Ethics statement
The study was approved by the Government of Lower Franconia, which nominated an external ethics committee that authorized the experiments. Studies were performed under the project license AZ 55.2.2-2532-2-1081.
Adenoviral vector vaccines
The replication-deficient (∆E1 ∆E3) adenoviral vectors are based on the human serotype Ad5. The encoded sequences for hemagglutinin (rAd-HA) and nucleoprotein (rAd-NP) are derived from H1N1 A/PR/8/34, and rAd-IL-1β contains the sequence for murine mature IL-1β as previously described23.
Mice
Five-weeks-old female BALB/cJRj mice were purchased from Janvier (Le Genest-Saint-Isle, France, ref: SC-BALBj-F) and housed in individually ventilated cages in accordance with German law and institutional guidelines under specific pathogen-free (SPF) conditions with constant temperature (20–24 °C) and humidity (45–65%) on a 12 h/12h-light/dark cycle. The housing took place in the animal facility of the University Hospital of Erlangen (Preclinical Experimental Animal Center, PETZ). The studies were conducted according to the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and the Society of Laboratory Animal Science (GV-SOLAS). For our study, we used exclusively female mice due to the advantages in regard to the housing conditions. However, since we aim for comparative analyses of different treatment or prevention strategies, the superiority of one strategy above another can be answered independently of the sex of individuals.
Immunization, viral infections and challenges
For priming, seven-weeks-old female BALB/cJRj mice were either immunized with 2 × 108 particles of each antigen- and adjuvant-encoding adenoviral vector or infected with a sublethal dose of 100 PFU of influenza H1N1 A/PR/8/34 both in a 50 µl volume via the intranasal route. For challenge experiments, mice received either 10,000 PFU influenza H3N2 A/HK/68, 1 × 106 PFU of the respiratory syncytial virus (RSV), or 10 µg lipopolysaccharide (LPS Merck, Cat: L4524) i.n. in a total volume of 50 µl.
Weight monitoring
To evaluate the morbidity of viral infections or the effect of LPS treatment, the percentage of body weight loss was determined daily until the mice reached their initial weight again or the pre-defined endpoints. Any mice that lost more than 25% of their initial body weight without gaining weight within the next 48 h were euthanized by an overdose of inhaled isoflurane.
Pulse-oximetry
Oxygen saturation in blood was measured via a MouseOxTM Pulse-oximeter as an indirect measure of lung function (Starr Life Science, Oakmont, PA). A mouse-adapted pulse-oximeter clip was positioned on the throat of conscious mice, and heart rate, oxygen saturation (SpO2), breath rate, breath distension, and pulse distension were monitored via the MouseOx Plus software.
Blood sampling and bronchoalveolar lavage fluid (BALF)
For serology, blood samples were collected through puncture of the retro-orbital sinus under light anesthesia. BALF samples were collected at postmortem by washing the lungs with 2 × 1 ml PBS (Cat: Gibco, 10x, Cat: 70011-036)) through the cannulated trachea.
Flow cytometric analyses of cellular infiltration
BALF from infected or immunized mice were centrifuged (5 min, 5000 × g) and one half of the cellular fraction was stained with anti-Gr1-AF488 (clone RB6-8C5, eBioscience, Cat: 53-5931-82, 1:300), anti-CD49b-PE (clone DX5, eBioscience, Cat: 12-5971-82, 1:300), anti-CD45-PerCP-Cy5.5 (clone 30-F11, BD Biosciences, Cat: 550994, 1:300), anti-CD19-PE-Cy7 (clone 1D3, BD Biosciences, Cat: 552854, 1:1000), anti-F4/80-APC (clone BM8, BioLegend, Cat: 123116, 1:300), anti-CD11b-APC-Cy7 (clone M1/70, BD Biosciences, Cat: 557657, 1:300), anti-CD11c-BV421 (clone HL3, BD Biosciences, Cat: 560521, 1:100), anti-CD4-BV605 (clone RM4-5, BioLegend, Cat: 100547, 1:300), anti-CD8α-BV711 (clone 53-6.7, BioLegend, Cat: 100747, 1:300) and anti-CD3e-BV510 (clone 145-2C11, BioLegend, Cat: 100353, 1:200). All antibodies are listed in Supplementary Table 2.
