Kaluza 1.3 analysis software

The following mAbs were used: anti-CD3 (17A2 and 145-2C11), anti-TCRβ (H57-597), anti-CD8 (53-6.7), anti-CD49b (DX5), anti-phospho-S6 (Ser-235/Ser-236), and anti-IL-18Rα (P3TUNYA) from eBioscience; anti-CD19 (1D3), anti-CD4 (RM4-5), anti-F4/80 (T45-2342), anti-NK1.1 (PK136), anti-Ki-67 (B56), and anti-BrdU (3D4) from BD Biosciences; anti-CD71 (RT7217), anti-CD98 (RL388) from Biolegend; and Live/Dead Fixable Yellow Dead Cell Stain from Invitrogen. Intracellular staining of Ki-67 was carried out using a Foxp3 staining kit (eBioscience). Mitochondrial mass was measured using nonylacridine orange (NAO) (Thermo Fisher Scientific). Cells were acquired using BD LSRFortessa or Thermofisher Attune NxT and analyzed using Kaluza 1.3 Analysis software (Beckman Coulter) or FlowJo (V10).

To measure the cytokines level in blood samples, cardiac puncture procedure was applied to collect blood samples. All sacrificed procedures were done under Schedule 1 of Animal Scientific Procedures Act 1986. Interferon gamma (IFN-γ), Monocyte chemoattractant protein 1 (MCP1) Interleukin 12 (IL-12p70), Interleukin 10 (IL-10), Interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) by using the BD Cytometric Bead (Biosciences.BD, UK). The blood samples were collected from infected male and female mice at day 6 post-infection in addition to uninfected male and female mice. Following the manufacturer’s recommended protocol, the assay was carried out. Acquisition was undertaken using a BD FACSDiva™ software. (BD Biosciences, Oxford, UK), and results determined using Kaluza 1.3 Analysis Software (Beckman Coulter, High Wycombe, UK).

For in vitro assays, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Lymphoprep) density-gradient centrifugation and incubated on plates coated with anti-CD3 (3 μg/ml) and anti-CD28 (1 μg/ml) mAbs (Biolegend) or with IL-2 (100 U/ml) and IL-15 (10 ng/ml)(PeproTech EC Ltd, London, UK) for activation of CD8⁺ T and NK cells, respectively. CD107a-FITC (clone H4A3) antibody was added to the wells followed by monesin (6 μg/ml, Biolegend) and cultured for 6 h before analysis on a Gallios Flow Cytometer using Kaluza 1.3 analysis software (Beckman Coulter, CA, USA). For IFN-γ production, activated CD8⁺ T lymphocytes were treated for 5 h with brefeldin A (10 μg/ml, Biolegend), and cell surfaces were stained with CD8-PE and CD3-PerCP mAbs, followed by intracellular staining with anti-INFγ-FITC for 30 min at 4°C. Similarly, PBMCs were incubated with CD8-PE, CD3-PerCP and CD16-APC mAbs, followed by intracellular staining with granzyme B-PE and perforin-FITC mAbs (Biolegend). In both cases, a fixation / permeabilization kit (Immunostep, Salamanca, Spain) was used and the 7-Aminoactinomycin D (7AAD) fluoresecent marker (Biolegend) was included to excluded dead cells from the analysis. Cells were further analyzed by flow cytometry.

The following mAbs were used: anti-CD3 (17A2 and 145-2C11), anti-TCRβ (H57-597), anti-CD49b (DX5), anti-CD43 (1B11), anti-Ly49H (3D10), anti-CD25 (PC61), CellTrace Violet stain, anti-phospho-S6 (S235/S236) (CUPK43K), anti-CD45.1 (A20), and anti-CD45.2 (104) from eBioscience; anti-NK1.1 (PK136), anti-Ki-67 (B56), anti-CD69 (H1.2F3), anti-CD120a (55R-286), anti-CD120b (TR75-89), anti-IFN-γ (HXG1.2), anti-CD107a (1D4B), anti-NFκB-p65 (pS529), and anti-BrdU (3D4) from BD Biosciences; anti-CD71 (RT7217), anti-CD98 (RL388) from BioLegend; and Live/Dead Fixable Yellow Dead Functional grade TNFα (MP6-XT22) and NKp46 (29A1.4) monoclonal antibodies were obtained from eBioscience. Intracellular staining of Ki-67 was carried out using a Foxp3 staining kit (eBioscience). Cells were acquired using BD LSRFortessa or ThermoFisher Attune NxT and analyzed using Kaluza 1.3 Analysis software (Beckman Coulter) or FlowJo (V10). Control anti-mouse IgG2a kappa isotype antibodies or unstained cells for a particular antibody were used as FMO. Intracellular staining with anti-phospho-S6 (S235/S236), anti-CD107a, and anti-IFN-γ antibodies was performed using BD Cytofix/Cytoperm protocols (BD Biosciences).