Fitc anti mouse cd8

T cells purified from lungs of day 9-infected CD45.2 BL/6J mice were stained with FITC anti-mouse CD8 and Alexa700 anti-mouse CD45.2 antibodies (BioLegend). The CD8⁺CD45.2⁺ T cell population was sorted on a BD FACSAria III sorter.

CpG ODN 2359 was obtained from Tsingke Biological Technology Co. (Beijing, China). Anti-OX40 agonistic antibody (αOX40, Clone OX-86) was provided by GenScript USA Inc. Murine anti-PD-1 antibody (G4C2) was provided by Shanghai Junshi Biosciences Co.,Ltd (Suzhou, China).Monoclonal antibodies (mAbs) used for flow cytometry were listed as flow: FITC anti-mouse CD45 (Biolegend, USA), PE/Dazzle™ anti-mouse CD3 (Biolegend, USA), FITC anti-mouse CD8 (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse CD4 (Biolegend, USA), FITC anti-mouse CD11c (Biolegend, USA), PE anti-mouse CD86 (Biolegend, USA), PE/Cyanine7 anti-mouse CD80 (Biolegend, USA), PE anti-mouse CD44 (Biolegend, USA), PE/Cyanine7 anti-mouse CD62L (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse PD-1 (Biolegend, USA), PE/Cyanine7 anti-mouse OX40 (Biolegend, USA), FITC anti-mouse CD11b (Biolegend, USA), PerCP/Cyanine5.5 anti-mouse F4/80 (Biolegend, USA), APC anti-mouse CD206 (Biolegend, USA), Mouse Regulatory T cell staining kit (eBioscience, USA).

The following antibodies were used in this study: CD81 rabbit monoclonal Ab (mAb; cat #10037, Cell Signaling Technology, Danvers, MA, USA), Alix rabbit mAb (cat #92880, Cell Signaling Technology), Akt rabbit mAb (cat #4691, Cell Signaling Technology), Phospho-Akt rabbit mAb (cat #4060, Cell Signaling Technology), PI3 Kinase rabbit mAb (cat #4257, Cell Signaling Technology), Phospho-PI3 Kinase rabbit mAb (cat #17366, Cell Signaling Technology), β-Actin rabbit mAb (cat #4970, Cell Signaling Technology), CD9 rabbit mAb (cat #ab92726, Abcam, Cambridge, UK), CD4 rabbit mAb (cat #ab183685, Abcam), CD8 rabbit mAb (cat #ab22378, abcam), CDCP1 rabbit pAb (cat #12754-1-AP, Proteintech, Rosemont, IL, USA), PerCP-anti-mouse CD8 (cat #101406, BioLegend, San Diego, CA, USA), FITC-anti-mouse CD3 (cat #100203, Biolegend), APC-anti-mouse CD4 (cat #103024, Biolegend), PE-anti-mouse IFNγ⁺ (cat #505808, Biolegend), FITC-anti-mouse CD8 (cat #100705, Biolegend).

Lung leukocytes (1 × 10⁶) were stimulated with CD3/CD28 anti-mouse antibodies (2.5 μg/mL ea.) in the presence of 1X brefeldin A (Biolegend) for 8–12 h prior to surface staining with anti-mouse CD45 Pacific blue (clone; S18009F, Biolegend, San Diego, CA, USA), anti-mouse CD3 APC (clone; 145-2C11, Tonbo Biosciences, San Diego, CA, USA), anti-mouse CD8 FITC (clone; 53–6.7, Biolegend, San Diego, CA, USA), anti-mouse CD4 BV605 (clone; GK 1.5, Biolegend, San Diego, CA, USA), anti-mouse CD69 PE/Cy7 (clone; H1.2F3, Invitrogen, CA), and anti-mouse CD103 BV711 (clone; BV711, Biolegend, San Diego, CA, USA). After two washes, cells were fixed in 2% paraformaldehyde and treated with permeabilization buffer (0.5% tween 20 in FACS buffer). Cells were stained with anti-mouse GzmB PE (clone QA18A28; Biolegend, San Diego, CA, USA) and anti-mouse IFN-γ APC-Cy7 (clone; B-XMG1.2, Biolegend, San Diego, CA, USA).

Tumors were extracted and processed as described above before re-suspension in PBS buffer containing 2% FBS and PBS for flow cytometric analysis. Zombie NIR™ Fixable Viability Kit (Biolegend, USA) was applied to cells for 30 min on ice in the dark. Cells were washed and incubated with fluorochrome-conjugated antibody (anti-mouse CD45 Birliant violet 605, BioLegend; anti-mouse CD3 APC, BioLegend; anti-mouse CD8 FITC, BioLegend) at the manufacturer’s recommended dilution for 30 min on ice in the dark. For samples requiring intracellular staining, cells were fixed with Fixation/Permeablization Diluent (eBioscience cat. 00–5223-56) for 30 min at room temperature, washed twice with Permeablization Buffer (eBioscience cat. 00–8333-56), and incubated with antibody (anti-mouse Granzyme B PE, BioLegend; anti-mouse Ki-67 Alexa Fluor 700, BioLegend) in permeabilization buffer for 30 min at room temperature in the dark. Following staining, cells were washed again with permeabilizaton buffer, subsequently washed with PBS, and re-suspended in PBS buffer for flow cytometric analysis on the CytoFLEX LX Flow Cytometer. 50,000–100,000 cells were analyzed per sample per mouse using Beckman CytExpert Software.