Both conventional APCs and nhAPCs are required for optimal CD8⁺ T-cell expansion and cytokine production. (A-B) B6→B6 and K^b−/−→B6 chimeras were immunized with 10⁷ pfu of rHuAd5-SIINFEKL-Luc intramuscularly and spleens, lungs and blood were harvested 12 or 42 days later. SIINFEKL-specific CD8⁺ T cells were identified using tetramer staining or intracellular cytokine staining. The percentage of tetramer-positive cells that produce IFN-γ (A) or TNF-α (B) was calculated by dividing the frequency of IFN-γ⁺ cells or TNF-α⁺ cells by the frequency of tetramer-positive cells and multiplying by 100. Each histogram represents the mean percentage (± SEM) for 8 mice. (C-D) K^b−/−D^b−/−→B6, K^b−/−D^b−/−→B6, and B6→B6 mice chimeras received 5 × 10⁴ Thy1.1⁺ OT-I CD8⁺ T cells intravenously. The next day, mice were immunized with 10⁸ pfu of rHuAd5-SIINFEKL-Luc intramuscularly. The frequency of SIINFEKL-specific CD8⁺ T cells were measured in the peripheral blood at day 8 by staining for Thy1.1 (C) or by intracellular cytokine staining for IFN-γ (D). Data points represent a total of 3 to 6 mice (± SEM) in each experiment. (D) Right panels show coexpression of IFN-γ and TNF-α by Thy1.1⁺ CD8⁺ T cells in each type of chimera. Value in each quadrant represents the mean frequency of Thy1.1⁺ cells in that quadrant. Frequency in the left panels represents the mean of Thy1.1⁺ cells of total lymphocytes. Frequency was calculated from a total of 3 to 6 mice per group. *P < .05, **P < .01.