Influenza infection fortifies local lymph nodes to promote lung-resident heterosubtypic immunity

Abstract

Influenza infection generates tissue-resident memory T cells (TRMs) that are maintained in the lung and can mediate protective immunity to heterologous influenza strains, but the precise mechanisms of local T cell-mediated protection are not well understood. In a murine heterosubtypic influenza challenge model, we demonstrate that protective lung T cell responses derive from both in situ activation of TRMs and the enhanced generation of effector T cells from the local lung draining mediastinal lymph nodes (medLNs). Primary infection fortified the medLNs with an increased number of conventional dendritic cells (cDCs) that mediate enhanced priming of T cells, including those specific for newly encountered epitopes; cDC depletion during the recall response diminished medLN T cell generation and heterosubtypic immunity. Our study shows that during a protective recall response, cDCs in a fortified LN environment enhance the breadth, generation, and tissue migration of effector T cells to augment lung TRM responses.

Figure 1.

Influenza infection generates memory CD4⁺ and CD8⁺ T cells in the lung tissue niche and draining LN. T cells were isolated from the lung and lung-draining medLNs of uninfected naive mice and memory mice (infected with X31 influenza 3–4 wk previously) following i.v. administration of anti-CD45.2 antibody (see Materials and methods). (A) Analysis of lung T cells shown in representative flow cytometry plots of CD44 expression by circulating labeled (red) and lung tissue niche (blue) CD4⁺ and CD8⁺ T cells (left) and graphs of numbers of lung niche CD44⁺CD4⁺ and CD44⁺CD8⁺ T cells in naive and memory mice (right). Data were compiled from two groups; n = 5 or 6 mice/group. (B) Schematic diagram depicting heterosubtypic challenge model in which uninfected naive mice and memory mice, generated from prior X31 infection, are simultaneously challenged with PR8 (H1N1) virus with concurrent FTY720 treatment. (C) Heterosubtypic protection in FTY720-treated mice. Shown are weight loss morbidity plots at indicated days post-infection (p.i.) during primary and recall influenza challenge of WT C57BL/6 mice (left) and viral titers in the BAL assessed on day 6 after challenge (right). Data were compiled from two independent experiments; n = 10–12 mice/group. (D) Lung-localized protection is independent of B cells. Weight loss morbidity (left) and BAL viral titers determined at day 7 after infection (right) after PR8 challenge of µMT naive and memory mice. Data were compiled from two experiments; n = 10–14 mice/group. Significance was determined using Student’s unpaired t test; ****, P < 0.0001; ***, P ≤ 0.001; **, P < 0.01; *, P ≤ 0.05. All error bars show mean ± SEM. n.s., not significant.

Figure S1.

Primary infection generates influenza-specific lung T_RMs. (A) Lung cells isolated from mice infected 3–5 wk after primary infection. Left: Flow cytometry plots gated on lung CD44⁺CD8⁺ showing NP tetramer expression by lung niche (blue) and labeled (red) cells and graph showing number of lung CD44⁺CD8⁺NP-Tet⁺ cells. Right: Flow cytometry plots gated on medLN CD44⁺CD8⁺ showing CD103 and NP tetramer expression and graph depicting absolute number of medLN CD44⁺CD8⁺NP-Tet⁺ T cells. Data are representative of two experiments; n = 4 mice/group. (B) Lung niche T cells from memory mice express surface T_RM markers. Surface expression of CD69, CD103, and CXCR6 by lung niche (blue) versus circulating (labeled; red) T cells shown as representative histograms (left) and graphs showing individual mice (right) treated with FTY720 for 2 d. Data were compiled from two independent experiments; n = 7 mice/group. Significance was determined by Student’s unpaired t test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Error bars are mean ± SEM.

Figure 2.

