Il 2 bv605

Frequencies of S-specific memory T cells in blood and BAL were assessed using a re-stimulation assay as described previously using 2 μg/mL PepMix SARS-CoV-2 Spike overlapping peptides in DMSO (15mers with 11 amino acid overlap, JPT Peptide Technologies)^{51 (link)}. The antibody panel used for surface staining was: CD103 FITC (2G5, Beckman Coulter, cat# B49222, 1:50 dilution), CCR7 BV421 (G043H7, Biolegend, cat# 353208, 1:50 dilution), CD8a BV711 (RPA-T8, Biolegend, cat# 301044, 1:80 dilution), CD4 PE-Cy5.5 (S3.5, Invitrogen, cat# MHCD0418, 1:80 dilution) and CD45RA BV650 (5H9, BD, cat# 740608, 1:500 dilution), and the intracellular proteins were stained using: IL-21 AF647 (3A3-N2.1, BD, cat# 560493, 1:20 dilution), IL-13 PE (JES10-5A2, BD, cat# 559328, 1:33 dilution), IL-2 BV605 (MQ1-17H12, BD, cat# 564165, 1:50 dilution), IL-17A BV785 (BL168, Biolegend, cat# 512338, 1:67 dilution), CD69 ECD (TP.1.55.3, Beckman Coulter, cat# 6607110, 1:67 dilution), CD3 APC-Cy7 (SP34-2, BD, cat# 557757, 1:200 dilution) and IFNγ AF700 (B27, Biolegend, cat# 506516, 1:200 dilution). Acquisition was performed using BD LSRFortessa cell analyzer, and the data were analyzed using FlowJo software v.10.7.1 (FlowJo).

For the intracellular cytokine staining, mouse spleen cells were stimulated with 2 μg/mL S1, S2 peptide pools spanning SARS-CoV-2 spike S1 and S2 respectively (15mers, overlapping by 11aa, GenScript, Nanjing, China) or equimolar amount of DMSO (negative control) in the presence of anti-mouse CD28 antibody (BD Bioscience, CA, USA) for 1 h. GolgiStop protein transport inhibitor (BD Bioscience) was added into the culture and further incubated for 5 h. After stimulation, cells were washed and stained with Fixable Viability Stain 510 (BD Bioscience). Cells were then blocked with anti-mouse CD16/32 and labeled with cell surface antibody cocktail including anti-mouse CD3-FITC, anti-mouse CD4-APC and anti-mouse CD8-Percp-cy5.5 (BD Bioscience) for 30 min at 4°C. Following surface staining, cells were incubated with fixing/permeabilizing solution (BD Bioscience) for 20 min at 4°C and stained with intracellular antibodies including anti-mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (BD Bioscience) for 30 min at 4°C. Cells were washed and resuspended in PBS buffer and analyzed on a CYTEK Aurora/NL.