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+ TRMs 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 TRM 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+ TRMs. 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+ TRMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8+ lung TRMs observed after immunization with recombinant Ad.

Cluster of differentiation (CD) 8+ tissue-resident memory T cells (TRMs) are a distinct subset of memory T cells that are established within barrier tissues, such as the lung, skin, and reproductive tract, where they provide a critical line of local defense against pathogen challenge. Canonically defined as extravascular cells that express surface markers known to promote retention (such as CD69 and/or CD103), TRMs share a core transcriptional signature that promotes their longevity and further distinguishes them from effector and central memory T cell (TEM and TCM, respectively) subsets (15). Within the lung and airways, CD8+ TRMs confer protection against a variety of respiratory pathogens, including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (24, 6, 7). Although they do not provide sterilizing immunity, lung-resident TRMs have been shown to significantly improve the immune response to heterologous influenza infection by rapidly reducing viral loads and limiting immunopathology (1, 811). However, although studies of TRM populations in the skin, intestinal tract, and reproductive tract indicate that CD8+ TRMs remain relatively stable within these tissues and provide long-lasting protection, the number of virus-specific CD8+ TRMs in the lung steadily declines over time to nearly undetectable levels (8, 1216). The mechanisms behind this loss of TRMs are not entirely understood, but it has been well established that the decline in lung TRMs greatly diminishes the protective capacity of cellular immunity against influenza virus (17).

Given the demonstrated importance of CD8+ lung TRMs in mediating protection against pulmonary challenge, identifying mechanisms governing their formation and longevity within the respiratory tract is of great interest. Despite many gaps in our current knowledge, several key factors, such as exposure to TGF-β and IL-15 and recognition of cognate Ag within the lung tissue, have been identified as important for the development and long-term survival of CD8+ lung TRMs (2, 1822). Several studies have also investigated the role of costimulatory molecules, such as 4-1BB/4-1BBL, in the formation and accumulation of TRMs, as well as their inclusion in vaccine platforms designed to target influenza virus (2328). Virus-based vectors, such as replication-deficient adenoviruses (Ads), are of particular interest as a vaccine platform candidate because they can be easily manipulated and have been shown to induce robust memory CD8+ T cell responses against viral and cancer Ags (2934). Most recently, Ad vectors have been used in the formulation of vaccines against the SARS-CoV-2 pandemic virus (3538). One key feature of Ad vectors that contributes to their success in inducing long-lasting cellular immunity is the ability of the vector to persist in vivo (3941). For example, a recent study demonstrated that Ad vectors can generate local Ag depots that support generation of local immunity (42). This finding complements prior work that showed that a combined systemic and local immunization strategy using an Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) results in formation of NP-specific CD8+ lung TRMs that provide protection against heterologous influenza virus for up to 1 y postimmunization, and that influenza NP Ag persists long term in the lungs of mice after immunization (31, 43). This starkly contrasts the dynamics of TRMs postinfection with influenza virus and could provide critical insight to the mechanisms of TRM generation and maintenance within the respiratory tract.

In this study, we further investigate mechanisms that contribute to the longevity of CD8+ lung TRMs and identify the cellular source of persistent Ag in AdNP-immunized animals. Prior findings suggested that circulating CD8+ T cells are pulled into the lung TRM pool in AdNP-immunized mice, potentially providing an explanation for the enhanced maintenance of lung TRMs and duration of protection (43). In this study, we found that CD8+ lung TRMs generated postinfection with influenza or immunization with AdNP are transcriptionally similar, indicating that cell-extrinsic factors are promoting TRM longevity. In addition, we found that help from CD4+ T cells is important for maintaining the TRM pool in the lungs and airways of mice immunized with AdNP. Using a combination of lineage tracing experiments and immunofluorescence microscopy, we identified alveolar macrophages as the cellular source of NP Ag in the lungs after intranasal (i.n.) immunization and confirmed that depletion of this cell subset reduces the number of CD8+ lung TRMs over time. These results provide further insight into the mechanisms driving enhancement of TRM in the respiratory tract after immunization with replication-deficient Ad vectors and will inform future design of vector-based vaccines against respiratory pathogens, including influenza virus.

