Cd8 bv421 53 6.7

Unspecific binding was blocked by incubating cells with Fc receptor-blocking monoclonal antibody (clone 2.4G2; BD Biosciences) for 10 min. Cells were then stained with the following antibodies specifically binding: CD3-PerCP/Cy5.5 (145-2C11; BioLegend), CD4-BV785 (RM4-5; BioLegend), CD8-BV421 (53-6.7; BioLegend), B220/CD45R-APC-Cy7 (RA3-6B2; BioLegend), CD138-BV605 (281-2; BioLegend), CD69-PE (H1.2 F3; BioLegend), and PD-1-PE-Cy7 (29 F.1A12; BioLegend). Dead cells were excluded by ZombieGreen™ (BioLegend) staining, and doublets by scatter analysis. Cells were measured on a LSRII flow cytometer (BD) and analyzed by FlowJo software. In most samples, a minimum of 1 × 10⁵ events were measured.

Antigen-specific OT-I splenocytes were isolated and homogenized into a single cell suspension. Red blood cells were lysed with 0.83% NH₄Cl-Tris Buffer with 1 ml for 5 min at room temperature. OT-I CD8 T cells were pulsed with SIINFEKL (N4) at 2 µg/ml and incubated for 3 days at 37 °C. SIINFEKL peptide was replaced with fresh media containing IL-2 (200-02, Peprotech, 1000 units/ml). Cells were cultured for an additional 3 days. CD8 T cells were isolated using a Ficoll gradient. Effector CD8 T cells were defined as CD8⁺ CD44⁺ cells which were ~95% of the cell population. CD8 T cell cultures were identified and validated using antibodies against CD8 BV421 (53–6.7, Biolegend), CD44 (103018, Biolegend), Vβ5 (MR9-4, Biolegend), and Vα2 (B20.1, Biolegend). For chronic stimulation assays, CD8 T cells were passaged onto plates coated with or without In Vivo Ready Anti-Mouse CD3e (145-2C11, Tonbo Biosciences, 40-0031-U100) and maintained in IL-2.