Figure 4. Antigen-specific antifungal CD8⁺ T cell responses to SIINFEKL-expressing vaccine yeast.

(A) Schematic diagram illustrating generation of recombinant yeast carrying the OT-I epitope SIINFEKL fused to truncated BAD-1 and fluorescent mCherry. N-term, N terminus; C-term, C terminus. (B) Microscopic visualization of recombinant vaccine yeast. Surface localization of model epitope-mCherry fusion (white arrow). Original magnification, ×60. (C and D) Primary response of OT-I cells after vaccination. Approximately 1 × 10⁶ Thy1.1⁺ OT-I cells were adoptively transferred to naive Thy1.2 mice. A day later, mice were vaccinated s.c. with SIINFEKL yeast (10⁶ CFU). After 14 days, DLN cells were harvested and incubated at 37°C with SIINFEKL peptide or anti-CD3 and anti-CD28 antibodies for 5 hours. Cells were then surface stained with anti-Thy1.1, anti-CD8, and anti-CD44 before staining for intracellular cytokines with anti–IFN-γ and anti–TNF-α antibodies. The percentage of activated (CD44^hi) and cytokine-producing OT-I T cells and polyclonal T cells was enumerated by flow cytometry (C). DLN cells were also directly surface stained using anti-CD8, anti-Thy1.1, anti-CD44, anti-CD62L, anti-CD127, anti–KLRG-1, anti-CD43, and anti-CD27 antibodies and analyzed by flow cytometry (D). Numbers in dot plots indicate the percentage of cells gated on activated (CD44^hi) OT-I and polyclonal T cells. Plots are representative of data collected from 4 mice per group.