C57BL/6J (wild-type) and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14) mice were bred in-house or purchased from Jackson Laboratory and were housed at Emory University under specific pathogen-free conditions. Mice were between 8 and 12 wk of age at time of infection, after which they were housed in specific animal biosafety level 2 conditions. Both male and female mice were used for experiments. All experiments were conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Emory University.

Replication-deficient AdNP was produced and titered as previously described (31, 44). Replication-deficient Ad serotype 5 expressing Cre recombinase (Ad-Cre) was obtained from SignaGen Laboratories. Before all infections, mice were anesthetized using isoflurane (Patterson Veterinary). For primary influenza infection, mice were inoculated i.n. with 30,000 egg ID50 influenza A/HKx31 (x31) in a 30-μl volume. For Ad immunizations, mice were inoculated with 2 × 107 PFUs of Ad via both i.n. and s.c. routes each in a 30-μl volume. For secondary infection experiments, mice received either 500 egg ID50 Sendai parainfluenza virus or 30,000 PFUs x31 NP N370Q (x31 NP) i.n. in a 30-μl volume. Control groups for challenge experiments received 30 μl i.n. of 1× PBS solution.

To distinguish tissue-resident cells from those in circulation, we i.v. labeled mice via tail-vein injection of fluorescent anti-CD3e (1.5 μg) or anti-CD45 Ab (4 μg) in 200 μl 1× PBS, and they rested for 5 min. Mice were subsequently euthanized by i.p. injection with Avertin (2,2,2-tribromoethanol) followed by brachial exsanguination. Spleen, lungs, and bronchoalveolar lavage (BAL) were then harvested. Lungs were enzymatically digested in Collagenase D (5 g/l; Roche) and DNase (2 × 106 U/l; Sigma) for 30 min at 37°C, with occasional mechanical dissociation. To enrich for lymphocytes, we centrifuged lung samples in a 40%/80% Percoll gradient. For Ad-Cre experiments, lungs were digested using Collagenase D (5 g/l), DNase (2 × 106 U/l), and Dispase (15 U/ml; Sigma) and then passed through a 70-μm filter without centrifugation over a Percoll gradient. Spleens were mechanically dissociated and then RBC lysed. For cell sorts, CD8+CD62L splenocytes were enriched for using a Miltenyi CD8a+ T cell isolation kit and biotinylated anti-CD62L Ab just before staining.

Single-cell suspensions were first FC blocked using murine 2.4G2 Ab. Samples were then stained with influenza-specific tetramer against NP366–374Db (provided by the National Institutes of Health Tetramer Core Facility at Emory University) for 1 h at room temperature, followed by extracellular staining for 30 min. Cell viability was determined using either Zombie fixable viability dye (BioLegend) or 7-aminoactinomycin D. All samples were run on either a Fortessa X20 or a Symphony A3 (BD Biosciences) flow cytometer. Flow cytometry data were analyzed using FlowJo v.10 software.

For each population, 100–2000 cells were sorted on a FACSAria II (BD Biosciences) directly into RLT buffer (Qiagen) containing 1% 2-ME and total RNA isolated using the Quick-RNA Microprep kit (Zymo Research). All resulting RNA was used as input for the SMART-seq v4 cDNA synthesis kit (Takara) with 12 cycles of PCR amplification. cDNA was quantitated and 200 pg of material was used with the NexteraXT kit and NexteraXT Indexing primers (Illumina, Inc.) in 12 cycles of PCR to generate libraries. Samples were quality checked on a bioanalyzer, quantitated by Qubit fluorometer, pooled at equimolar ratios, and sequenced on a NextSeq500 using 75-bp paired-end chemistry at the University of Alabama, Birmingham Helfin Genomics Core. Raw sequencing reads were mapped to the mm10 version of the mouse genome using STAR v2.5.3a (45), and duplicate reads were flagged using PICARD (http://broadinstitute.github.io/picard/) filtered based on the uniquely mappable and nonredundant reads. Reads mapping to exons for all unique ENTREZ genes were summarized using GenomicRanges v1.34.0 (46) package in R v3.5.2, and data were normalized using custom R/Bioconductor scripts. Differentially expressed genes (DEGs) were determined using edgeR v3.24.3 (47), and genes that displayed an absolute log2 fold change > l and a Benjamini–Hochberg false discovery rate corrected p < 0.05 were considered DEGs. Principal-component analysis (PCA) was performed using the vegan package v2.5.6 using the indicated set of DEGs. The sequencing dataset can be accessed in the GEO repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198980) under accession number GSE198980.

