Figure 3. Migratory CD103⁺ DCs are the major cell type carrying infectious virus particles to the MLNs.

A–C. Isolation of different MLN cell populations from influenza infected mice by cell sorting. A. Live gate for total MLN leukocytes shown as CD45⁺ DAPI^- cells (gate II). B. Sorting strategy to obtain total migratory DCs (gate V), individual CD103⁺ DCs (gate VI), or CD11b^high DCs (gate VII), and non-DC MLN cells by pooling gates i, ii, and iii during collection. All these cell populations were selected from the total CD45⁺ live cell gate I. C. Alternative sorting strategy starting with gate II, to obtain B220⁺ cells (gate B), Gr1⁺ cells (gate G), and pDCs (gate P2). D. Total migratory DCs (gate V) comprised of CD103⁺ DCs and CD11b^high DCs, or MLN non-DC cells (gates I, ii, and iii pooled) sorted at 3 dpi, were layered over MDCK cells in a 1∶2 serial dilution, starting with 14,000 and 500,000 cells respectively, and were co-cultured in the presence of TPCK-trypsin for 3 days. Supernatants were assayed for infectious virus particles by hemagglutination of RBCs. D'. Supernatants from MLN-DC/MDCK co-cultures were assayed for infectious virus by plaque immunostaining on MDCK cells. E. Immunofluorescence of intracellular viral NP of sorted CD103⁺DCs and CD11b^high DCs from infected mice at 3 dpi. F. Individual cell populations, isolated by sorting at day 3 and 4 post-infection, as described above, were injected into 10-day old embryonated eggs, and 2 days later, allantoic fluid was assayed for infectious virus particles by hemagglutination of RBCs. Each cell type at each time point was assayed in triplicate. F’. Allantoic fluid from eggs injected with CD103⁺ DCs was assayed for infectious virus particles by a plaque immunostaining assay on MDCK cells.