Tissue-localized protection to heterosubtypic challenge is mediated by rapid lung niche T cell expansion. Lung T cells were isolated from naive and memory mice following in vivo antibody labeling at the indicated days post-infection (p.i.), as in Fig. 1. (A) Accumulation of CD4⁺CD44⁺ (upper) and CD8⁺CD44⁺ (lower) T cells in the lung following primary and recall challenge is shown in representative flow cytometry plots (left) and in graphs depicting absolute numbers of T cells in the lung niche (middle) and labeled (right) CD44⁺ T cells. (B) Accumulation of CD103⁻ and CD103⁺CD4⁺CD44⁺ and CD8⁺CD44⁺ T cells in the lung during the recall response is shown in representative flow cytometry plots (left), and graphs show absolute numbers of lung niche CD4⁺CD44⁺ (middle) and CD8⁺CD44⁺ (right) T cells. Data from A and B are from one experiment with three to five mice per group, representative of three independent experiments. (C) Accumulation of lung niche (left) and lung labeled (right) influenza-specific tetramer⁺ (NP-Tet⁺) CD8⁺ T cells over the course of a primary or recall response. (D) Accumulation of CD103⁻ and CD103⁺CD8⁺CD44⁺ NP-Tet⁺ lung niche T cells during the recall response. Data from C and D were compiled from two independent experiments; n = 2–6 mice/group. (E) Absolute numbers of medLN CD4⁺CD44⁺ (left) and CD8⁺CD44⁺ (right) T cells during primary and recall responses to PR8 challenge. (F) Absolute numbers of spleen CD4⁺CD44⁺ (left) and CD8⁺CD44⁺ (right) T cells during primary and recall responses to PR8 challenge. Data from E and F are compiled from three experiments; n = 6–8 mice/group. All error bars show mean ± SEM.

Figure S2.

Lung niche T cell migration during influenza infection and FTY720 treatment. (A) T cells were isolated from the lungs of memory and naive mice 5 d after PR8 challenge with or without daily FTY720 treatment. Left: Representative flow plot of lung CD4⁺ and CD8⁺ T cells showing CD44 expression versus in vivo administered CD45.2 antibody labeling. Right: Histograms depicting number of CD44⁺CD4⁺ or CD8⁺ T cells either labeled or protected by in vivo fluorescent antibody with or without FTY720 treatment. n = 4 or 5 mice per group. (B) CD44^loCD45.1⁺ OT-II indicator population is transferred into both naive and memory mice and challenged with PR8-OVA (PR8-OTII). Lung cells were isolated 5 d after PR8-OVA challenge. Graph depicts number of lung CD4⁺CD45.1⁺ OT-II T cells during the primary and recall responses with or without FTY720 treatment. Data were compiled from two independent experiments; n = 4–9 mice/group. (C) BrdU incorporation by lung OT-II cells and CD45.1⁻ host polyclonal cells 5 d after recall challenge with PR8-OVA. Data are representative of two independent experiments; n = 3–5 mice/group. (D) OT-II cells in the lung, medLN, and spleen of μMT hosts 4 d after primary or recall challenge shown in representative flow cytometry plots (left) and graphs depicting total numbers of OT-II cells in each tissue site in primary or recall responses. Data are representative of two independent experiments; n = 3–5 mice/group/experiment. Significance was determined by Student’s unpaired t test; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Error bars show mean ± SEM. n.s., not significant.

Figure 3.

Heterosubtypic recall promotes rapid proliferation of CD103⁻ T cells in the lung niche and medLN. Lung, medLN, and spleen T cells were isolated from naive and memory mice challenged as in Fig. 2 A following administration of BrdU i.p. and i.n. 2 h before tissue harvest. (A) BrdU incorporation by lung niche and labeled T cells is shown in representative flow cytometry plots (left) and graphs depicting absolute numbers of CD4⁺CD44⁺ (middle) and CD8⁺CD44⁺ lung niche (blue) and labeled (red) BrdU⁺ T cells. (B) BrdU incorporation of lung niche T cells as a function of CD103 expression depicted in representative flow cytometry plots (left) and graphs showing the number of BrdU⁺ lung niche CD103⁻ and CD103⁺CD44⁺CD4⁺ and CD8⁺T cells (right). A and B were compiled from three independent experiments; n = 7–11 mice/group. (C) Graphs showing frequency of BrdU expression by CD103⁺ and CD103⁻ lung niche CD44⁺CD62L⁻CD4⁺ (left) and CD8⁺ (right) T cells, with paired values shown for each mouse. Data were compiled from three independent experiments; n = 7–11 mice/group. Significance was determined by paired t test between CD103⁻ and CD103⁺ groups. (D) Graph depicting medLN and spleen CD4⁺ and CD8⁺ T_EM (CD44⁺CD62L⁻) BrdU incorporation during day 5 of the recall response. C and D were compiled from two independent experiments; n = 5–7 mice/group. Significance was determined by Student’s unpaired t test except for C. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. All error bars show mean ± SEM. n.s., not significant.

