Figure 2. LAIV increases frequency of influenza-specific TNF-α– and IFN-γ–producing CD4⁺ and TRM CD4⁺ T cells in the lung.

Frequencies of cytokine-producing CD4⁺ and TRM CD4⁺ T cells were measured in human BAL samples by intracellular staining flow cytometry analysis with and without (mock) in vitro influenza antigen stimulation. Volunteers were divided by vaccine and colonization status in TIV/Spn^– (n = 6), TIV/Spn⁺ (n = 8), LAIV/Spn^– (n = 10), LAIV/Spn⁺ (n = 9), and unvaccinated (n = 8, 3 Spn^– and 5 Spn⁺) groups. (A) Production of TNF-α by total CD4⁺ T cells in each group (paired unstimulated [mock] and stimulated condition [flu]). (B) Influenza-specific production of TNF-α by total CD4⁺ T cells (difference between influenza-stimulated and unstimulated) in each group. (C) Production of TNF-α by CD4⁺CD69⁺ T cells in each group. (D) Production of influenza-specific TNF-α by CD4⁺CD69⁺ T cells in each group. (E) Production of IFN- γ by total CD4⁺ T cells in each group. (F) Production of influenza-specific IFN-γ by CD4⁺ T cells in each group. (G) Production of IFN-γ by CD4⁺CD69⁺ T cells and (H) influenza-specific IFN-γ by CD4⁺CD69⁺ T cells in each group. Each individual dot represents a single volunteer, and the conditions from 1 individual are connected. Medians with IQR are depicted for influenza-specific responses (B, D, F, and H). *P < 0.05, **P < 0.01 by Wilcoxon’s test for comparisons within the same group and by Mann-Whitney U test for between-group comparisons.