Figure 1.

Lung T_RM cells are reactivated more rapidly but less efficiently than T_LN cells in medLNs. (A) P14/OT-I Nur77-GFP immune chimeras were generated by cotransferring naive P14⁺ (Thy1.1⁺) and OT-I⁺ (Ly5.1⁺) Nur77-GFP CD8⁺ T cells (5 × 10⁴ cells each) into C57BL/6 (Th1.2⁺/Ly5.2⁺) mice 1 d before i.n. coinfection with recombinant influenza X31-gp33 and X31-ova. 30 d later, the P14/OT-I Nur77-GFP immune chimeras were reinfected with PR8-gp33 i.n., and reactivation of P14⁺ lung T_RM and medLN T_LN cells was assessed at 0, 12, 24, 48, and 120 h.p.i. (B) Representative flow plots of Nur77-GFP expression in P14⁺ lung T_RM cells and medLN T_LN cells. In vivo labeling with αCD8β antibody was used to distinguish CD8⁺ T_RM cells in the lung parenchyma from CD8⁺ T cells in the vasculature. (C and D) Frequency of Nur77-GFP⁺ CD8β⁻ P14⁺ cells (i.e., top left quadrant of flow plots) in B was quantified. Data are expressed as mean ± SD. (E and F) As negative controls, Nur77-GFP expression in OT-I⁺ and P14⁺ cells was examined after PR8-gp33 and PR8 2° infection, respectively. (G) To limit the contribution of T_CIRC cells, a low dose of αThy1.1 (clone 19E12)–depleting antibody was administered i.p., and the frequency of Nur77-GFP⁺ P14⁺ T_RM cell reactivation was examined. N.D., not detected. Antigen-activated P14⁺ lung T_RM (blue) and medLN T_LN (red) cells are distinguished from the bystander-activated OT-I⁺ lung T_RM (green) and medLN T_LN (gold) cell samples by color. Data shown are representative of two independent experiments (n = 3–5 mice/group).