70 m strainer

Cell suspensions from murine brachial lymph nodes and spleens were generated by mashing through a 70 µM strainer (Thermo Fisher). In spleen suspensions, erythrocytes were lysed by 2 min of incubation in ammonium chloride-based lysis buffer (BD Biosciences). Cells were then counted and plated (1–2) × 10⁶ per well in a flat-bottom 96-well microtiter plate. Cells were incubated with or without recombinant RSV pre-F antigen (1 μg/mL) in 200 μL RPMI 1640 + 10% (v/v) heat-inactivated fetal calf serum (Hyclone), 5 × 10⁻⁶ M β-mercaptoethanol, 1% (v/v) penicillin-streptomycin, 1% (v/v) sodium pyruvate, 1 mM L-glutamine, 100x dilution of non-essential amino acids and 10 mM HEPES (all from Life Sciences). Cells were either incubated for 72 h for measurement of secreted cytokines, or for 24 h in the presence of 0.1% (v/v) BD GolgiPlug (BD Biosciences) for flow cytometry.

After sacrifice, spleen and lung samples were mashed through 70 µm strainer (22363548, Fischer scientific) to a new well. The strainer was washed with MAC buffer (2mM EDTA, 0,5% BSA, 1 x PBS). Isolated cells were incubated for 5 min RT with red blood lysis buffer (420301, Biolegend). The reaction was stopped with 1 x PBS. 1 x 10⁶ cells were spined down (5 min, 500 x G) and resuspend in 2% BSA, 1 x PBS, 2 µL TruStain FcX (101320, Biolegend). Total cellular fraction isolated from lungs were analyzed from the presence of lymphocytes. Specifically, the isolated cells were incubated with anti-CD8 and anti-CD19 antibodies according to manufacturer’s recommendations (Table S2). Isolated spleen cells were incubated with conjugated CD19/CD3 antibodies (Table S2) for 1 h at 4°C in dark. Blood was drawn with intracardiac punctures into anti-coagulated K2E tubes (BD Microtainer, 1307939). Whole blood was stained with conjugated anti-CD19 antibody for 1 h at 4°C in dark (Table S2). All samples were washed with cell staining buffer (BioLegend, 420201) and centrifugated for 5 min at 500 g. Cell pellet was resuspended in 500 µL of cell staining buffer. The presence of CD19-positive cells was analyzed using flow cytometry (BD LSRFortessa, BD Biosciences). The data was analyzed with Flowing Software 2.5.1 (Turku, Finland).

Briefly, peritoneal fluid was collected in cold FACS and processed into single cell suspension by using RBC lysis buffer (Sigma-Aldrich, USA) for about 60 seconds and then washed with cold FACS. Then all samples were filtered with 70µm strainer (ThermoFisher, USA), centrifuged at 4°C, 1000 RPM for 10 minutes and then the cell pellets were resuspended in cold FACS. To determine the T cell and natural killer (NK) subset, isolated cells were stained with anti-CD3-APC and NK1.1-BB515 antibodies (Biolegend, USA), respectively. Finally, the stained samples were analyzed by using BD-FACS Celesta flow cytometry system (BD, USA) and acquired data were visualized by using built-in Diva software (BD, USA) as described before (41 (link)).

PDX-bearing mice were euthanized, and tumors harvested. Tumors were minced and incubated in collagenase (225 U/mL; BioShop Canada Inc., Burlington, ON, Canada) in HBSS, Thermo Fisher Scientific, Mississauga, ON, Canada) at 37 °C on an end-over-end shaker. After 2 h, cell suspension was passed through a 70 µm strainer (Fisher Scientific) and centrifuged for 5 min at 500× g. Cells were resuspended in red blood cell lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA). After 5 min, cells were centrifuged, resuspended in PBS, and passed through a 70 µm strainer. Cells were centrifuged, resuspended in Aldefluor buffer (Stem Cell Technologies, Inc., Vancouver, BC, Canada), and passed through a 70 µm strainer. Approximately 1 × 10⁷ cells were incubated with anti-H-2Kd (1:1000 SF1-1.1, BioLegend, San Diego, CA, USA) at 37 °C with shaking. After 1 h, cells were centrifuged and resuspended in Aldefluor buffer with 7-AAD (1:10, Biolegend). Stained cells were gated on SSC and FSC to eliminate doublets. 7-AAD^- H-2Kd^- cells were sorted into ice-cold PBS with 5% BSA (Sigma-Aldrich). H-2Kd purity was assessed (FACS Aria, BD Bioscience, San Jose, CA, USA).

CD19 magnetic‐bead‐selection and enrichment of cells of interest was performed with the RoboSep‐S instrument (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's protocol. Briefly, the sample was diluted 5‐fold using RoboSep dilution buffer (STEMCELL Technologies, Vancouver, Canada), mixed gently, and spun at 300 g for 10 minutes, followed by the aspiration of the supernatant. The sample was then diluted to the original volume with RoboSep buffer and mixed well. If required, the sample was filtered through a pre‐wetted 70 µm strainer (Fisher Scientific, Waltham, MA) to remove bone fragments and cell aggregates or debris. The sample was diluted 1:1 in EasySep RBC buffer (STEMCELL Technologies, Vancouver, Canada), transferred to a 14 ml centrifuge tube, and processed on the RoboSep‐S instrument using the RoboSep HLA Chim WB CD19 Positive Selection Kit (STEMCELL Technologies, Vancouver, Canada). In‐house magnetic‐bead selection achieved CD19+ cell purities within the stated range of the manufacturer (94.3%–99.6%).