Lymphocyte isolation and intracellular cytokine staining (ICS)
To differentiate between tissue-resident and circulating lymphocytes, intravascular (iv)-staining was performed70. For this purpose, 2 μg anti-CD45-BV510 (clone 30-F11, BioLegend, Cat: 103138) were injected intravenously in a total volume of 150 µl PBS three min before euthanizing the animal. After euthanasia, BALF and lungs were harvested for evaluation of the T-cell responses at the indicated time points. First, lungs were cut into small pieces and enzymatically digested in collagenase D (250 µ/ml,Merck, Cat: C7657) and DNase I (80 µ/ml, AppliChem, Cat: A3778) diluted in 2 ml R10 medium (RPMI 1640 (gibco, Cat: 31870-025) supplemented with 10% FCS (Anprotec, Cat: AC-SM-0027), 2 mM L-Glutamine (Gibco, Cat: 35050-038), 10 mM HEPES (Applichem, Cat: A3724), 50 µM β-mercaptoethanol (Gibco, Cat: 31350-010) and 1% penicillin/streptomycin (Gibco, Cat: 15140-122) for 45 min at 37 °C. Disaggregated lung tissues were filtered through 70-μm cell strainers, and red blood cells were lysed by resuspension in ammonium-chloride-potassium (ACK Lysing Buffer, Gibco, Cat: A10492). For in vitro restimulation, 100 µl lung cell suspension was plated in a 96-well plate and 100 μl R10 medium containing monensin (2 μM, Sigma, Cat: M5273), anti-CD28 (1 μg/ml, Invitrogen, Cat: 14-0281), anti-CD107a-FITC (clone 1D4B, BD Bioscience, Cat: 553793, 1:100), and 5 μg/ml of the respective MHC-I/II peptides: MHC-II peptide HA110-120 (SFERFEIFPKE), MHC-I peptide HA518-526 (IYSTVASSL), or MHC-II peptide NP55-69 (RLIQNSLTIERMVL), MHC-I peptide NP147-155 (TYQRTRALV), or MHC-I peptide M282-90 (SYIGSINNI) were added and incubated for 6 h at 37 °C. Positive controls were stimulated in an antigen-independent manner by using anti-CD28 (1 μg/ml, Invitrogen, Cat: 14-0282) and anti-CD3ε (2 μg/ml, BD Bioscience, Cat: 553057). Non-stimulated samples were used for subtraction of background cytokine production (negative values were set at zero). After stimulation, the cells were stained extracellularly with anti-CD8α-Pacific Blue (clone 53-6.7, BioLegend, Cat: 100725, 1:300), anti-CD4-PerCP-eFluor710 (clone RM4-5, Invitrogen, Cat: 46-0042-82, 1:2000), and a Fixable Viability Dye-eFluor780 (Thermo Fisher, Cat: 65-0865-14, 1:100). After fixation, permeabilization, and Fc receptors blockage (anti-CD16/-CD32 (2 μg/ml, Invitrogen, Cat: 14-0161-86)), cells were stained intracellularly with anti-IFNγ-PE (clone XMG1.2, BioLegend, Cat: 505808), anti-IL-2-APC (clone JES6-5H4, BioLegend, Cat: 503810), and anti-TNFα-PE-Cy7 (clone MP6-XT22, BioLegend, Cat: 506324) (all 1:300). Data were acquired on an Attune NxT Flow Cytometer and analyzed using FlowJo v.10 software (Tree Star Inc.). The gating strategy is shown in Supplementary Fig. 3 (Supplementary Fig. 3). All antibodies are listed in Supplementary Table 2.
