Cd8 t cell

Groups of mice (5/group) were IP vaccinated with znBMZ, S19, or DPBS, as described above, and evaluated 42 days after vaccination. Ex vivo intracellular IFN-γ expression by immune T cells were measured by flow cytometry similarly to previously described [35 (link),36 (link)]. Lymphocytes were then stained for TCRβ, CD4, CD8 T cell, CD44, CX3CR1, and KLRG1 mAbs (BioLegend, San Diego, CA, USA; eBioscience, San Diego, CA, USA), washed, and then fixed with IC Fixation Buffer (eBioscience). Subsequently, cells were permeabilized with Permeabilization Buffer (eBioscience), and stained with labeled anti-IFN-γ (eBioscience). Fluorescence was acquired on Cytek Auora flow cytometer using SpectroFlo^® software version 2.0 (Fremont, CA, USA). All samples were analyzed using FlowJo software version 10.8.1 (Tree Star, Ashland, OR, USA).
To measure soluble proinflammatory cytokine production by each treatment group, isolated splenic mononuclear cells (3 × 10⁶) in 1.0 mL of CM were restimulated with 10⁹ CFUs of heat-killed RB51 (HKRB51) at 37 °C under 5% CO₂. After three days of in vitro stimulation, culture supernatants were harvested for cytokine analyses. IFN-γ, TNF-α, and IL-17 were measured by cytokine-specific sandwich-based ELISAs using the relevant monoclonal Abs for detection as previously described [37 (link),38 (link)].

Different subtypes of mononuclear cells in the tonsil of each participant were separated by gradient centrifugation at 400 × g for 30 min at 4°C and enriched using magnetic beads. In brief, the tonsil tissues were cut into small pieces and ground gently with the plunger of a 5 ml syringe until single-cell suspensions were obtained. Samples were filtered through nylon mesh to remove debris, then the tonsil cell suspensions were centrifuged at 300 × g for 10 min at 4°C. The erythrocytes in the cell suspensions were eliminated using a lysis solution (Solarbio, Beijing, China). The CD4⁺ T cells, CD8⁺ T cells, B cells and monocytes from the tonsil single-cell suspensions were isolated by using the MojoSort™ Human CD4 T cell, CD8 T cell, pan B cell, and Pan Monocyte Cell Isolation Kit (Cat No. 480009, 480011, 480081, 480059, BioLegend Inc. San Diego, CA, USA), respectively, according to the manufacturer’s instructions.