Cd8 apc h7

Freshly isolated PBMC from CCLm group were stimulated with PMA (50 ng/ml)/ionomicyn (10⁻⁶M) for 6 h (positive control) or SLA (10 μg/ml) or rLmlRAB or rLmlRABC (10 μg/ml) for 120 h, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 h of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 min at 4 °C. For intracellular IFN-γ detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer’s instructions and labelled with PE-anti-IFN-γ mAb (intracellular formulation) (BD Biosciences). BD™ CompBeads Set Anti-Mouse Ig, κ (Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) was used for compensation controls. Analysis was performed with DIVA software.

PE-IL-4, PE-IL-17A, APC-IFN-γ, PE-granzyme B, APC-perforin (all from BioLegend, San Diego, CA 92121, USA), PerCP-anti-CD3, PerCP-anti-CD4, APC-H7-CD8, PE-TCRαβ, APC-TNF-α, FITC/PE -TCRγδ and PE-TCRVδ2 (all from BD Pharmingen), APC-NKG2D (R&D, Minneapolis, MN 55413, USA), FITC-TCRVγ9 and PE-TCRVδ2 (both from Thermo Scientific; Darmstadt, Germany), APC-FasL (Miltenyi Biotec), and FITC-Fas (Immunotech, Praha, Czech Republic) were used according to the manufacturer’s instructions. For intracellular staining of cells, the FIX & PERM^® Cell Permeabilization Kit (ADG Bio Research, Diepoldsau, Switzerland) was used. Cells were cultured at 37 °C for 1 h in 96-well plates in 200 μL culture medium in the presence of 1 μg Brefeldin A before staining. Samples were measured using a BD FACS CantoII flow cytometer and analyzed using Flow Jo Software.

Freshly isolated PBMC were stimulated with PMA (50 ng/ml)/ionomicyn (10⁻⁶ M) or PHA at 10 µg/ml for 6 h (positive control) or with TSLA at 10 µg/ml or with LaPSA-38S at 10 µg/ml for 120 h and, or kept with medium alone. Cells were treated with Golgistop (BD Biosciences) for the last 6 hours of culture, then washed and incubated with antibodies: FITC CD3, PerCPcy5.5 CD4, APC-H7 CD8 or PerCPcy5.5 CD8, and PE-Cy7 CD69 or PE CD69 (BD Biosciences), for 20 minutes at 4°C. For intracellular IFN-γ detection, cells were fixed and permeabilized using BD Cytoperm/cytofix plus kit (BD Biosciences) according to manufacturer's instructions and labeled with PE-anti-IFN-γ mAb (intracellular formulation) (BD Biosciences). Analysis was performed with FACSCalibur using CellQuest Pro software or FACS canto II flow cytometer using DIVA software. FITC mouse IgG1, PerCPCy5.5 mouse IgG1, mouse IgG1PE, PE mouse IgG2a (intracellular formulation) (Biosciences) were used as isotype controls for acquisition by FACSCalibur. For acquisition by FACSCanto, BD CompBeads Set Anti-Mouse Ig, κ (Anti-Mouse Ig, κ/Negative Control (FBS) Compensation Particles Set) (BD Biosciences) were used.

The characterization of PBMCs that were co-cultured for 1 hour with Vero E6 and TZM-bl cell monolayers was performed by staining with the following conjugated antibodies purchased from BD Biosciences (San Jose, CA): CD3-PE, CD8-APC-H7, CD56-BV605, TCRγδ-FITC, and CD107a-PE-Cy7. CD107a was used as a degranulation marker and its expression at the cell surface peaks within 1 hour of target cell engagement (35 (link)), being afterwards actively recycled from the cell surface (36 (link)). CD3+CD8- were assumed to be CD4+ T cells, which included those cells with downregulated CD4 expression caused by HIV-1 infection (37 (link)). Isotype controls were used to determine the background signal. Data acquisition was performed with BD LSRFortessa X-20 flow cytometer (BD Biosciences) and data analysis with FlowJo software v10.0.7 (Tree Star Inc.). Gating strategy is shown in Supplementary Figure 1.

Vero E6 cells were infected with equal amounts (100 ng p24 Gag/well) of the most important variants of SARS-CoV-2 within clade 19B that were circulating in Spain at the time of the study: D614 and G614 [24 (link)]. Both pseudotyped viruses pNL4-3Δenv_SARS-CoV-2-SΔ19(G614)_Ren and pNL4-3Δenv_SARS-CoV-2-SΔ19(D614)_Ren were incubated with Vero E6 cells for 48 h. Then, Vero cells were co-cultured for 1 h with PBMCs isolated from the participants (ratio 1:10). Vero cells were detached with trypsin-EDTA solution (Sigma Aldrich-Merck, Darmstadt, Germany), and induction of cytotoxicity was measured using Caspase-Glo 3/7 Assay system (Promega) to evaluate the PBMCs-induced activation of caspace-3 in the monolayer. Viral infection in Vero E6 cells was also determined by measuring Renilla with Renilla Luciferase Assay kit (Promega) in Centro XS3 LB 960 luminometer (Berthold Technologies).
PBMCs were collected previous to detach the Vero E6 monolayer to evaluate the presence of the following cytotoxic cell populations: Natural Killer (NK) (CD3⁻CD56⁺CD16^±), NKT-like (CD3⁺CD56⁺), and TCRγδ (CD3⁻CD8^±TCRγδ⁺) cells by using specific conjugated antibodies: CD3-PE, CD8-APC H7, TCRγδ-FITC, CD56-BV605, and CD16-PercP (BD Biosciences). Analyses were performed using a BD LSRFortessa X-20 flow cytometer and FlowJo software version 10.7.1 (Tree Star Inc.).