Figure 5. Evidence that antigen in the... | Rockefeller University Press

Figure 5.

$Evidence that antigen in the lung is required for medLN TRM formation. (A and B) Staggered (A) and contemporaneous (Contemp.; B) approach for recruiting LCMV-specific cells into the inflamed lung. (C) Representative flow cytometry plots of LCMV-specific (gray) or influenza-specific (red) CD8+ T cells gated on the i.v.neg fraction isolated from lung or medLN after staggered infection. (D) Frequency of LCMV-specific (gray) and influenza-specific (red) cells within the i.v.neg fraction of the lung or medLN that coexpressed integrin-β7 and CD103 following staggered or contemporaneous infection modalities. Numbers above the bars represent days after infection for either LCMV-specific (gray) or PR8-ova–specific (red) CD8+ T cells. Data collected over two independent experiments (n = 8). Bars represent mean ± SEM. **, P < 0.01; ****, P < 0.0001 as determined by unpaired Student’s t test. (E–G) Dynamics of CD69 and CD25 expression on P14 and OT-1 cells within the medLN following infection with PR8-gp33 (E) or PR8-ova (F) at the indicated time points. Expression of CD69 and/or CD103 in the indicated tissue among transgenic i.v.neg CD8+ T cells after PR8-gp33 (E, right plots, day 5) or PR8-ova (G, day 8). Mice received 5 × 105 transgenic CD8+ T cells before infection to detect cells in the medLN at early time points. (H–K) Mice were contemporaneously challenged with LCMV i.p. and PR8-ova i.n. (as in B), and OT-I (red) and P14 (gray) cells were examined 18 d later. Spec., specific. (I) Phenotype by flow cytometry. Numbers in quadrants represent mean ± SEM from a three-mouse experiment performed twice with similar results (Fig. S2). (J and K) Distribution of influenza- and LCMV-specific cells by IF microscopy in the lung (J; enlarged images on right) and medLN (K). Scale bars represent 50 µm in J and 300 µm in K.$

Evidence that antigen in the lung is required for medLN T_RM formation. (A and B) Staggered (A) and contemporaneous (Contemp.; B) approach for recruiting LCMV-specific cells into the inflamed lung. (C) Representative flow cytometry plots of LCMV-specific (gray) or influenza-specific (red) CD8⁺ T cells gated on the i.v.^neg fraction isolated from lung or medLN after staggered infection. (D) Frequency of LCMV-specific (gray) and influenza-specific (red) cells within the i.v.^neg fraction of the lung or medLN that coexpressed integrin-β7 and CD103 following staggered or contemporaneous infection modalities. Numbers above the bars represent days after infection for either LCMV-specific (gray) or PR8-ova–specific (red) CD8⁺ T cells. Data collected over two independent experiments (n = 8). Bars represent mean ± SEM. **, P < 0.01; ****, P < 0.0001 as determined by unpaired Student’s t test. (E–G) Dynamics of CD69 and CD25 expression on P14 and OT-1 cells within the medLN following infection with PR8-gp33 (E) or PR8-ova (F) at the indicated time points. Expression of CD69 and/or CD103 in the indicated tissue among transgenic i.v.^neg CD8⁺ T cells after PR8-gp33 (E, right plots, day 5) or PR8-ova (G, day 8). Mice received 5 × 10⁵ transgenic CD8⁺ T cells before infection to detect cells in the medLN at early time points. (H–K) Mice were contemporaneously challenged with LCMV i.p. and PR8-ova i.n. (as in B), and OT-I (red) and P14 (gray) cells were examined 18 d later. Spec., specific. (I) Phenotype by flow cytometry. Numbers in quadrants represent mean ± SEM from a three-mouse experiment performed twice with similar results (Fig. S2). (J and K) Distribution of influenza- and LCMV-specific cells by IF microscopy in the lung (J; enlarged images on right) and medLN (K). Scale bars represent 50 µm in J and 300 µm in K.