Cd4 bv711 clone rm4 5

TIICs were stained with 5 μM live/dead fixable violet dye (Thermofisher) followed antibody staining with Tim3-PE (clone-RMT3-23; biolegend; Catalog number-119703); CD4-BV711 (clone-RM4-5, biolegend, Catalog number-100549), CD8-PerCP (clone-53-6.7, biolegend, catalog number-100731) at pretitrated concentrations. Stained cells were fixed with 4% paraformaldehyde prior to running them on BD Fortessa. FCS files were analyzed on FlowJo (version 10.3.1).

Leukocytes from previously immunized (NP, NP-Tag, CpG + Tag, and CpG-NP-Tag) mice were restimulated with tumor cells. A total of 5 × 10⁴ 4T1 tumor cells were seeded in a flat-bottom 24-well plate overnight, and subsequently, 1 × 10⁶ of leukocytes were added and incubated at 37 °C with 5% CO₂. After one hour of incubation, Golgi plug (BD biosciences)—the protein transport inhibitor containing brefeldin A, was added. Unstimulated pneumocytes were used as the negative control. After 5 h of incubation, pneumocytes were harvested and suspended in FACS buffer to stain with the fluorochrome antibody cocktail: CD3-APC (clone 145-2C11; Tonbo Biosciences, San Diego, CA), CD4-BV711 (clone RM 4-5; Biolegend, San Diego, CA, USA), CD8-BV650 (clone 53-6.7; Biolegend, San Diego, CA, USA), CD69-APC-CY7 (clone H1.2F3; Tonbo Biosciences, San Diego, CA, USA), CD103-FITC (clone QA17A24; Biolegend, San Diego, CA, USA). For the staining of intracellular granzyme B (GZMB), cells were fixed and permeabilized with BD cytofix/cytoperm (BD biosciences), according to the manufacturer’s instructions, followed by intracellular staining with granzyme B-PE (clone QA18A28; Biolegend, San Diego, CA, USA) and data acquired as outlined in Section 2.10.