Flow cytometric analysis of T-cell phenotypes and pentamer staining
Single-cell suspensions were stained with influenza-specific H-2KD HA533-541 pentamer or H-2KD NP147-155 pentamer or RSV-specific H2KD M282-90 pentamer (all ProImmune, 1:40) for 20 min at 4 °C. Cells intended for CD4+ T-cell analysis were incubated without pentamer staining. In a second extracellular staining step, cells provided for CD8+ T-cell analysis were incubated with anti-CD127-FITC (clone A7R34, BioLegend, Cat: 135008, 1:300), anti-CD103-BV605 (clone 2E7, BioLegend, Cat: 121433, 1:200), anti-CD69-PerCP/Cy5.5 (clone H1.2F3, BioLegend, Cat: 104522, 1:200), anti-CD45.2-PE/Dazzle594 (clone 104, BioLegend, Cat: 109846, 1:500), anti-CD8α-Pacific Blue (clone 53-6.7, BioLegend, Cat: 100725, 1:300), anti-KLRG1-PE-Cy7 (clone 2F1, invitrogen, Cat: 25-5893-82, 1:300), anti-P2X7R-PE (clone 1F11, BioLegend, Cat: 148704, 1:300), and anti-IFITM3-Biotin (clone aa2-57, R&D Systems, Cat: BAF3377, 1:300). Phenotypic characteristics of CD4+ T cells were specified by staining with anti-CD44-APC (clone IM7, BioLegend, Cat: 103018, 1:200), anti-CD4-AF488 (clone GK1.5, BioLegend, Cat: 100423, 1:200), anti-CD103-BV605 (clone 2E7, BioLegend, Cat: 121433, 1:200), anti-CD69-PerCP/Cy5.5 (clone H1.2F3, BioLegend, Cat: 104522, 1:200), anti-CD45.2-PE/Dazzle594 (clone 104, BioLegend, Cat: 109846, 1:500), anti-CD11a-eFluor710 (clone M17/4, invitrogen, Cat: 48-0111-82, 1:300), anti-CXCR3-APC-Fire750 (clone CXCR3-173, BioLegend, Cat: 126539, 1:200), anti-P2X7R-PE (clone 1F11, BioLegend, Cat: 148704, 1:300), and anti-IFITM3-Biotin (clone aa2-57, R&D Systems, Cat: BAF3377, 1:300). Subsequently, streptavidin-BV711 (BioLegend, Cat: 405241, 1:300) was used for flow cytometric detection of IFITM3. All samples were aquired on an Attune NxT Flow Cytometer, and flow cytometry data were analyzed using FlowJo v.10 software. Supplementary Fig. 4 (CD8) and Supplementary Fig. 6 (CD4) represent the respective gating strategy (Suppl. Figs. 4, 6). All antibodies are listed in Supplementary Table 2.
FACS-based antibody analysis
Doxycycline-inducible HEK293A cell lines stably transduced with lentiviral particles encoding either HA or NP of H1N1/PR/8/34 or HA of H3N2 A/HK/68 were used for the detection and quantification of specific antibodies in serum and BALF samples. The respective antigen was overexpressed by doxycycline (Sigma, Cat: D9891) stimulation (HA: 100 ng/ml, NP: 400 ng/ml) for 24 h. Following, 1 × 105 cells/well were seeded in a 96-well round-bottom plate, and HEK293A-NP cells were fixed and permeabilized before antibody staining. For binding of the surface antigen HA, cells were incubated with sera (1:200) or BALF (1:20) diluted in FACS buffer (PBS with 0.5% BSA (Merck, Cat: A4503)) and 1 mM sodium azide (Sigma, Cat: 71289). To bind intracellular NP, samples were diluted in permeabilization buffer (FACS buffer supplemented with 0.5% saponin (Sigma, Cat: 47036-250G-F). Specifically bound antibodies were detected using the polyclonal anti-mouse IgG-FITC (poly4060, BioLegend, Cat: 406001, 1:300) detection antibody or with an antibody mixture of anti-mouse IgA-FITC (polyclonal, Fortis Life Sciences, Cat: A90-103F), anti-mouse IgG1-APC (clone RMG1-1, BioLegend, Cat: 406610), and anti-mouse IgG2a-PerCP-eFluor710 (clone m2a-15F8, invitrogen, Cat: m2a-15F8) (all 1:300). The median fluorescence intensity (MFI) of each fluorophore was measured on an Attune NxT Flow Cytometer and analyzed using FlowLogic TM (Inivai). The exact concentration of each antibody subtype was extrapolated using a standard serum with known antibody concentration. All antibodies are listed in Supplementary Table 2.
Influenza microneutralization assay
Neutralizing antibodies in sera and BALF were determined in a microplate neutralization assay. Briefly, the samples were incubated with 2000 PFU of influenza H1N1 A/PR/8/34 or H3N2 A/HK/68 for 1 h at 37 °C before adding the mix to confluent MDCK-II cells in a 96-well plates. On day 4 after infection, the medium was removed, and plaques were identified by crystal violet (AppliChem, Cat: 131762.1608) staining. The highest sample dilution, which completely inhibited an infection, determines the neutralization titer. This titer is given as the reciprocal dilution level.
Quantitative reverse-transcription real-time PCR (qRT-PCR) for viral RNA detection
Viral RNA was isolated from lung and BALF samples using the Nucleo Spin RNA Virus kit (Machery-Nagel, Cat: 740956.250) according to the manufacturer’s instructions. Samples were quantified by qRT-PCR (Go Taq 1-Step RT-qPCR kit, Promega, Cat: A6020) for the IAV M gene using the 7500 Real-Time PCR System (Applied Biosystems) and 7500 software v2.3. For M viral RNA analysis, the following primers were used:
for 5’-AGATGAGTCTTCTAACCGAGGTCG-3’, rev 5’-TGCAAAAACATCTTCAAGTCTCTG-3’, and rev 5’-TGCAAAGACATCTTCCAGTCTCTG-3’. Results were expressed as absolute RNA copy numbers calculated according a standard curve of RNA preparations with known copy numbers.