To deplete CD4+ T cells, we first injected mice i.p. with 200 μg of anti-CD4 mAb (clone GK1.5; BioXCell) or isotype control in 1× PBS, and then injected them with 100 μg i.p. every 3–4 d afterward for a total of 1 mo of treatment.

High-potency anionic liposomal clodronate and empty liposomes were obtained from FormuMax Scientific. Mice were anesthetized via injection with Avertin and then given 2 mg in a 100-μl volume intratracheally.

BAL was collected and resuspended in 1× PBS. A total of 30,000–50,000 cells were then concentrated onto a glass slide using a Thermo Shandon Cytospin 4 cytocentrifuge. Slides were subsequently H&E stained using standard protocols or fixed using 75:25 acetone/ethanol. Fixed slides were blocked using FACS buffer containing 1 μg/ml murine 2.4G2 Ab, 10% mouse serum, 10% rat serum, and 10% donkey serum. Staining was done in blocking buffer using anti-mouse CD11c-A594 (clone N418; BioLegend), anti-mouse influenza A nucleoprotein-FITC (clone 431; Abcam), rabbit anti-fluorescein-A488 (Life Technologies), and DAPI. Coverslips were applied using ProLong Gold antifade reagent, and samples were imaged the following day using a Zeiss Axio Observer Z1 immunofluorescence microscope with an Axiocam 506 monochromatic camera. Image processing was performed with Zen 2 software.

Cell counts were determined either manually using a hemocytometer or with a LUNA-II automatic cell counter (Logos Biosystems). Statistical analyses were performed using the GraphPad Prism Software.

To determine whether persistent Ag in AdNP-immunized mice has any potential cell-intrinsic effects on the genetic program of lung TRMs that result in their enhanced longevity, we performed RNA sequencing to compare the transcriptional profiles of influenza NP-specific lung TRMs (CD8+ i.v. AbNP+CD69+CD103+) and splenic TEMs (CD8+CD62LNP+) from mice either infected with x31 influenza or immunized with AdNP at 1-mo (35 d postinfection [d.p.i.], x31 and AdNP) and 1-y (365 d.p.i., AdNP only) time points (Fig. 1A, 1B). PCA revealed that TEMs and lung TRMs cluster separately, as expected, at both 1 mo (Fig. 1C) and 1 y (Fig. 1D) postinfection regardless of whether mice were given influenza or AdNP. Interestingly, we identified very few DEGs between lung TRMs from AdNP-immunized and x31-infected mice at 1 mo, suggesting that there is no significant transcriptional difference between lung TRMs formed after influenza infection or AdNP immunization (Fig. 1E). In contrast, we identified several DEGs between lung TRMs on days 35 and 365 postimmunization with AdNP (Fig. 1F). Notably, lung TRMs from AdNP-immunized mice had similar expression of genes from a known core TRM transcriptional program, including Itgae, Cdh1, Klf2, and S1pr1, confirming that these cells are bona fide TRMs at both time points postimmunization (4, 48) (Fig. 1G). However, the DEGs observed at 365 d postimmunization were enriched for TGF-β signaling (including Slc20a1, Smad3, and Cdh1) (Fig. 1G). Nevertheless, overall, we did not identify any transcriptional differences that would suggest the persistence of CD8+ lung TRMs in AdNP-immunized mice is due to a distinct genetic program that confers increased durability.

FIGURE 1.

