Anti cd8 percp cy5

Splenocytes were prepared as described above, plated in 96-well round-bottom plates, and stimulated using peptide pools for EBOV GP, SUDV GP, or MARV GP (as described above) at a final concentration of 5 µg/mL or media only. Stimulation and staining was then performed as described previously [33 (link)] except that the following antibodies were used: anti-CD4-Qdot605, anti-CD127-APCef780 (Invitrogen), anti-CD62L-PeCy7, and anti-CD8-PerCP/Cy5.5 antibodies (eBioscience), as well as LIVE/DEAD^® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific), anti-TNF-Alexa488, anti-IL-2-PE, and anti-IFN-γ-e450 antibodies (eBioscience). Antigen-specific cells were identified by gating based on doublet negative, size, live cells, and either CD4⁺ or CD8⁺ surface expression. Background responses in unstimulated control samples were subtracted from responses of peptide stimulated T cells.

Cultured T cells were stimulated with the cognate SARS-CoV-2 OPPs (ORF1ab-C, ORF3a, N, S, nsp12) and incubated for 4 h in the presence of Golgiplug (BD Biosciences). Following stimulation, cells were washed and stained with anti-CD8-PerCP-Cy5.5 (eBioscience) and anti-CD4-PE-Cy7 or anti-CD4-Pacific Blue (BD Biosciences) for 30 min before being fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences). After 20 min of fixation, cells were washed in BD Perm/Wash buffer (BD Biosciences) and stained with anti-IFN-γ-Alexa Fluor700 or IFN-γ- PE (BD Biosciences) for a further 30 min. Finally, cells were washed again and acquired using a BD LSRFortessa with FACSDiva software. Post-acquisition analysis was performed using FlowJo software (TreeStar; FlowJo LLC, Ashland, Oregon). Cytokine detection levels identified in the no-peptide control condition were subtracted from the corresponding test conditions to account for nonspecific, spontaneous cytokine production.

EBV-associated cancer cells were plated at a density of 1×10⁵ cells/well. After 24 hours, T cells were added at an effector-to-target ratio of 50:1 and the culture was incubated for 24 hours at 37°C and 6.5% CO₂. To assess the impact of T cells on cancer cells, the cultured cells were then incubated at 4°C with the following antibodies: human anti-CD45-V500 (clone HI30, BD Biosciences), anti-CD3-AF700 (clone HIT3a, BioLegend), anti-CD56-BV421, anti-CD8-PerCP-Cy5.5, anti-CD19-APC-Cy7, anti-perforin-PE (clone dG9, eBioscience), anti-granzyme K-FITC (clone G3H69, BD Biosciences), anti-granzyme B-BV711 (clone GB11, BD Biosciences), Ki67-BV421 (clone B56, BD Biosciences), anti-active caspase-3-BV605 (clone C92-605, BD Biosciences) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher Scientific, MA). Flow cytometry was performed using a BD LSRFortessa with FACSDiva software and postacquisition analysis was performed using FlowJo software.

To analyze cell surface proteins levels, 5 × 10⁵ lymphocytes were labeled with the following mAntibodies: anti-B220 FITC, anti-CD4 PE, anti-IFNγ PE (BD Pharmingen™), anti-CD122 PE, anti-CD127 PE, anti-CD25 APC, anti-CD25.AF488, anti-CD3 APC, anti-CD44 FITC, anti-CD62L PE, anti-CD62L PErCP.Cy5.5, anti-CD8 PerCP.Cy5.5, anti-Granzyme B FITC, anti-IgG2a K FITC, anti-IgG2a K PE, anti-Klrg1 FITC (all eBioscience™). For intracellular staining, 1 × 10⁶ cells/ml were stimulated in vitro for 6 h with 10 nM of Phorbol 12-myristate 13-acetate (PMA) plus 1 μM of ionomycin (both from Calbiochem^®). Brefeldin A (1:1000; BD Pharmingen™) was added to the culture for the last 2 h. Cells were harvested and stained with anti-CD44 APC and anti-CD62L PErCP.Cy5.5 antibodies. Then, the cells were fixed, permeabilized, and stained with anti-Granzyme B FITC, anti-IFN-γ PE or anti-IL-2 PE antibodies and analyzed by flow cytometry on a FACSCalibur. For the memory subtype analysis, day 10 memory cells generated in vitro were sorted using a MoFlo Cell Sorter (Beckman Coulter, Inc) based on the CD62L level. All the flow cytometry data were analyzed using FlowJo^® software.

Cultured T cells (5 × 10⁵ per test) or PBMC (2 × 10⁶ per test) were stimulated separately with the SARS-CoV-2 overlapping peptide pools (1 µg/mL of each peptide), individual defined epitopes (1 µg/mL) or with a cytokine stimulation cocktail (eBioscience), and incubated for 4 h (T cells) or 6 h (PBMC) at 37 °C in the presence of GolgiPlug (Brefeldin-A), GolgiStop (Monensin) and anti-CD107a-FITC (BD Biosciences). Following stimulation, cells were washed and stained with anti-CD8-PerCP-Cy5.5 (eBioscience), anti-CD4-Pacific Blue (BD Biosciences) and live/dead fixable near-IR dead cell stain (Life Technologies) for 30 min at 4 °C before being fixed and permeabilized with Fixation/Permeabilization solution (BD Biosciences). After 20 min of fixation, cells were washed in BD Perm/Wash buffer (BD Biosciences) and stained with anti-IFN-γ-Alexa Fluor 700, anti-IL-2-PE and anti-TNF-APC (all from BD Biosciences) for a further 30 min at 4 °C. Finally, cells were washed again and acquired using a BD LSRFortessa with FACSDiva software. Post-acquisition analysis was performed using FlowJo software (TreeStar). Cytokine levels detected in the no-peptide control condition were subtracted from the corresponding test conditions to account for non-specific, spontaneous cytokine production.