DC subset positioning influences in vivo antigen uptake and MHC II presentation. (A–C) Additional quantitative analysis of data presented in Fig. 1. (A) Relative EαGFP uptake by LN-resident cDC1 and cDC2 with respect to distance to the LS in LNs isolated 4 h after immunization. (B) Comparison of the frequency of EαGFP-positive DCs at the indicated time points. (C) EαGFP-positive cDC1 and cDC2 cells located in close proximity to the LS (LS-Prox), as defined by the top left quadrant in A, were examined for the geometric mean fluorescence intensity (gMFI) of EαGFP 1–6 h after immunization. (D and E) Flow cytometry analysis of EαGFP uptake and MHC II presentation (pMHC II) by DC subsets 4 h after immunization. (F and G) Isolated DCs were cultured in vitro with EαGFP for 1 h, washed, and further cultured for 3 h, after which they were examined for EαGFP uptake and MHC II presentation by flow cytometry. (H) Mice were injected with EαGFP or EαGFP-APC, and DC subsets were examined for antigen uptake by flow cytometry. (I) Frequency of antigen-positive cDC1 and cDC2 cells was enumerated (left). Frequencies of antigen-positive DCs, macrophages (Macs), and B cells were compared between EαGFP and EαGFP-APC after administration of 10–20 µg antigen (right). (J) EαGFP MFI was quantified for EαGFP-positive DCs after administration of the indicated quantities of EαGFP and EαGFP-APC antigen. Data represent at least two independent experiments. Dashed lines in B, C, E, and I connect cells within the same draining LNs. Paired t test was used for comparing cells within the same LNs. Unpaired t test was performed in I. Numbers in flow cytometry and histo-cytometry plots represent frequencies. Error bars are mean ± SD. **, P ≤ 0.01; *, P ≤ 0.05; and NS, P > 0.05.