Immunization with AdNP generates CD8+ TRMs that are transcriptionally alike those generated during a primary infection with influenza. (A) Experimental design. (B) Example gating strategy to sort for influenza NP (FluNP366–374)-specific splenic TEMs and CD69+CD103+ lung TRMs 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, two independent sorts. For AdNP, n = 10 mice per sort, two independent sorts per time point. (C) PCA plot of 2250 DEGs identified in influenza-infected and AdNP-immunized mice on day 35 postinfection. (D) PCA of 3234 genes identified on days 35 and 365 postimmunization with AdNP. (E) Volcano plot illustrating DEGs identified when comparing CD69+CD103+ lung TRMs from AdNP-immunized mice with those from x31 influenza-infected mice on day 35 postinfection. (F) Volcano plot illustrating DEGs between CD69+CD103+ lung TRMs from AdNP-immunized mice on days 35 and 365 postimmunization. (G) Heatmaps of selected genes from FluNP-specific splenic TEMs and CD69+CD103+ lung TRMs from AdNP-immunized mice related to TGF-β signaling and a core TRM signature.

FIGURE 1.

Immunization with AdNP generates CD8+ TRMs that are transcriptionally alike those generated during a primary infection with influenza. (A) Experimental design. (B) Example gating strategy to sort for influenza NP (FluNP366–374)-specific splenic TEMs and CD69+CD103+ lung TRMs 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, two independent sorts. For AdNP, n = 10 mice per sort, two independent sorts per time point. (C) PCA plot of 2250 DEGs identified in influenza-infected and AdNP-immunized mice on day 35 postinfection. (D) PCA of 3234 genes identified on days 35 and 365 postimmunization with AdNP. (E) Volcano plot illustrating DEGs identified when comparing CD69+CD103+ lung TRMs from AdNP-immunized mice with those from x31 influenza-infected mice on day 35 postinfection. (F) Volcano plot illustrating DEGs between CD69+CD103+ lung TRMs from AdNP-immunized mice on days 35 and 365 postimmunization. (G) Heatmaps of selected genes from FluNP-specific splenic TEMs and CD69+CD103+ lung TRMs from AdNP-immunized mice related to TGF-β signaling and a core TRM signature.

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CD4+ T cells are important for proper maintenance and recall of influenza-specific CD8+ memory T cells in the lungs and airways (49, 50). Furthermore, IFN-γ produced by CD4+ T cells is critical for formation of protective CD103+ CD8+ TRMs in the lung after infection with influenza virus (51). To investigate whether CD4+ T cell–dependent signals are required for long-term maintenance of CD8+ TRMs in AdNP-immunized mice, we treated mice with anti-CD4–depleting Ab starting 30 d postimmunization (Fig. 2A). After administering depleting Ab for a total of 1 mo, we confirmed depletion of CD4+ T cells in all tissues (data not shown) and evaluated the number of influenza NP-specific CD8+ TRMs (Fig. 2B). As expected, there was no change in the number of CD8+ splenic TEMs on depletion of CD4+ T cells. However, within the lung and airways, depletion of CD4+ T cells resulted in a significant reduction in the number of NP-specific CD8+ TRMs when compared with mice that received an isotype control Ab. Furthermore, the decrease in the overall number of CD8+ TRMs in the lungs and airways correlated in both tissues with a reduction in CD69+CD103+ NP-specific CD8+ TRMs (Fig. 2C, 2D). These results show that help from CD4+ T cells plays an important role in the long-term maintenance of CD8+ TRMs in the lungs and airways of mice immunized with AdNP.

FIGURE 2.

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

FIGURE 2.

