Figure 8.

Visualizing retrograde migration. (A and B) Representative example of the medLN (blue shade) and medLN-associated lymphatics (red shade) dissected out for flow cytometric analysis 16 d after P14 transfer and i.n. PR8 infection. (C) Frequency of P14 cells expressing a CD103⁺ Ly6C^lo phenotype isolated from medLN (left) or associated lymphatic vessels (right) 12–21 d after infection with PR8-gp33 (n = 5 or 6 mice per time point). Bars represent mean ± SEM. ***, P < 0.001 as determined by Student’s t test comparing day 12 to day 21. (D) 18 d after P14 cell transfer and i.n. PR8-gp33 infection, IF microscopy of the medLN and associated lymphatic vessels. Arrows indicates Ly6C^lo P14 cell in lymphatic vessels. Scale bar represents 500 µm. (E) C_e3D and two-photon microscopy of the medLN and associated lymphatics 16 d after PR8 infection (Video 1). Scale bar represents 200 µm. (F) Mice infected with influenza virus 16 d earlier were treated with α-Thy1.1–depleting antibody and compared with untreated (No Tx) controls 4 d later. Numbers in top right image indicate regions enlarged in the panels below. Scale bars represent 200 µm. (G) As in F, Ly6C^lo P14 cells could be identified at the medLN periphery and directly within medLN-associated lymphatics (marked by asterisk) in both untreated mice (top) and mice treated 4 d earlier with α-Thy1.1–depleting antibody (bottom). Arrows highlight influenza-specific cells that are Ly6C^lo/neg. Scale bars represent 50 µm. Representative images are from at least three sections per tissue, per mouse, of three or more individual mice.