Cross-presentation induced by TLR7 is mediated by LCs and CD8α⁺ DCs. B6 mice were immunized in the footpad with 50 μg of the OVA-TLR7a conjugate. Twenty-four hours after immunization, popliteal LN were harvested as before, the cell suspensions from each mouse pooled, and the DCs were purified using a flow sorter (MoFlow) based on CD11c expression and the markers outlined in Figure 5A and supplemental Figure 3. (A) DCs were incubated at the indicated cell number and or (C) at 2.5 × 10⁵ cells per well with 0.5 × 10⁶ effector OTI T cells stimulated in 5 days of peptide-pulsed culture. The cells were coincubated for 4 hours in the presence of brefeldin A and then stained for intracellular IFNγ. Production of IFNγ by OTI T cells was assessed by gating on expression of the congenic marker CD45.1. The level of IFNγ response by the OTI T cells was expressed as a percent of the maximal production of IFNγ induced by DCs pulsed with the SIINFEKL peptide. (B) Sorted DCs were available in limited and varied frequencies across the 4 DC subsets as indicated. To normalize for differing range of cell frequencies and to enable comparison of OTI stimulation by various DC subsets, linear regression curves of the OTI response to titrating numbers of DCs for each subset were determined and the magnitudes of the corresponding OTI IFNγ response were normalized to a unified range of DC numbers. To compare the level of cross-presentation by DCs across 3 independent experiments, each DC subset incubated at a given cell concentration with OTI T cells was assigned a color in a heat map representing relative levels of OTI stimulation by each of the 4 DC subsets indicated. A graphical representation of the magnitudes of OTI IFNγ response is also shown for the highest concentration of DCs used in each experiment. The data shown are representative of (A) 6 and (C) 2 independent experiments. Statistical analyses (*) were performed as described in “Statistical analyses.”