(A) On D14, T cells were harvested and stained with anti-CD8 and M1 tetramer, then assayed for inhibitory receptor expression. Representative histograms shown were gated on CD8⁺, M1 tetramer⁺ population. Red: 10 μM. Blue: 10 nM. Yellow: 10 fM. High antigen concentration–induced M1-specific T cells (10 μM) showed higher inhibitory receptor expression except for BTLA. (B) Real-time qPCR analysis of BATF expression level in D14 sorted M1-specific cells. (C) Blockade of PD-1 signaling by anti–PD-1 antibody (clone EH12.2H7) during high antigen concentration stimulation did not improve polyfunctionality. The lack of any effect of anti–PD-1 treatment was seen at the standard dose used of 10 μg/ml (see above) as well as at 10-fold higher concentrations of anti–PD-1 treatment. Isotype control antibody (clone MG1-45) was added at the same concentration. (D–F) Ex vivo CD8⁺ T cells stimulated with different concentrations of aAPCs weekly for 2 weeks showed different levels of T cell polyfunctionality. aAPCs were made by conjugating biotinylated HLA-A2–M1 and biotinylated anti-CD28 complex onto anti-biotin microbeads (Miltenyi Biotec). (D) Percentage of tetramer-positive cells and (E) total M1-specific cell expansion. Higher concentrations of aAPCs induced more robust cell expansion, similar to results seen in Figure 1 with peptide-pulsed moDC-induced T cells. (F) Polyfunctionality assessment of aAPC-stimulated M1-specific T cells. High concentration aAPCs induced M1-specific T cells with lower polyfunctionality even in the absence of inhibitory receptors on the aAPCs. The results are representative of 3 independent experiments performed on at least 3 different subjects. *P < 0.05.