Figure 4.

Recall response enhances local LN T cell priming and migration to the lung tissue niche. (A) Experimental schematic for tracking new effector and circulating T cell responses: CD44^lo OT-II CD4⁺ or OT-I CD8⁺ transgenic T cells were transferred into naive and memory mouse hosts, which were subsequently challenged (in the presence of FTY720 treatment) with PR8 as a control and/or recombinant PR8-OVA expressing the OT-II or OT-I peptide epitope, respectively; tissues were harvested 4–5 d after infection following in vivo antibody labeling. (B) The presence of OT-II (CD45.1⁺) CD4⁺ T cells in the indicated sites of mice at day 5 of the primary or recall challenge with PR8 or PR8-OVA is shown in representative flow cytometry plots (left, OT-II–containing quadrants outlined in green) and in graphs (right) depicting total numbers of OT-II cells in each tissue site for each challenge condition in individual mice. FACS plots are gated on total CD4⁺ cells. Data are representative of two independent experiments with n = 3–5 mice/group. (C) The presence of OT-I (CD45.2⁺) CD8⁺ T cells in the indicated sites of mice at day 5 of the primary or recall challenge with PR8-OVA is shown in representative flow cytometry plots (left, OT-I–containing quadrants outlined in green) and in graphs (right) depicting total numbers of OT-I cells in each tissue site for individual mice. FACS plots are gated on total CD8⁺ T cells. Data are compiled from two independent experiments; n = 4–8 mice/group. Significance between primary and recall groups was determined by Student’s unpaired t test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. All error bars show mean ± SEM. n.s., not significant.

Figure 5.

Enhanced naive T cell recruitment, activation, and proliferation in the medLN during the recall response. (A) Prior influenza infection results in increased numbers of naive and memory T cells in the medLN. Graphs show numbers of indicated CD4⁺ and CD8⁺ T cell subsets (T-naive, CD44^loCD62L^hi; T_EM, CD44^hiCD62L^lo; central memory T cells [TCM], CD44^intCD62L^hi) in the medLN of uninfected (naive) mice and memory mice 3–5 wk after infection. Data are representative of two experiments compiled from three to five mice per group. (B) Recruitment and/or retention of naive CD45.1⁺ OT-II CD4⁺ T cells in medLN 2 d following transfer of equal numbers (50,000 cells/mouse) into naive and memory congenic mice is shown in representative flow cytometry plots (left) and a graph (right) showing absolute numbers of OT-II cells, compiled from four or five mice per group. (C and D) Enhanced priming in the medLN of memory mice during recall. Proliferation dye–labeled (eFluor 450) OT-II/Nur77-GFP cells were transferred into naive and memory mouse hosts and subsequently challenged with PR8-OVA 1 d later. (C) Nur77-GFP expression by T cells isolated from medLN and spleen 2 d after challenge in the recall response is shown in representative flow cytometry plots gated on OT-II cells (left) and graphs (right) showing frequency of GFP⁺ cells. (D) Proliferation of OT-II T cells in the medLN 3 d after PR8-OVA challenge measured by proliferation dye dilution is shown in representative flow cytometry plots (left) and graphs depicting the percentage of OT-II cells divided (middle) and total number of OT-II cells (right). Data shown are representative of two independent experiments; n = 3–5 mice/group. Significance was determined by Student’s unpaired t test; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Error bars show mean ± SEM.

Figure 6.