Immunofluorescence microscopy
Mice were euthanized, the trachea was cannulated, and 1 ml of 20% sucrose (Roth, Cat: 9097.1) in PBS and O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA, Cat: 4583) (1:2 mixture) was injected to prevent the collapse of the pulmonary airways. The trachea was tied with a string. Lungs, trachea, and heart were removed and submerged into 20% sucrose/PBS over night at 4 °C. Following, the lungs were washed in sodium chloride and snap frozen using liquid nitrogen. 15 µm thick cryosections were prepared at the Institute of Pathology of the University Hospital Erlangen and stored at − 80 °C until use. To get a general overview about the tissue architecture and immune cell infiltration, lung tissue was stained with hematoxylin and eosin (HE). Whole slide images of HE-stained lung sections were scanned using an S210 digital slide scanner (Hamamatsu) and digitally analyzed with QuPath version 0.4.2, open-source software for digital pathology, and whole slide image analysis71. Figures were created using ImageJ software. Histopathological changes were confirmed by a pathologist from the University Hospital Erlangen. The precise localization of various immune cells was determined by immunofluorescence staining. Prior to primary staining, the lung sections were fixed in ice-cold acetone/methanol (Acetone: AppliChem, Cat: 211007.1212 / Methanol: Merck, Cat: 1.06008.6025) (1:1 mixture) for 5 min, air-dried, and tissue sections were surrounded using a PAP pen (Science Services, Cat: PAP-Pen Mini). Next, the sections were rehydrated and blocked with 5% FBS in PBS-T (0.05% Tween, Sigma, Cat: P7949) solution, containing anti-CD16/-CD32 (clone 93, invitrogen, Cat: 14-0161-86, 10 µg/ml). For detection of different immune cell specificities, lung sections were stained with CD4-AF488 (clone GK1.5, BioLegend, Cat: 100423, 5 µg/ml), anti-CD8α-AF647 (clone 53-6.7, BioLegend, Cat: 100727, 5 µg/ml), and anti-B220-BV711 (clone RA3-6B2, BioLegend, Cat: 103255, 5 µg/ml) overnight at 4 °C. Nuclei were stained by mounting with ProLong Glass antifade, which contains NucBlue (Hoechst 33342; Thermo Fisher Scientific, Cat: P36981). For the identification of pentamer-specific cells, lung sections were initially stained with anti-CD8α-AF488 (clone 53-6.7, BD Biosciences, Cat: 557668, 5 µg/ml) overnight at 4 °C, before the APC-conjugated Pro5 MHC HA533-541 or NP147-155 Pentamer (both ProImmune, 1:20) was added overnight at 4 °C. APC fluorescence intensity was amplified by anti-APC-AF647 (clone 936809, R&D Systems, Cat: FAB8927R, 10 µg/ml) and anti-mouse-IgG2b-AF647 (polyclonal, Invitrogen, Cat: A21242, 30 µg/ml) (each 1 h at 4 °C). Stained sections were mounted as described before. To stain and localize antigen-specific cells of various virus specificities, lung sections were stained with anti-B220-AF488 (clone RA3-6B2, BioLegend, Cat: 103225, 5 µg/ml) overnight at 4 °C. Biotin-conjugated Pro5 MHC RSV-M80-92 Pentamer (ProImmune, 1:20) was pre-incubated with streptavidin-PE (Miltenyi, Cat: 130-106-790, 1:2000) for 30 min at 4 °C. Similarly, APC-conjugated Pro5 MHC NP147-155 Pentamer (ProImmune, 1:20) was pre-incubated with anti-APC-AF647 (clone 936809, R&D Systems, Cat: FAB8927R, 10 µg/ml). Both complexes were mixed together, added to the tissue slice, and incubated 24 h at 4 °C. Finally, fluorescence intensity for the detection of NP-specific cells was intensified by incubation with anti-mouse-IgG2b-AF647 (polyclonal, Invitrogen, Cat: A21242, 30 µg/ml) for 1 h at 4 °C. Stained lung sections were mounted with ProLong Glass antifade including NucBlue (see above) and images were collected using a Leica SP5X laser scanning confocal microscope (Leica Camera AG, Wetzlar, Germany) with a 40x oil objective (PL APO, NA1.75). For image acquisition, LAS AF software was used and image processing was performed with ImageJ software. All antibodies are listed in Supplementary Table 2.
