Transfected mature DCs signaled through CD40 increase H-2K^b loading of transgenic peptides and antigen presentation to specific CD8⁺ T cells. (A) H-2K^b-SIINFEKL complex expression by: (1) mature DCs after transfection with pCMV-LacZ (negative control); (2) nontransfected mature DCs incubated with a saturating dose of synthetic peptide SIINFEKL (positive control); or (3) mature DCs after transfection with pCMV-OVA-TR and cultured in medium alone (control), with irrelevant IgM (control), or with agonist anti-CD40 (IgM) mAb. Numbers represent percentages of positive mature DCs and MFI (in parentheses). Results from 1 of 3 representative experiments are shown. (B) Induction of OT-1 CD8⁺ T-cell proliferation after stimulation with syngeneic mature DCs at different cell ratios. • Mature DCs transfected with pCMV-OVA-TR and cultured for 24 hours with anti-CD40 mAb induced significantly higher proliferation of OT-1 CD8 ⁺ T cells than □ mature DCs transfected with pCMV-OVA-TR and cultured in medium alone (P < .0001). Negative controls were ▵ nontransfected mature DCs cultured with anti-CD40 mAb and ▵ nontransfected mature DCs cultured in medium alone. Positive controls were mature DCs pulsed with a saturating dose of SIINFEKL and cultured for 24 hours with ♦ or without ▴ anti-CD40 mAb. (C-D) High OT-1 proliferation induced by mature DCs transfected with the gene gun and CD40 signaled corresponded with high and sustained expression of H-2K^b-SIINFEKL complex in the surfaces of mature DCs. (C) Percentage of mature DCs expressing H-2K^b-SIINFEKL complex in the cell membrane. (D) MFI after gene gun transfection with pCMV-OVA-TR or after incubation with OVASIINFEKL peptide in the presence or in the absence of the agonist CD40 mAb. Means ± SD of 3 independent experiments are displayed.