Programming Multifaceted Pulmonary T Cell Immunity by Combination Adjuvants

Abstract

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of T_H1/T_C1/T_H17/T_C17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 T_RM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 T_RM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2.

Keywords: CD4; CD8; T1 and T17 programming; adjuvants; heterosubtypic; influenza virus; mucosal subunit vaccines; respiratory immunity; tissue-resident memory.

Graphical abstract

Figure 1

Effector CD8 T Cell Response to Adjuvanted Vaccines C57BL/6 mice were vaccinated intranasally (IN) twice (3 weeks apart) with influenza virus nucleoprotein (NP) formulated in the indicated adjuvants. At day 8 post-booster vaccination (PV), cells in the lungs and bronchoalveolar lavage (BAL) were stained with D^b/NP366 tetramers along with antibodies to cell surface molecules, granzyme B, and transcription factors directly ex vivo. (A, B, C, and E) Fluorescence-activated cell sorting (FACS) plots show percentages of gated tetramer-binding CD8 T cells in respective gates/quadrants. (D) Median fluorescence intensities (MFIs) for transcription factors in NP366-specific CD8 T cells. Data are pooled from two independent experiments or represent one of two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 2

Effector CD4 T Cell Response to Adjuvanted Vaccines Groups of C57BL/6 mice were vaccinated IN, as in Figure 1. At day 8 PV, cells from lungs and BAL were stained with I-A^b/NP311 tetramers along with antibodies to cell surface molecules and transcription factors. (A) FACS plots show the percentages of I-A^b/NP311 tetramer-binding cells among CD4 T cells. (B) Percentages of the indicated cell population among NP311-specific, tetramer-binding CD4 T cells. (C) FACS plots are gated on I-A^b/NP311 tetramer-binding cells, and the numbers in each quadrant are the percentages of cells among the gated population; MFIs for transcription factors in NP311-specific CD4 T cells are plotted in the adjoining graphs. (D) FACS plots in (C) were used to quantify the percentages of T-bet^LOEOMES^HI cells (quadrant 4) among NP311-specific CD4 T cells. (E) Percentages of CD103^HI and CD69^HI cells among NP311-specific CD4 T cells. Data are representative of two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 3

Functional Polarization of Effector CD8 and CD4 T Cells C57BL/6 mice were vaccinated as in Figure 1. On the 8^th day PV, lung cells were stimulated with NP366 or NP311 peptides for 5 h. The percentages of NP366-specific CD8 T cells (A–C) or NP311-specific CD4 T cells (D–F) that produced IFN-γ, IL-17, TNF-α, and IL-2 were quantified by intracellular cytokine staining. (A) Percentages of cytokine-producing cells among the gated CD8 T cells. (B) To demonstrate relative dominance of T_C1 versus T_C17 in different groups, we calculated the relative proportions of these cells among total cytokine-producing CD8 or CD4 T cells (IL-17+IFN-γ-producing cells following stimulation with NP366 peptide). (C) Plots are gated on IFN-γ-producing CD8 T cells, and the numbers are the percentages among the gated cells. (D) Percentages of cytokine-producing cells among the gated CD4 T cells. (E) Calculated percentages of IFN-γ and/or IL-17-producing CD4 T cells among NP311-specific, cytokine-producing CD4 T cells. (F) Plots are gated on IFN-γ-producing CD4 T cells. Data are representative of two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 4