Cell labeling and sorting
Single-cell suspensions of lung tissues of rAd-NP/IL-1β or H1N1 A/PR/8/34 treated mice were prepared as described above. Cells of each sample were counted (Automated Cell Counter Luna, Logos Biosystems) and labeled with TotalSeq-C anti-mouse hashtag antibodies (TotalSeq-C0301 anti-mouse Hashtag 1 Antibody (Cat: 155861), TotalSeq-C0302 anti-mouse Hashtag 2 Antibody (Cat: 155863), TotalSeq-C0303 anti-mouse Hashtag 3 Antibody (Cat: 155865), TotalSeq-C0304 anti-mouse Hashtag 4 Antibody (Cat: 155867), BioLegend; 0.5 µg for 2×106 cells) for multiplex single-cell sequencing analysis and stained with APC-labeled H-2KD NP147-155 Pentamer (ProImmune, 1:40) for 30 min at 4 °C. Afterwards, cells from one group of mice were pooled together and stained with anti-CD8α-BV421 (clone 53-6.7, BioLegend, Cat: 100737, 1:300) and anti-CD45.2-PE/Dazzle594 (clone 104, BioLegend, Cat: 109846, 1:300) for 20 min at 4 °C (Supplementary Table 2). After resuspension in FACS buffer supplemented with 0.05 M EDTA (Lonza, Cat: 51201), NP147-155-specific CD8+ T cells were sorted at a MoFlo Astrios Cell Sorter (Beckmann Colter) in the core unit for cell sorting and immunomonitoring of the Friedrich-Alexander-Universität Erlangen-Nürnberg.
10x Genomics library preparation and sequencing
For each group, 2 × 105 barcoded NP147-155-specific CD8+ T cells were sorted in the FACS buffer. Samples were centrifuged, resuspended in nuclease-free water, and a master mix containing RT Reagent B, Poly-dt RT Primer, and RT Enzyme C was added This master mix and cell suspension mixture was loaded into Chromium Chip K (10x Genomics) and partitioned into Gel Beads In-Emulsions (GEMs) in a chromium controller (10x Genomics). Single-cell RNA libraries, as well as those for scTCR-sequencing, were prepared according to the Chromium Next GEM Chip K Single Cell Kit (Cat: PN-1000286), the Chromium Next GEM Single-Cell 5′ Kit v2 Kit (Cat: PN-1000263), the Dual Index Kit TT Set A (Cat: PN-1000215), the 5’ Feature Barcode Kit (Cat: PN-1000541), and the Chromium Single-Cell Mouse TCR Amplification Kit (Cat: PN-1000255) (all 10x Genomics, CA, USA). The single-cell RNA-sequencing (scRNA-seq) and VDJ libraries were sequenced by Novogene UK.
scRNA-seq data processing
Cell-gene matrices were generated via Cell Ranger v6.1.2 (10x Genomics), and the scRNA-seq reads were aligned to the mm10 genome (UCSC, CA, USA) using Cell Ranger count. Cell Ranger v6.1.2 multi-pipeline was applied for scRNA-seq and VDJ-seq analysis. The generated count matrix was loaded into Scanpy v1.9.372. Doublets were removed with HashSolo, invoked via Scanpy73. Cells with a mitochondrial content higher than 10% were filtered out. Raw counts were normalized using size factors calculated by DESeq2 v1.34.074 and subsequently scaled with Scanpy’s log1p function. Data dimensions were reduced using the UMAP algorithm75. Based upon the location of the cells on the two-dimensional UMAP plot, the cells were annotated as TRM (Cd69+/- Itgae+/-) and TCM (Ccr7+ Sell+). Scirpy v0.11.2 was employed to analyze the T-cell receptor (TCR) data76. Calculation of transcripts per million (TPM), as well as selection of cell populations, was performed using scSELpy v1.1.977. For the differential expression (DE) analysis, the data was treated as pseudo-bulk. Per annotation and sample, the raw Unique Molecular Identifier (UMI) count of all cells for each gene was summed. At this point, the data matrix looks like a bulk-seq UMI matrix. The pseudo-bulk matrix was passed to DESeq2 in order to calculate the log2 fold change and the adjusted p-value. In order to compare differential expression between single individuals of the same treatment group, we employed MAST (Model-based Analysis of Single Cell Transcriptomics)78 on the normalized matrix that was also used for dimension reduction with UMAP.
Statistical analysis
Statistical analyses were performed using GraphPad Prism (version 9.5.1, GraphPad Software, Inc.). In addition, the volcano plot and the heatmap were created via GraphPad Prism.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
