x10⁵ cells were washed in PBS, resuspended in 100 μl PBS containing 0.1 μl Zombie Yellow fixable viability dye (Biolegend) and incubated for 15 mins at 4°C. Cells were then washed, resuspended in 20 μl of staining buffer containing surface antibodies at previously determined optimum dilution along with 1 μl of human Fc receptor blocking solution for 20 mins at 4°C, fixed in 50 μl of 1% paraformaldehyde solution for 15 mins at room temperature, washed, resuspended in 15 μl of BD Perm/Wash buffer 1x (BD Biosciences) containing intracellular antibodies at previously determined optimum dilution for 30 mins at 4°C, washed with BD Perm/Wash 1x solution, and resuspended in staining buffer. Samples were acquired using a LSR Fortessa 4 (BD Biosciences) with Diva software, and analyzed using FlowJo software (version 6.0).

FACS analysis of peritoneal lavage cells used the LSR Fortessa 4 (BD Biosciences) and FACS sorting the MOFLO XDP (Beckman Coulter, Inc.), with analysis undertaken using FLowJo vX.0.7 software. Antibodies used were APC anti-mouse CD11b (Cat 101212, clone M1-70, IgG2b, Biolegend), isotype control APC Rat IgG (Cat 400612, clone RTK4530, IgG2b, Biolegend), FITC anti-mouse F4/80 (Cat MCA497FB, clone CI:A3-1, IgG2b, AbD Serotec), and isotype control FITC Rat IgG (Cat 400505, clone RTK2758, Biolegend).

200 μl of whole blood was stained with anti-human CD4-BV510, anti-human CD8-APC-H7 and anti-human CD38-APC at previously determined optimum dilution for 15 mins at 37°C with or without recombinant human IFN-α2 (Biolegend) or recombinant human IFN-γ (ProSpec) at 100 ng/mL. Whole blood was lysed and fixed with Phosflow Lyse/fix 1x solution (BD Biosciences) and lymphocytes permeabilized with BD Perm III solution (BD Biosciences) according to the manufacturer’s instructions. Cells were then resuspended in 50 μl of staining buffer containing anti-human pSTAT1-PECF594, anti-human pSTAT4-AF488 and anti-human pSTAT5-PE at previously determined optimum dilution for one hour at room temperature. Cells were washed and resuspended in staining buffer. Samples were acquired using a LSR Fortessa 4 (BD Biosciences) with Diva software, and analyzed using FlowJo software (version 6.0).

Whole blood collected in Lithium Heparin vacutainers was lysed and fixed with BD FACS lysing solution (BD Biosciences) and lymphocytes permeabilized with BD FACS permeabilizing solution 2 (BD Biosciences) according to the manufacturer’s instructions. Cells were then resuspended in 50 μl of staining buffer containing surface and intracellular antibodies at previously determined optimum dilution along with 1 μl of human Fc receptor blocking solution (Human TruStain FcX, Biolegend) for 30 mins at room temperature, washed and resuspended in staining buffer. Samples were acquired using a LSR Fortessa 4 (BD Biosciences) with Diva software, and analyzed using FlowJo software (version 6.0).

Sorted CD38⁺ and CD38^- CD4⁺ T cells were plated at 50,000 cells/well in RPMI 1640 containing 25 mM Hepes, 2 mM L-glutamine, supplemented with 10 units/mL of Penicillin, 10 μg/mL of Streptomycin and 10% fetal bovine serum and stimulated with 5 ng/mL of phorbol myristate acetate and 500 ng/mL Ionomycin for 5h at 37°C in an atmosphere of 5% C0₂. After one hour, Brefeldin A (GolgiPlug, BD Biosciences) was added at 1 μg/ml. Following stimulation, cells were washed, resuspended in 20 μl of staining buffer containing anti-human CD4-BV510 and anti-human CD8-APC-H7 at previously determined optimum dilution for 20 mins at 4°C, fixed in 50 μl of 1% paraformaldehyde solution for 15 mins at room temperature, washed, resuspended in 15 μl of BD Perm/Wash buffer 1x (BD Biosciences) containing anti-human IFN-γ-FITC at previously determined optimum dilution for 30 mins at 4°C, washed with BD Perm/Wash 1x solution, and resuspended in staining buffer before acquisition on LSR Fortessa 4 (BD Biosciences) with Diva software. FlowJo version 6.0 was used for gating.

Sorted CD38⁺ and CD38^- CD4⁺ T cells were plated at 50,000 cells/well in RPMI 1640 containing 25 mM Hepes, 2 mM L-glutamine, and supplemented with 10 units/mL of Penicillin, 10 μg/mL of Streptomycin and 10% fetal bovine serum in a 96-well plate pre-coated overnight with 10 μg/mL of anti-human CD3 OKT3 antibody (Biolegend) and incubated for 5 h at 37°C in an atmosphere of 5% C0₂. Following stimulation, cells were resuspended in 20 μl of staining buffer containing anti-human CD4-BV510, anti-human CD8-APC-H7, anti-human CD69-AF700 at previously determined optimum dilution for 20 mins at 4°C, washed and resuspended in staining buffer before acquisition on LSR Fortessa 4 (BD Biosciences) with Diva software. FlowJo version 6.0 was used for gating.