Single-cell analysis reveals lasting immunological consequences of influenza infection and respiratory immunisation in the pig lung
The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. We employed scRNA-seq and flow cytometry to characterize the major leucocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing haemagglutinin and nucleoprotein with or without IL-1ß. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1ß reduced the number of functionally active Treg. Second, influenza infection upregulated IFI6 in BAL cells, decreasing their susceptibility to virus replication?in vitro. Our data provide a reference map of BAL cells following respiratory infection or immunization in a highly relevant large animal model for respiratory virus infection. Overall design: Samples obtained from a previous experiment conducted on inbred Babraham pigs were utilized (Schmidt et al., 2023). Briefly three groups of 5 pigs each, were treated as follows: 1) infected intranasally with 8x106 PFU of A/swine/England/1353/2009 (pH1N1) using a mucosal atomization device (MAD); 2) immunized intranasally with 1x109 particles of recombinant adenoviral vectors expressing NP from H1N1/PR8/34 virus and HA from pH1N1/Texas/05/2009 (Ad-HA/NP) using a MAD and 3) immunized intranasally with a mix of 1x109 particles of Ad-HA/NP and 1x109 particles recombinant adenoviral vector encoding porcine IL-1ß (Ad-HA/NP+Ad-IL-1ß) using a MAD. Control pigs (n=3) received PBS intranasally. The animals were culled three weeks later, and bronchoalveolar lavage (BAL), tracheobronchial lymph nodes and blood collected. Virus shedding and immune responses in respiratory tissues and peripheral blood were described in (Schmidt et al., 2023). Cryopreserved BAL cells were thawed, washed and resuspended in PBS. Cells were incubated with antibodies against CD4 (clone 74-12-4, PerCP-Cy5.5-conjugated, BD Biosciences), CD8ß (clone PPT23, FITC-conjugated, Bio-Rad), CD3 (clone PPT3, APC-conjugated, Southern Biotech), CD16 (clone G7, AF647-conjugated, Bio-Rad), CD172a (clone 74-22-15, PE-conjugated, Bio-Rad) and Fixable Viability Dye eFluor780 (ThermoFisher). After two washes in PBS, cells were fixed, washed again and sorted on a FACSAria UIII (BD Biosciences), equipped with 405, 488, 561 and 640nm lasers and BD FACSDiva 8 software. After gating on FSC-AdimSSC-Adim cells and exclusion of doublets and dead cells, CD4+, CD8ß+ and CD3-CD16- (to enrich for B cells) cells were sorted (12,000 cells per population). In addition, after gating on FSC-AbrightSSC-Abright cells, doublet and dead cell exclusion and gating on CD16+CD172a+ cells, 12,000 macrophages were sorted. Cells from each fraction were pooled and subjected to partitioning and barcoding on a 10x Genomics Chromium iX Controller. For 10X, single cells were processed with the Chromium iX controller, using the Next GEM chip G, and single cell 3' kit v3.1 library preparation kit (10X Genomics, Pleasanton, CA). An estimated 10,000 cells were targeted for recovery during partitioning. Samples were randomly grouped and run with two libraries per sequencing run on three NextSeq High Output 150 cycle reagent kits (Illumina, San Diego, CA), with 1% PhiX, targeting approximately 200 million reads per sample. Bcl files were demultiplexed using 'cellranger mkfastq' and aligned/counted using 'cellranger count' (cellranger-7.0.0) using default parameters and Sus scrofa genome (genome assembly 11.1, Ensembl release 107).