Figure 7.

Regulation of Vaccine-Induced CD8 T Cell Memory and Protective Immunity by CD4 T Cells

Groups of C57BL/6 mice were vaccinated with NP+ADJ+GLA, as in Figure 1, and treated with isotype control antibodies (non-depleted) or anti-CD4 antibodies (CD4-depleted) i.v. and IN on days −1, 0, and 1 relative to prime and boost vaccination. T cell memory in lungs (A–F) and protective immunity to influenza A virus (G–P) was determined at 80 days PV.

(A–F) T cell memory in lungs at day 80 PV. To stain for vascular cells, mice were injected i.v. with anti-CD45.2 antibodies, 3 min prior to euthanasia. Lung cells were stained directly ex vivo with D^b/NP366 or I-A^b/NP311 tetramers along with the indicated antibodies. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 h before intracellular staining.

(A) FACS plots are gated on CD4 T cells and show NP311-specific, tetramer-binding memory CD4 T cells only in non-depleted mice.

(B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-depleted and CD4 T-cell-depleted mice.

(C) Expression of tissue residency markers on NP366-specific, tetramer-binding memory CD8 T cells in lungs.

(D) Percentage of vascular (CD45.2^+ve) and non-vascular (CD45.2^−ve) cells among NP366-specific, tetramer-binding memory CD8 T cells in lungs.

(E) Percentages of IFN-γ- and IL-17-producing, NP366-specific cells among CD8 T cells in lungs.

(F) Calculated proportions of IFN-γ- and/or IL-17-producing cells among cytokine-producing, peptide-stimulated, IFN-γ+IL-17 NP366-specific CD8 T cells.

(G–P) At day 80 after booster vaccination, non-depleted and CD4 T-cell-depleted mice were challenged IN with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge.

(G) Percentages of NP366-specific, tetramer-binding cells among CD8 T cells in lungs.

(H) Percentages of NP366-specific, tetramer-binding CD8 T cells in vascular and non-vascular lung compartment.

(I) Percentages of NP311-specific, tetramer-binding cells among CD4 T cells in lungs.

(J) Expression of tissue residency markers on NP366-specific, tetramer-binding CD8 T cells.

(K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs.

(L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo.

(M) Percentages of IFN-γ- and IL-17-producing, NP366-specific CD8 T cells.

(N) Relative proportions of IFN-γ- and/or IL-17-producing cells among total IFN-γ plus IL-17-producing, peptide-stimulated, NP366-specific CD8 T cells.

(O) Viral titers in lungs at day 6 after challenge.

(P) Body weight measured as a percentage of starting body weight prior to challenge.

Data are pooled from two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.1, ∗∗p < 0.01, and ∗∗∗p < 0.001.