Regulation of the Effector T Cell Response by Antigen Receptor Signaling (A) Ly5.1 Nur77-GFP OT-I CD8 T cells were adoptively transferred into congenic Ly5.2 C57BL/6 mice and vaccinated IN next day with OVA protein formulated in the indicated adjuvants. At days 2, 5, or 8 PV, cells from lymph nodes and lungs were stained with K^b/SIINFEKL tetramers, anti-Ly5.1, anti-Ly5.2, anti-CD8, and anti-CD44 antibodies. The GFP MFIs in donor Ly5.1^+ve OT-I CD8 T cells were quantified by flow cytometry. (B) Wild-type non-transgenic (WT) and transgenic KLF2-GFP mice were vaccinated with NP protein formulated in adjuvants, as in (A). At day 8 PV, lung cells were stained with anti-CD8, anti-CD44, and D^b/NP366 tetramers. The overlay histogram shows GFP fluorescence (MFI) for the gated tetramer-binding CD8 T cells from WT (black) and KLF2-GFP transgenic (red) mice. (C) B6 mice were vaccinated with NP protein formulated in various adjuvants, as in (A). At day 8, PV lung cells were stained with anti-CD8, anti-CD44, anti-PD-1, and D^b/NP366 tetramers. Plots show the percentages of PD-1^+ve cells among the gated D^b/NP366 tetramer-binding CD8 T cells. (D) Statistical correlation analysis between the percentages of PD-1^+ve CD8 T cells and the percentages of tetramer^+ve CD8 T cells at day 8 PV. Data are pooled from two independent experiments or represent one of two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons. For (D), we used two-way ANOVA, Student’s t test, and simple regression analysis; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5

Mucosal CD8 and CD4 T Cell Memory in Vaccinated Mice At 100 days after booster vaccination, NP366-specific memory CD8 T cells (A–D) and NP311-specific CD4 T cells (E–H) were characterized in lungs, airways (BAL), and spleen. To stain for vascular cells, mice were injected intravenously with fluorescent-labeled anti-CD45.2 antibodies, 3 min prior to euthanasia. Cells from lungs and BAL were stained with D^b/NP366 tetramers, I-A^b/NP311 tetramers, and anti-CD4, anti-CD8, anti-CD44, anti-CD103, and anti-CD69 antibodies. (A) Percentages and total numbers of NP366-specific CD8 T cells in lungs, BAL, and spleen. (B) FACS plots are gated on NP366-specific, tetramer-binding CD8 T cells; numbers are the percentages of vascular and non-vascular cells in the gated population. (C) Percentages of CD69^+veCD103^+ve T_RM cells among NP366-specific CD8 T cells. (D) Total numbers of vascular and non-vascular CD103^+ve NP366-specific CD8 T cells in lungs. (E) Percentages and total numbers of NP311-specific CD4 T cells in lungs. (F) Percentages of vascular and non-vascular cells among NP311-specific CD4 T cells in lungs. (G) Percentages of IFN-γ- or IL-17-producing cells among CD4 T cells. (H) Calculated percentages of IFN-γ- and/or IL-17-producing CD4 T cells among total NP311-specific, cytokine-producing (IFN-γ + IL-17) peptide-stimulated CD4 T cells. Data are pooled from two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 6

Vaccine-Induced Protective Immunity to H1N1 and H5N1 Influenza Viruses (A–C) Groups of C57BL/6 mice were vaccinated twice IN, as in Figure 1. At 100 days after the booster vaccination, mice were challenged IN with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6^th day after virus challenge. (A) Viral titers in the lungs on the 6^th day after virus challenge. (B) Percentages of NP366-specific, IFN-γ- and IL-17-producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ- and/or IL-17-producing cells among total IFN-γ+IL-7-producing, peptide-stimulated, NP366-specific T cells (pie charts). (C) Percentages of NP311-specific, IFN-γ- and IL-17-producing cells among CD4 T cells and calculated proportions of IFN-γ- and/or IL-17-producing cells among total IFN-γ+IL-7-producing, peptide-stimulated, NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. 180 days after the last vaccination, mice were challenged IN with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control immunoglobulin G (IgG) or anti-IL-17A antibodies (intravenously [i.v.] and IN) at −1, 0, 1, 3, and 5 days relative to virus challenge. On the 6^th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice, as above. 50 days after booster vaccination, vaccinated and unvaccinated mice were challenged IN with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival were monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. In (E), we used non-linear regression for analyzing weight loss data. For the rest, we used one-way ANOVA test with Tukey-corrected multiple comparisons; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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.