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

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Although it has been established that influenza NP Ag is still present in mice immunized with AdNP for at least several months after immunization, the cellular source of this persistent Ag reservoir has not yet been identified (43). To investigate this, we used a replication-deficient Ad-Cre to immunize Ai14 (tdTomato) reporter mice, in which cells express the reporter protein tdTomato after Cre-mediated recombination. Within the lung, tdTomato fluorescence was predominantly observed in alveolar macrophages up to at least a year postimmunization with Ad-Cre (Fig. 3A, 3B). Minimal, if any, fluorescence was observed in dendritic cells (Fig. 3B). Even as early as day 4 postimmunization with Ad-Cre, tdTomato fluorescence was mostly limited to alveolar macrophages and was not detected in any other cell type, including fibroblasts, epithelial cells, and monocytes (Supplemental Fig. 1). In addition, the frequency of tdTomato+ alveolar macrophages was varied at all time points examined (Fig. 3C). To confirm this finding, we obtained a cytospin of BAL samples from naive and AdNP-immunized mice (90 d.p.i.). Immunofluorescent staining revealed colocalization of CD11c and influenza nucleoprotein at memory after immunization with AdNP (Fig. 3D). Combined, these experiments identify alveolar macrophages as the cellular source of persistent influenza NP Ag after i.n. immunization with recombinant Ad.

FIGURE 3.

Alveolar macrophages are the cell source of persistent influenza NP Ag 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 (naive) or Ad-Cre at indicated time points. (C) Frequency of tdTomato+ alveolar macrophages at indicated time points. n = 3–5 mice per time point, two experiments per time point. (D) H&E staining and immunofluorescence microscopy of BAL samples from mice that were naive (top row) or 90 d postimmunization with AdNP (bottom row) showing CD11c (red), influenza nucleoprotein (FluNP, green), and DAPI (blue). Original images were taken at ×100 magnification.

FIGURE 3.

Alveolar macrophages are the cell source of persistent influenza NP Ag 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 (naive) or Ad-Cre at indicated time points. (C) Frequency of tdTomato+ alveolar macrophages at indicated time points. n = 3–5 mice per time point, two experiments per time point. (D) H&E staining and immunofluorescence microscopy of BAL samples from mice that were naive (top row) or 90 d postimmunization with AdNP (bottom row) showing CD11c (red), influenza nucleoprotein (FluNP, green), and DAPI (blue). Original images were taken at ×100 magnification.

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Alveolar macrophages are among the first responders to influenza infection, and their depletion has been shown to result in increased morbidity and mortality during infection (52, 53).

Given that our data show that alveolar macrophages are a source of prolonged influenza NP Ag after i.n. immunization with AdNP, we hypothesized that depletion of this population would result in decreased maintenance of NP-specific CD8+ TRMs over time. We therefore depleted alveolar macrophages by administering liposomal clodronate at 1 mo postimmunization with AdNP (Fig. 4A). Treatment resulted in a significant reduction of alveolar macrophages in both lung and airways, when compared with injection of empty liposomes or mock treatment (Supplemental Fig. 2). Although depletion of alveolar macrophages had no effect on the number of influenza NP-specific CD8+ TRMs in the lung, we did observe a significant decrease in the number of TRMs within the airways, including CD69CD103, CD69+CD103, and CD69+CD103+ NP-specific CD8+ TRMs (Fig. 4B, 4C). These data further support the observations that alveolar macrophages provide a source of persistent influenza NP Ag in animals after immunization with recombinant Ad.

FIGURE 4.

Depletion of alveolar macrophages results in reduced longevity of influenza NP-specific CD8+ TRMs. (A) Experimental design. (B) Number of influenza NP-specific CD8+ TRMs in the spleen, lung, and BAL in mice immunized with AdNP and then intratracheally administered empty liposomes or liposomes containing clodronate. (C) Example staining of influenza NP-specific CD8+ TRMs 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 CD69CD103, CD69+CD103, and CD69+CD103+ influenza NP-specific CD8+ TRMs in the BAL. n = 3–8 mice per group, three 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. ns, not significant.

FIGURE 4.

Depletion of alveolar macrophages results in reduced longevity of influenza NP-specific CD8+ TRMs. (A) Experimental design. (B) Number of influenza NP-specific CD8+ TRMs in the spleen, lung, and BAL in mice immunized with AdNP and then intratracheally administered empty liposomes or liposomes containing clodronate. (C) Example staining of influenza NP-specific CD8+ TRMs 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 CD69CD103, CD69+CD103, and CD69+CD103+ influenza NP-specific CD8+ TRMs in the BAL. n = 3–8 mice per group, three 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. ns, not significant.

