Ccr7 bv421

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 phenotypic analysis of NK cells, PBMC were stained with the following fluorochrome conjugated antibodies or isotype matched controls: CD3-Cy5.5/PerCP or CD3/Pe-Cy7, (eBioscience, Hatfield, UK), CD56-PE Texas Red (Beckman Coulter, High Wycombe, UK), CD56-FITC, CD16-APC Cy7, HLA-DR V500, CXCR3-Cy.5./PerCP, CD57-BV605, NKp46-V450, TRAIL-PE, (BD Biosciences, Oxford, UK), TRAIL-BV421, CD62L-AF700, CXCR6-PE, CD69-APC, CCR7-BV421, KLRG1-FITC, (Biolegend, London, UK), NKG2A-APC, NKG2C-Cy5.5/PerCP, NKG2D-PE (R&D systems, Abingdon, UK), NKp30-APC, NKp44-PE (Miltenyi Biotec, Surrey, UK), in the presence of fixable live/dead stain (Invitrogen, Paisley, Scotland). For phenotypic analysis of T cells PBMC were stained with the fluorochrome conjugated antibodies or isotype matched controls: CD3/Pe-Cy7, CD8-AF700, CD4-APC Cy7 (eBioscience, Hatfield, UK), CD38-PE Texas Red, CD14-V500, CD19-V500 (Biolegend, London, UK). Flourescence minus-one (FMOs) were used for gating purposes for all flourochromes; an example of the gating strategy is shown in S1A Fig. Cells were acquired on a FACS LSRII multicolour flow cytometer (Beckton Dickinson) and analysed using Flow Jo analysis software (Tree star, Ashland, OR, USA). In addition to percentage, absolute numbers of NK cell subsets were calculated by multiplying their percent by total lymphocyte count.

Directly conjugated antibodies were acquired from the following: (1) BD Biosciences: CD3-H7APC, CXCR5-Alexa488 (RF8B2), CCR7-Alexa700, IgG-APC, IFN-γ-Alexa700 and IL-21-Alexa647 (3A3-N2.1) (2) Beckman Coulter: CD45RO-ECD and CD27-PC5 (3) Biolegend: CCR7-BV421, CCR6-PE (TG7/CCR6), CCR6-Alexa647 (TG7/CCR6), CD20-BV570, CD150-PE, IL-2-BV605, IL-17a-Cy5.5PerCP and CD154-Cy5PE (4) Invitrogen: CD4-Cy5.5PE, CD27-QD655, CD27-QD605 and CD19-PacBlue (5) Southern Biotech: IgD-FITC and IgD-PE (6) eBioscience: cmaf-eFluor660 (sym0F1), CXCR5-PerCP-efluor710 (MU5UBEE). Biotinylated anti-PD-1 was from R&D and streptavidin-Cy7PE (or QD655) was from Molecular Probes. The following antibodies were conjugated in our lab: CD19-QD705 and CD57-QD565. Quantum dots and Aqua amine viability dye were obtained from Invitrogen.

Cells isolated from spleen, lungs or peripheral blood were cultured with 2 µg/ml of two immunodominant peptides (Pepscan, Lelystad, The Netherlands), [SSTHEANTMAMMARDT] and [AGYAGTLQSLGAEIAV] of the TB10.4 protein, previously demonstrated to stimulate both CD4⁺ & CD8⁺ T cell responses (Kaveh & Hogarth, unpublished); 1 µg/ml anti-CD28 (BD Biosciences) and 10 µg/ml Brefeldin A (Sigma) for 16 h at 37 °C/5% CO₂. Cells were washed at 300 g for 5 min and surface stained with combinations of CD62L-FITC, CD27-PerCP-Cy5.5, CD8-AF700, CD44-BV421, CD127-PE-Cy7, CD69-FITC, CCR7-BV421, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). Cells were then washed, treated with BD Biosciences Cytofix/Cytoperm as per manufacturer’s instructions and stained intracellularly with combinations of IFN-γ-PE-Cy7, IL-2-APC (both eBioscience), IFN-γ-BV605 and TNF-α-BV605 (both BioLegend). Cells were washed again and analysed using an LSRFortessa™ analyser utilising a 532 nm laser for PE and PE-conjugate excitation with FACSDiva™ software (BD Biosciences). Final analysis was performed using FlowJo® software (Tree Star Inc.) on a minimum of 100,000 live lymphocytes (50,000 for peripheral blood).

Directly after Ficoll-Paque isolation, between 5∙10⁷ and 20∙10⁷ PBMCs were stained with CD95-FITC, CD4-APC-eF780 (both eBioscience), CD3-PerCP, CCR7-BV421, CD8a-BV510, and CD45RO-PE-Cy7 (all from BioLegend), and either CD56-APC, CD56-PE, or CD56-PE/Dazzle-594 (the first purchased from BD Biosciences and the latter two from BioLegend). T-cell subpopulations were defined as follows: truly naive, T_TN (CCR7⁺CD45RO^-CD27⁺CD95⁺), central memory, T_CM (CD45RO⁺CD27⁺), effector memory, T_EM (CD27^-CD45RO⁺) and effector memory re-expressing RA, T_EMRA (CD27^-CD45RO^-). The full gating strategy can be found in S1 Fig. Cells were sorted on a FACSAria II or FACSAria III sorter (BD Biosciences), and data was analyzed with the BD FACSDiva v8.0.1 and FlowJo V10 software.