Antigen-specific CD4+ and CD8+ effector memory T-cells in lung.

Lung lymphocytes were isolated from 5 mice of each group 2 weeks post final immunization and assayed for M2e-specific and virus-specific CD4+ and CD8+ T effector memory cells (Tem). (A) The gating strategy of single or double cytokine-secreting antigen-specific CD4+, CD8+ Tem. FSC-A/FSC-H: removal of cell doublets; FSC-A/SSC-A: gating of the lymphocyte population; LD/SSC-A: gating of the live lymphocyte population; CD8/CD3: gating of the live CD3+CD8+ lymphocyte population; CD4/CD3: gating of the live CD3+CD4+ lymphocyte population; CD62L/CD44: gating of the CD4+Tem and CD4+Tcm cell population; IFNg/TNF: gating of CD4+Tem cells (producing TNFa, IFNg or both). For the CD8+ cell population, the gating is similar. (B) Antigen-specific CD4+ and CD8+ Tem cells after re-stimulation with M2e or virus (A/H3N2 for mice immunized with FlgH2-2-4M2e; A/H1N1pdm09 for mice immunized with FlgH2-1-4M2e). (C) Antigen-specific cytokine-producing CD4+ Tem after re-stimulation with M2e and virus (A/H3N2 or A/H1N1pdm09). (D) Antigen-specific cytokine-producing CD8+ cells after re-stimulation with M2e and virus (A/H3N2 or A/H1N1pdm09). Data are presented as the mean±SEM. Statistical significance was determined using the Mann-Whitney U-test. The P values between groups are indicated.