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Given our identification of alveolar macrophages as an Ag source after i.n. immunization with AdNP, we next investigated the impact of subsequent, antigenically distinct, respiratory infections known to deplete alveolar macrophages on the maintenance of pre-existing NP-specific CD8+ TRMs. To do so, we first infected AdNP-immunized mice with Sendai virus, a murine parainfluenza virus, and then subsequently infected them with an x31 influenza strain that does not present NP Ag on MHC class I (x31 NP) and would therefore not boost the pre-existing influenza NP-specific TRM population (54) (Fig. 5A). We then examined the impact of these infections on the number of NP-specific CD8+ TRMs generated during the initial AdNP immunization. After initial infection of AdNP-immunized mice with Sendai virus, we observed no significant effect on the number of NP-specific CD8+ TRMs (Fig. 5B, 5D). However, the number of NP-specific CD8+ TRMs was significantly reduced in the lungs and airways after the second unrelated infection with x31 NP influenza when compared with mock infection (Fig. 5C). Unsurprisingly, subsequent infection with x31 NP had no effect on the number of NP-specific memory CD8+ T cells in the spleen (Fig. 5C). After both Sendai virus and x31 NP infections, the number of CD69+ CD103+ NP-specific CD8+ TRMs also declined in both the lung and airways (Fig. 5E). Lastly, infecting Ad-Cre–immunized reporter mice with Sendai virus and x31 NP also resulted in an overall decline in the percentage of tdTomato+ alveolar macrophages when compared with animals that were mock infected (Fig. 5F). These findings underscore the importance of alveolar macrophages for the long-term maintenance of NP-specific CD8+ TRMs generated after i.n. immunization with AdNP.

FIGURE 5.

Subsequent respiratory viral infections impact the maintenance of influenza NP-specific CD8+ TRMs in AdNP-immunized mice. (A) Experimental design. (B) Number of influenza NP-specific CD8+ TRMs 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, two independent experiments. (C) Number of influenza NP-specific CD8+ TRMs 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, two independent experiments. (D) Number of CD69+CD103+ influenza NP-specific CD8+ TRMs in the lung and BAL after Sendai parainfluenza infection of AdNP-immunized mice. (E) Number of CD69+ CD103+ influenza NP-specific CD8+ TRMs in the lung and BAL postinfection 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 (A). n = 8–14 mice per group, two 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+ TRMs in AdNP-immunized mice. (A) Experimental design. (B) Number of influenza NP-specific CD8+ TRMs 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, two independent experiments. (C) Number of influenza NP-specific CD8+ TRMs 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, two independent experiments. (D) Number of CD69+CD103+ influenza NP-specific CD8+ TRMs in the lung and BAL after Sendai parainfluenza infection of AdNP-immunized mice. (E) Number of CD69+ CD103+ influenza NP-specific CD8+ TRMs in the lung and BAL postinfection 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 (A). n = 8–14 mice per group, two 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.

