Anti cd4 bv650

Spleens were harvested, dissociated with collagenase (Sigma), and processed to single-cell suspensions by using a 70-μm cell strainer (BD Biosciences). Total cell numbers were quantified by using a hemocytometer. Cells were then stained with the following antibodies: anti-CD45-BUV395, anti-CD3e-peridinin chlorophyll protein (PerCP)-Cy5.5, anti-CD4-BV650, anti-CD8a-Alexa 700, anti-CD62L-phycoerythrin (PE)-Cy7, anti-CD44-BV510 (all from BD Biosciences), and anti-NK1.1-PE (BioLegend). Flow cytometry data were acquired with an LSRFortessa cell analyzer (BD Biosciences) and analyzed by using FlowJo software (FlowJo, LLC).

CD3⁺ cells were depleted from the freshly thawed PBMCs using EasySep Positive Selection Kits II (Stemcell). Flowthroughs consisting of CD3⁻ cells were collected. The cells (5 × 10⁵) were then pulsed with 5 μg/ml of peptides or DMSO for 1 h in 37°C. After incubation, pulsed cells were washed twice before addition of autologous nasal cells together with anti-CD40L-PE (RRID: AB_314828; BioLegend) and anti-CD107a-APC (RRID: AB_1727417; BD). After 3 h of incubation at 37°C, the cells were washed in PBS stained with Zombie NIR Fixable Viability Kit to exclude dead cells in subsequent analysis. Then, they were washed in FACS buffer and stained with surface markers anti-CD3-BV605 (RRID: AB_2561911; BioLegend), anti-CD4-BV650 (RRID: AB_2744425; BD), anti-CD8-PE-Cy7 (RRID: AB_396852; BD), anti-CD69-AF700 (RRID: AB_493775; BioLegend), and anti-CD103-FITC (RRID: AB_10597744; eBioscience) diluted in FACS buffer for 30 min on ice. After two more washes in FACS buffer, cells were resuspended in PBS before acquisition with a Beckman Coulter CytoFLEX S analyzer.

CD8⁺ T‐cell lines were tetramer stained for 1 h at room temperature. Cells were washed and surface stained with anti‐CD8‐PerCP‐Cy5.5 (BD Biosciences, Melbourne, Australia), anti‐CD4‐BV650 (BD Biosciences), anti‐CD14‐APCH7, anti‐CD19‐APCH7 and Live/Dead Fixable Near‐IR Dead Cell Stain (Life Technologies, Melbourne, Australia). Cells were either fixed with 1% paraformaldehyde and acquired on a CytoFLEX Flow Cytometer or were directly single‐cell sorted for clonal expansion. Plates were centrifuged and stored at −80°C until use.

CD8⁺ T cell lines were generated as previously described^{25 (link),58 (link)}. In brief, PBMCs were incubated with 1 μM of individual peptide (NQK-Q8 or NQK-A8) and cultured for 10–14 days in RPMI-1640 supplemented with 2 mM MEM non-essential amino acid solution (Sigma-Aldrich), 100 mM HEPES (Sigma-Aldrich), 2 mM l-glutamine (Sigma-Aldrich), penicillin–streptomycin (Life Technologies), 50 mM 2-ME (Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (Bovogen). The cultures were supplemented with 10 IU IL-2 2–3 times weekly. CD8⁺ T cell lines were used fresh for subsequent analysis. For the double tetramer staining experiments 0.5 × 10⁶ cells from the CD8⁺ T cell lines were stained with a single PE-conjugated tetramer (HLA-B*15:01-NQK-Q8 or HLA-B*15:01-NQK-A8) or double stained with both tetramers (PE-conjugated NQK-A8 and APC-conjugated NQK-Q8 tetramer) for 1 h at room temperature. Cells were washed and surface-stained with anti-CD3-BV480 (dilution 1:100), anti-CD8-PerCP-Cy5.5 (1:50), anti-CD4-BV650 (1:100), anti-CD14-APCH7 (1:200) and anti-CD19-APCH7 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were single-cell sorted into PCR plates (Eppendorf) using the BD Aria Fusion system.

Cells were resuspended in PBS and stained with Zombie NIR Fixable Viability Kit to exclude dead cells in subsequent analysis. The cells were next washed in FACS buffer with 2 mM EDTA and stained with surface markers anti-CD3-BV605 (RRID: AB_2561911; BioLegend), anti-CD4-BV650 (RRID: AB_2744425; BD), anti-CD8-PE-Cy7 (RRID: AB_396852; BD), anti-CD69-AF700 (RRID: AB_493775; BioLegend), anti-CD103-APC (RRID: AB_10669816; eBioscience), anti-CD45RA-FITC (RRID: AB_395879; BD), and anti-CCR7-BV421 (RRID: AB_2728119; BD) diluted in FACS buffer for 30 min on ice. After two more washes in FACS buffer, cells were resuspended in PBS before acquisition.

Expanded A68/NP₁₄₅⁺ CD8⁺ T cells were stimulated with 1 µM peptide (DATYQRTRALVR, DTTYQRTRALVR, DVTYQRTRALVR or pool of DATYQRTRALVR, DTTYQRTRALVR, and DVTYQRTRALVR) and cultured for 5 h in the presence of 10 U/ml rIL2 and Golgi Stop (BD Biosciences). Following activation, cells were surface stained for 30 min with human anti-CD3-BV510 (1:200, Biolegend #317332), anti-CD4-BV650 (1:200, BD Horizon #563875), anti-CD14-APC-H7 (1:100, BD Pharmingen #560180), anti-CD19-APC-H7 (1:100, BD Pharmingen #560177), anti-CD8-PerCPCy5.5 (1:100, BD Pharmingen #565310), Live/Dead near-infrared (1:800, Invitrogen), and PE-streptavidin-conjugated A68/NP₁₄₅ (DATYQRTRALVR) tetramer. Cells were fixed with BD Fix-Perm buffer (BD Biosciences) for 20 min, before intracellular staining for 30 min with human anti-IFN-γ-V450 (1:100, BD Horizon #560371) in perm wash buffer (BD Biosciences). Cells were washed, acquired on the BD Fortessa (BD Biosciences) and analyzed using the Flowjo software (Treestar, OR, USA).

pHLA class-I (pHLA-I) tetramers were generated by refolding each peptide with its restricted HLA α-heavy chain-BirA and β2-microglobulin^{53 (link),85 (link),86 (link)} before 8:1 conjugation with PE- or APC-streptavidin (BD, cat #554061 and #554067 respectively) to generate tetramers; B7/NP₃₀, B7/NS1₁₉₆, B8/NP₃₀, B8/NP₉₂, B8/NP₄₇₉, B35/HA₂₃₁ and B35/NS1₂₆₀. To test newly generated tetramers, 0.2–0.4 × 10⁶ frozen IBV pool-specific CD8⁺ T cells were thawed in RF10 supplemented with 2 µg/ml DNAse. Cells were incubated with anti-human FcR block (Miltenyi Biotec) for 15 min and stained with tetramer at room temperature for 1 h before surface staining on ice for 30 min with anti-CD3-BV510 (1:200, BioLegend #317332), anti-CD4-BV650 (1:200, BD Horizon #563875), Live/Dead NIR (1:800, Invitrogen #L34976) and anti-CD8-PerCP-Cy5.5 (1:50, BD Pharmingen #565310). Cells were then fixed with 1% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 20 min on ice, acquired on an LSRII Fortessa and analyzed with FlowJo v10.8.1.