Persistent Antigen Harbored by Alveolar Macrophages Enhances the Maintenance of Lung-Resident Memory CD8+ T Cells

Abstract

Lung tissue-resident memory T cells are crucial mediators of cellular immunity against respiratory viruses; however, their gradual decline hinders the development of T cell-based vaccines against respiratory pathogens. Recently, studies using adenovirus (Ad)-based vaccine vectors have shown that the number of protective lung-resident CD8⁺ T_RMs can be maintained long term. In this article, we show that immunization of mice with a replication-deficient Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) generates a long-lived lung T_RM pool that is transcriptionally indistinct from those generated during a primary influenza infection. In addition, we demonstrate that CD4⁺ T cells contribute to the long-term maintenance of AdNP-induced CD8⁺ T_RMs. Using a lineage tracing approach, we identify alveolar macrophages as a cell source of persistent NP Ag after immunization with AdNP. Importantly, depletion of alveolar macrophages after AdNP immunization resulted in significantly reduced numbers of NP-specific CD8⁺ T_RMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8⁺ lung T_RMs observed after immunization with recombinant Ad.

Figure 1.. Immunization with AdNP generates CD8⁺ T_RM that are transcriptionally alike those generated during a primary infection with influenza.

(A) Experimental design. (B) Example gating strategy to sort for influenza NP (FluNP_366–374)-specific splenic T_EM and CD69⁺ CD103⁺ lung T_RM from mice either infected with x31 influenza or immunized with AdNP. Final sorted populations are highlighted in red. For x31, n = 10–20 mice per sort, 2 independent sorts. For AdNP, n = 10 mice per sort, 2 independent sorts per timepoint. (C) Principal component analysis (PCA) plot of 2250 differentially expressed genes (DEGs) identified in influenza-infected and AdNP-immunized mice on day 35 post-infection. (D) PCA of 3234 genes identified on day 35 and 365 post-immunization with AdNP. (E) Volcano plot illustrating DEGs identified when comparing CD69⁺ CD103⁺ lung T_RM from AdNP-immunized mice to those from x31 influenza-infected mice on day 35 post-infection. (F) Volcano plot illustrating DEGs between CD69⁺ CD103⁺ lung T_RM from AdNP-immunized mice on days 35 and 365 post-immunization. (G) Heatmaps of selected genes from FluNP-specific splenic T_EM and CD69⁺ CD103⁺ lung T_RM from AdNP-immunized mice related to TGF-ß signaling and a core T_RM signature.

Figure 2.. CD4⁺ T cells are important for the maintenance of CD8⁺ T_RM following immunization with AdNP.

(A) Experimental design. (B) Number of FluNP-specific CD8⁺ T_RM in the spleen, lung, and bronchoalveolar lavage (BAL) following depletion of CD4⁺ T cells. For isotype control, n = 9 mice total, 2 independent experiments. For anti-CD4, n = 10 mice total, 2 independent experiments. (C) Example staining for CD69 and CD103 subsets amongst FluNP-specific CD8⁺ T_RM in the lung and BAL. (D) Number of CD69⁺ CD103⁺ FluNP-specific CD8⁺ T_RM in the lung and BAL following depletion of CD4⁺ T cells. n = 4–5 mice per group, 2 independent experiments. Significance was determined using a Mann-Whitney test. Data represent mean ± SEM. P values are as follows: * = p<0.05, ** = p<0.01, *** = p<0.001, ns= not significant.

Figure 3.. Alveolar macrophages are the cell source of persistent influenza NP antigen in AdNP-immunized mice.

(A) Example gating strategy for alveolar macrophages and dendritic cells in the lung of Ai14 reporter mice immunized with Ad-Cre. (B) Expression of tdTomato by alveolar macrophages (top row) and dendritic cells (bottom row) from mice immunized with either PBS (naïve) or Ad-Cre at indicated timepoints. (C) Frequency of tdTomato⁺ alveolar macrophages at indicated timepoints. n = 3–5 mice per timepoint, 2 experiments per timepoint. (D) H&E staining and immunofluorescence microscopy of BAL samples from mice that were naïve (top row) or 90 days post-immunization with AdNP (bottom row) showing CD11c (red), influenza nucleoprotein (FluNP, green), and DAPI (blue). Original images were taken at 100X magnification.

Figure 4.. Depletion of alveolar macrophages results in reduced longevity of influenza NP-specific CD8⁺ T_RM

(A) Experimental design. (B) Number of influenza NP-specific CD8⁺ T_RM in the spleen, lung, and BAL in mice immunized with AdNP and then intra-tracheally administered empty liposomes or liposomes containing clodronate. (C) Example staining of influenza NP-specific CD8⁺ T_RM based on expression of CD69 and CD103 from the BAL of mice immunized with AdNP and then treated with empty or clodronate liposomes. (D) Number of CD69⁻ CD103⁻, CD69⁺ CD103⁻, and CD69⁺ CD103⁺ influenza NP-specific CD8⁺ T_RM in the BAL. n = 3–8 mice per group, 3 independent experiments. Significance was determined using a Mann-Whitney test. Data represent mean ± SEM. P values are as follows: * = p<0.05, ** = p<0.01, *** = p<0.001, ns= not significant.

Figure 5.. Subsequent respiratory viral infections impact the maintenance of influenza NP-specific CD8⁺ T_RM in AdNP-immunized mice

(A) Experimental design. (B) Number of influenza NP-specific CD8⁺ T_RM in the spleen, lung, and BAL of AdNP-immunized mice subsequently infected with Sendai parainfluenza or mock infected with PBS. n = 5 mice per group, 2 independent experiments. (C) Number of influenza NP-specific CD8⁺ T_RM in the spleen, lung, and BAL of AdNP-immunized mice subsequently infected with Sendai parainfluenza followed by x31 NP⁻ influenza. n = 5–8 mice per group, 2 independent experiments. (D) Number of CD69⁺ CD103⁺ influenza NP-specific CD8⁺ T_RM in the lung and BAL following Sendai parainfluenza infection of AdNP-immunized mice. (E) Number of CD69⁺ CD103⁺ influenza NP-specific CD8⁺ T_RM in the lung and BAL following infection of AdNP-immunized mice with both Sendai parainfluenza and x31 NP⁻ influenza. (F) Frequency of tdTomato⁺ alveolar macrophages in Ai14 reporter mice immunized with Ad-Cre and then either mock infected (PBS) or infected with Sendai parainfluenza and x31 NP⁻ influenza as described in part A. n = 8–14 mice per group, 2 independent experiments. Significance was determined using a Mann-Whitney test. Data represent mean ± SEM. P values are as follows: * = p<0.05, ** = p<0.01, *** = p<0.001, ns= not significant.