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Establishment of a robust memory T cell response is critical to the success of T cell–based vaccines. However, in the case of respiratory infections, the steady decline of lung-resident CD8+ TRMs over time presents a concern in generating long-term immunity. Although the mechanism behind this decline is not entirely understood, a recent study has shown that the harsh environment of the lung and airways leads to a high rate of apoptosis among CD8+ TRMs (55). However, when mice are immunized with a replication-deficient AdNP using a combination of i.n. and systemic routes, the number of pulmonary CD8+ TRMs is maintained long term (31). These CD8+ lung TRMs are protective for up to at least a year postimmunization and are replenished by circulating TEMs being recruited into the TRM pool after encountering Ag locally within the lung tissue (43). In this study, we demonstrate that CD8+ lung TRMs generated after immunization with AdNP or infection with influenza virus have similar transcriptional profiles, indicating that immunization with AdNP does not result in cell-intrinsic differences responsible for the improved longevity of CD8+ lung TRMs. In addition, we found further support for the model of TEM recruitment to the lungs after immunization with AdNP when we deplete CD4+ T cells postimmunization and observe a significant decrease in the number of CD8+ TRMs in the lungs and airways. A prior study showed that CD4+ T cells are required for generation of CD103+ CD8+ lung TRMs and promote their migration to the airways via an IFN-γ–dependent mechanism (51). CD4+ T cell help was also found to be associated with lower expression of the transcription factor T-bet, thereby allowing for TGF-β–mediated induction of CD103 (51). We hypothesize a similar mechanism is occurring in our model. Lastly, lineage tracing using an Ad-Cre identified alveolar macrophages as a primary cell source of Ag in the respiratory tract. We were able to confirm this finding by observing colocalization of CD11c and influenza nucleoprotein in airway cells isolated from mice immunized with AdNP. Combined with prior findings, our data support a model in which an Ag reservoir maintained in long-lived alveolar macrophages helps promote differentiation of CD8+ lung TRMs from the circulating TEM pool.

It has been well established that anatomic location directly impacts the development and maintenance of TRMs (56). Because generation of CD8+ TRMs in the lung is largely dependent on recognition of local cognate Ag, establishing a reliable Ag depot within the tissue can be critical to the success of immunization against respiratory pathogens. Interestingly, a recent report showed fibroblastic stromal cells in the lung can also serve as long-lived Ag depots after i.v. administration of an Ad5-based vector and support inflationary memory CD8+ T cells in an IL-33–dependent manner (42). Although this seemingly contrasts our finding, we believe both studies emphasize the importance of the route of immunization in defining unique mechanisms and cell types that can support the maintenance of lung TRMs by acting as Ag depots. For example, alveolar macrophages may be shielded from infection by blood-borne vectors, whereas i.n. delivery of the vectors does not allow for efficient infection of cells within the lung parenchyma, such as fibroblastic stromal cells. In addition to lung fibroblastic stromal cells, IL-33 is produced by activated macrophages in both humans and mice and has been shown to influence CD8+ TRM formation by downregulating expression of KLF2 and inducing expression of CD69 and CD103 (5760). It is therefore conceivable that IL-33 could also be playing a role in our system. Importantly, i.m. injection remains the most popular route of vaccination and is currently in use for the mRNA- and Ad vector–based SARS-CoV-2 vaccines, but it can induce suboptimal mucosal immune responses (61). Alternatively, i.n. administration is widely accepted as the ideal route for targeting the respiratory tract because it most accurately mimics the natural route of infection. However, prior work showed that combined i.n. and s.c. injections of AdNP were superior compared with i.n. immunization alone in establishing a long-lived CD8+ lung TRM population, suggesting that local Ag supply on its own is not sufficient for maximal T cell responses (31).

As some of the first immune cells to encounter pathogens within the airways, alveolar macrophages have long been appreciated as important players in respiratory immunity, with their depletion resulting in increased viral load, pulmonary damage, and mortality after infection with influenza virus (52, 53, 62, 63). They have also been shown to be important for the establishment and reactivation of memory CD8+ T cells in the lung (21, 64, 65). One likely reason why i.n. immunization with AdNP results in prolonged maintenance of the CD8+ lung TRM pool is the longevity of alveolar macrophages (66, 67). In both humans and mice, macrophage populations are maintained for several months to years after formation (6871). However, after infection with influenza virus, alveolar macrophages undergo high levels of cell death (72, 73). In contrast, replication-deficient Ad vectors are capable of transducing macrophages without causing their elimination (7480). Indeed, we show in this study that Ad-Cre–transduced alveolar macrophages persist in the lung at varying frequencies for up to a year postimmunization. Furthermore, loss of alveolar macrophages through depletion using liposomal clodronate resulted in a significant decline in the number of CD8+ TRMs in the airways, despite no significant change in the number of CD8+ TRMs within the lung tissue. We hypothesize that this is likely due to alveolar macrophages residing predominantly within the airway spaces. Another possibility is that a small frequency of interstitial macrophages harbors persistent Ag and supports maintenance of CD8+ TRMs within the lung interstitium; however, we are not able to distinguish this using our system. Lastly, it is also important to note that although the use of liposomal clodronate is widely considered the standard method of depleting alveolar macrophages, its effects are not exclusive to this population, and it is known to target dendritic cells as well. Although we cannot ensure that dendritic cells were not impacted during our depletion experiments, our Ad-Cre data clearly demonstrate that dendritic cells are transduced by Ad-Cre at very low frequency and do not persist over time.

