Cd8 apc h7 clone sk1

Cell surface staining for flow cytometry was performed using the following antibodies: CD45-BUV395 (clone HI30, BD), CD3-AF700 (clone UCHT1, BD), CD4-APC-H7 (clone RPA-T4, BD), CD8-APC-H7 (clone SK1, BD), TIGIT-PE/Dazzle™ 594 (clone A15153G, Biolegend), TIGIT-BV421 (clone A15153G, Biolegend), and PD1-BV421 (clone EH12.2H7, Biolegend). These antibodies were used to analyze surface receptors in two different panels. Isotype-matched antibodies, labeled with the proper fluorochromes, were used as negative controls. Extracellular staining was performed according to the manufacturer’s instructions. Briefly, an antibody cocktail was added to whole blood or BM samples, which were then incubated at room temperature for 15 minutes in the dark, followed by red cell lysis with lysis buffer (BD; Cat: 555899). The lysed cells were washed twice with phosphate buffer saline (PBS), and then 350 μl stain buffer was added for further flow cytometer analysis. Twenty microliters of absolute count microsphere (Thermo; Cat: C36950) were added to the samples for absolute number analysis. Cells were analyzed using a BD Fortessa flow cytometer (BD Biosciences), and data analysis performed with Flowjo 10.6 software.

Cell surface staining for flow cytometry was performed using the following antibodies: CD3-AF700 (clone UCHT1, BD), CD4-APC-H7 (clone RPA-T4, BD), CD8-APC-H7 (clone SK1, BD) and PD-1-BV421 (clone EH12.2H7, Biolegend). Isotype-matched antibodies, labeled with the proper fluorochromes, were used as negative controls. Cells were analyzed using a BD Fortessa flow cytometer (BD Biosciences), and data analysis was performed with Flowjo 10.6 software as previously described (22 (link)).

The cytokine production profile of HCV-specific T-cell lines was determined by intracellular cytokine staining (ICS) after restimulation with NS3-peptides. For T-cell epitope mapping 0.2×10⁶ expanded cells were either restimulated with NS3-peptide-pool (1 µg/ml/peptide) or without NS3-peptide, in medium containing co-stimulatory αCD28 and αCD49d molecules (2 µg each, BD-Biosciences) and 5% FCS (MP-Biomedicals). After 2 hrs, Brefeldin A (Golgiplug 1∶1000, BD-Biosciences) was added and 16 hours later the surface markers were stained with a panel of fluorochrome labeled antibodies, containing CD3-PacificBlue (clone SP-34-2, (BD-Pharmingen), CD14-PE-TexasRed (clone RMO52, BeckmanCoulter), CD20-PE-TexasRed (clone B9E9, BeckmanCoulter), CD4-PE-Cy7 (clone SK3, BD-Pharmingen) and CD8-APC-H7 (clone SK1, BD-Pharmingen). Stained cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences), followed by staining of accumulated intracellular cytokines with IFNγ-APC (clone B27, BD Pharmingen), IL-2-PE (clone MQ1-17H12, BD Pharmingen) and TNFα-FITC (clone MAb11, BD Pharmingen). Cell staining was analyzed on a FACSAria (BD Bioscience) and DIVA software Version 6.1.1.
To evaluate antigen sensitivity, T-cell lines were restimulated with increasing concentrations of individual peptide ranging from 0.05 to 10 µg/ml before intracellular cytokine production was measured.

The following antibodies were used: CD161-BV605 (HP-3G10, BioLegend), CD11c-BV650 (clone B-ly6, BD Biosciences), CD107a-BV711 (clone H4A3, BioLegend), CD4-BV786 (clone SK3, BD Biosciences), Vα7.2-AF-647 (clone 3C10, BioLegend), HLA-DR-AF-700 (clone L243, BioLegend), CD8-APC-H7 (clone SK1, BD Biosciences), IFNγ-V450 (clone B27, BD Biosciences), CD40-PerCp-Cy5.5 (clone 53C, BioLegend), CD3-ECD (clone UCHT1, Beckman Coulter), and PD1-BV711 (Clone EH12.2H7, BioLegend). Aqua fluorescent dye (Invitrogen) was used as a viability dye.
Following stimulation, cells were stained for viability with the Aqua fluorescent dye and incubated for 10 min at room temperature. After viability staining, surface staining was performed with CD161, Vα7.2, CD4, and CD40. Cells were then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) followed by intracellular staining with CD3, CD8, CD14, CD11c, HLA-DR, and IFNγ. The stained samples were acquired on a BD LSR Fortessa FACSDiva Software. A total of 500,000 events were collected and results analyzed using FlowJo (version 9.9.6, FlowJo LLC). We defined MAIT cells as CD3⁺CD161⁺Vα7.2⁺ T cells, after excluding doublets and dead cells.

For the suppression assay, the following antibodies were used: CD3 (Pacific Blue, clone UCHT1, BioLegend), CD8 (PE, clone RPA-T8,BDPharmingen) and CD69 (APC, clone FN50, BioLegend). The percent infection was assessed by quantifying GFP expression using the following formula:
$\left[1-\left(\%\text{GFP}+\text{CD}4+\text{T}\text{cells}\text{cultured}\text{with}\text{CD}8+\text{Tcells}\right)/\left(\%\text{GFP}+\text{CD}4+\text{T}\text{cells}\text{without}\text{effectors}\right)\text{x}100\%\right].$
The following markers were used to assess cell death, immune activation, and exhaustion: CD3 (PE, clone UCHT1, BD Pharmingen), CD8 (APC-H7, clone SK1, BD Biosciences), CD69 (APC, clone FN50, BioLegend), 7AAD (Read in PerCP/Cy5.5, BD Pharmingen), Annexin V (Read in V450, BD Biosciences) and PD1 (PerCP/Cy5.5, clone EH12.2H7, BioLegend)CD25 (FITC, clone M-A251, BD Pharmingen), HLA-DR (PE, clone L243, Biolegend) and Ki67 (APC, clone Ki67, Biolegend). Proliferation was assessed using the CellTrace cell proliferation kit from Invitrogen, and read in the FITC channel.