Unmanipulated medLN T_RM proliferate after secondary lung challenge. (A) Influenza-immune mice were depleted of circulating P14 cells 16 d after infection by injection of αThy1.1-depleting antibody. 75 d later, mice received infection-matched congenically distinct circulating memory P14 cells. Mice were then rechallenged i.n. or transcervically with VV-gp33 1 d later. (B) P14 Thy1.1 cell abundance within blood 1 d before, 1 d after, and 30 d after αThy1.1 antibody depletion. (C) Phenotype of medLN P14 cells 75 d after αThy1.1 antibody depletion. Mice had received circulating CD45.1 P14 cells 1 d earlier. (D) Percentage of Ki67⁺ cells among CD103^neg, CD103⁺, and CD45.1 (circulating) medLN P14 cells 4 d after i.n. or t.c. VV-gp33 challenge. *, P < 0.05, as determined by an unpaired Student’s t test comparing cells of the indicated phenotype after i.n. or transcervical rechallenge; ns, not significant. (E) As in D, representative flow cytometry plots displaying the degree of proliferation among medLN P14 cells bearing a CD103⁺, CD69⁺, or Ly6C^lo phenotype. Data are pooled from two independent experiments (n = 9 for i.n. rechallenge; n = 6 for transcervical rechallenge). Error bars represent mean ± SEM.