Recombinant Ad vectors (rAds) display broad tissue tropism and have been shown to transduce a variety of immune and nonimmune cell types both in vitro and in vivo. Although murine macrophages do not express the classical Coxsackie and Ad receptor, rAd vectors, including human Ad5 and Ad26, two vectors currently being developed as vaccines for SARS-CoV-2, have been shown to transduce macrophages using scavenging receptors (38, 8185). Less is known about the entry methods used by non-human rAd vectors; nevertheless, given the ease of altering rAd vectors, targeting Ad vectors to specific tissues and cell types is possible and is an important consideration for future vaccine design (86). However, given the sensitive nature of the lung tissue, careful consideration must also be taken when designing any immunization strategy that creates a persistent Ag source. First, although i.n. + s.c. immunization with AdNP results in prolonged protection against challenge with influenza virus, we show in this article that subsequent unrelated infection(s) also results in a decline in the number of NP-specific CD8+ TRMs. This presents a potential limitation to targeting Ag to alveolar macrophages, because recurrent respiratory infections from diverse pathogens are likely to deplete the reservoir. In addition, the possibility of prolonged inflammation and immunopathology that accompany persistent Ag must be more thoroughly evaluated.

In summary, our results identify alveolar macrophages as a persistent cellular source of Ag after i.n. immunization with a recombinant AdNP. Transduced alveolar macrophages are maintained for at least a year postimmunization and are essential for continual replenishment of the CD8+ TRM pool. Furthermore, we show that persistent Ag does not induce T cell–intrinsic changes that account for the longevity of CD8+ TRMs. These results further define mechanisms that promote CD8+ lung TRM generation and maintenance and have important implications for the design of T cell–based vaccines against respiratory pathogens.

This work was supported by National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH) Grant R35HL150803. J.L.L. was supported by NHLBI NIH Grant F31HL156639. We thank the NIH Tetramer Core Facility (Contract 75N93020D00005) for providing class I tetramers. This research project was supported by the Emory University School of Medicine Flow Cytometry Core and the Pediatric/Winship Flow Cytometry Core.

The online version of this article contains supplemental material.

J.L.L. and J.E.K. designed the study; J.L.L. performed experiments with input from I.U., J.M.B., and J.E.K.; J.L.L., C.D.S., and T.M. analyzed the data; J.L.L. wrote the manuscript; and J.M.B., A.R.T., J.P.C., and J.E.K. edited the manuscript.

The sequencing dataset has been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE198980) repository under accession number GSE198980.

Abbreviations used in this article:

     
  • Ad

    adenovirus

  •  
  • Ad-Cre

    Ad serotype 5 expressing Cre recombinase

  •  
  • AdNP

    Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein

  •  
  • BAL

    bronchoalveolar lavage

  •  
  • CD

    cluster of differentiation

  •  
  • DEG

    differentially expressed gene

  •  
  • d.p.i.

    days postinfection

  •  
  • i.n.

    intranasally

  •  
  • PCA

    principal-component analysis

  •  
  • rAd

    recombinant Ad vector

  •  
  • SARS-CoV-2

    severe acute respiratory syndrome coronavirus 2

  •  
  • TCM

    central memory T cell

  •  
  • TEM

    effector memory T cell

  •  
  • TRM

    tissue-resident memory T cell

  •  
  • x31

    influenza A/HKx31

  •  
  • x31 NP

    x31 NP N370Q

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The authors have no financial conflicts of interest.

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