Influenza infection promotes long-term quantitative increases of DCs in the medLN. (A) Persistence of lung-migratory DCs in the medLN following influenza infection. Left: Frequency of total DCs (CD11c⁺MHC class II⁺; top) and lung-migratory cDCs (CD11b⁻CD103⁺; bottom) in the medLN at indicated time points after primary X31 infection shown in representative flow cytometry plots (left) and in graphs depicting absolute numbers of medLN CD45⁺CD11c⁺ I-A/I-E^hi DCs (top) and ratio of CD11b⁻CD103⁺/CD11b⁺CD103⁻ DCs (bottom) over time after infection. Data are compiled from two independent experiments; n = 4–8 mice per group. (B) Quantification of cDCs using zbtb46-GFP reporter mice. Left: Flow cytometry plots show zbtb46-GFP expression gated on CD45⁺ cells from indicated sites of naive or memory mice. Right: Graphs show absolute numbers of zbtb46⁺CD11c^hi I-A/I-E^hi cDCs in these sites compiled from four experiments; n = 6–12 mice/group. (C) CD103 expression is enhanced in cDCs from memory mice. CD103 and CD11b expression by zbtb46-GFP⁺ cDCs in indicated sites of naive and memory mice is shown in representative flow cytometry plots (left) and graphs displaying proportion of CD11b⁻CD103⁺zbtb46-GFP⁺ cDCs (right) compiled from four experiments; n = 6–12 mice/group. (D) Whole-transcriptome profiling by population RNA-seq of CD45⁺zbtb46-GFP⁺CD11c⁺ I-A/I-E⁺ cDCs sorted from medLNs of naive and memory mice as in B. Volcano plot depicts genes by the absolute value of log₁₀P_adj and the relative fold change comparing memory cDCs with naive cDCs. Genes with differential gene expression of at least twofold change are shown in green, and genes with an absolute value of log₁₀P_adj >4 are shown in red. For all experiments, significance between naive and memory mice was determined by Student’s unpaired t test; ****, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Significance in A was determined by comparison with number of medLN DCs at day 0. Error bars show mean ± SEM. n.s., not significant.

Figure S3.

Qualitative analysis of cDCs in the medLN of memory compared with naive mice. (A) CD103⁺ lung migratory cDCs exhibit elevated CD86 expression. Analysis of CD86 expression by CD103-expressing cDC subsets in naive and memory mice is shown in representative flow cytometry plots of medLN zbtb46⁺ cDCs (left) and graph depicting percentage CD86 expression by CD11b⁻CD103⁺ and CD11b⁺CD103⁻ medLN cDCs (right). Data were compiled from three experiments; n = 9 mice/group. (B) Whole-transcriptome profiling of medLN cDCs from Fig. 6 D. Heat map of top differentially expressed genes between naive and memory cDCs based on transcripts per million (TPM) and determined by P_adj ≤ 0.05 and mean fold difference ≥2. z-Score is based on SD from mean TPM value per gene. Error bars show mean ± SEM. n.s., not significant.

Figure 7.

medLN cDCs are required for enhanced T cell priming and protection during the recall response. (A) Schematic for cDC depletion experiments: B6 CD45.1^+/+ mice were lethally irradiated and reconstituted with 10⁷ CD45.2^+/+ zbtb46-DTR bone marrow cells to create B6.zDTR bone marrow chimeras. After 10 wk, B6.zDTR chimeras were infected with X31 to generate B6.zDTR memory mice. B6.zDTR memory mice were treated with FTY720 and treated with either i.p. DT (40 µg/g body weight) or PBS as a vehicle control. 1 d later, mice were administered i.n. DT (20 µg/g body weight) or PBS vehicle control, and eFluor 450 proliferation dye–labeled CD45.1⁺CD45.2⁺ OT-II or OT-I cells were transferred into FTY720-treated B6.zDTR memory mice. 1 d later, mice were challenged with PR8-OVA (see Materials and methods), and recall protection and OT-II/OT-I priming was assessed 3 d after infection. (B) Preferential reduction in medLN cDCs following DT treatment in B6.zDTR memory mice depicted in representative flow cytometry plots (left) gated on CD45⁺ cells showing CD11c⁺ cDCs in the medLNs and lungs of PR8-OVA–challenged mice and in graphs of cDC numbers in each site (right). Data are representative of two independent experiments; n = 3 or 4 mice/group. (C) Weight loss morbidity of DT-treated and untreated memory mice at indicated times after PR8-OVA challenge. Data were compiled from two independent experiments; n = 6 mice/group. (D) DT treatment abrogates enhanced priming of medLN OT-II cells. Left: Flow cytometry plots show medLN and spleen OT-II proliferation and CD69 expression with or without DT treatment. Right: Graph showing proportion of medLN and spleen OT-II cells that express diluted proliferation dye. (E) Same as D but showing medLN OT-I cells. Data are compiled from two independent experiments; n = 7–9 mice/group in D and n = 4–10 mice/group in E. Significance was determined by Student’s unpaired t test; ****, P ≤ 0.0001; **, P ≤ 0.01. Error bars exhibit mean ± SEM. n.s., not significant.