Single-Cell Antigen Receptor Sequencing in Pigs with Influenza

Understanding the pulmonary adaptive immune system of pigs is of importance as respiratory pathogens present a major challenge for swine producers and pigs are increasingly used to model human pulmonary diseases. Single-cell RNA sequencing (scRNAseq) has accelerated the characterization of cellular phenotypes in the pig respiratory tract under both healthy and diseased conditions. However, combining scRNAseq with recovery of paired VJ and VDJ T cell receptor (TCR) as well as heavy (IGH) and light (IGL) chains of B cell receptors (BCR) to interrogate receptor repertoires has not to our knowledge been demonstrated for pigs. Here, we developed primers to enrich porcine TCR and BCR chains that are compatible with the 10x Genomics VDJ sequencing protocol. Using these pig-specific assays, we sequenced the T and B cell receptors of cryopreserved lung cells from CD1D-expressing and -deficient pigs after one or two infections with influenza A virus (IAV), a major swine and human respiratory pathogen, to examine whether natural killer T (NKT) cells alter pulmonary TCR and BCR repertoire selection. We also performed paired single-cell RNA and TCR sequencing of FACS-sorted T cells longitudinally sampled from the lungs of IAV-vaccinated and -infected pigs to track clonal expansion in response to IAV exposure. All pigs presented highly diverse repertoires. Pigs re-exposed to influenza antigens from either vaccination or infection exhibited higher numbers of expanded CD4 and CD8 T cell clonotypes with activated phenotypes, suggesting potential IAV reactive T cell populations. Our results demonstrate the utility of high throughput single-cell TCR and BCR sequencing in pigs. Six CD1D-/- and six CD1D-/+ pigs were transferred to biocontainment rooms at 4 weeks of age after being confirmed seronegative for IAV nucleoprotein antibodies by ELISA. At day 0, 3 CD1D-/- and 3 CD1D-/+ pigs were intratracheally infected with 1x106 tissue culture infectious dose (TCID50) of H1N1 A/California/04/2009 (pdmH1N1) in 2mL of DMEM after sedation with a combination of midazolam, butorphanol, and xylazine. Fourteen days later, all 12 pigs were sedated and infected with 1x106 TCID50 of H1N1 A/Missouri/CS20N08/2020 IAV. Five days later all 12 pigs were euthanized by pentobarbital sodium IV injections (150 mg/kg of body weight). At necropsy, the lungs were removed from the thoracic cavity for tissue collection. and euthanized. For the study to collect T cells through successive lung lavages, two pigs (FLU1 and FLU2) were vaccinated with a combination of 1x106 TCID50 of ultraviolet-inactivated pdmH1N1 virus and an oil-in water adjuvant (Emulsigen, 1:5 vaccine volume) at 28 days of age and boosted 15 days later. Both pigs were intratracheally infected with 1x106 TCID50 live pdmH1N1 virus 2 weeks after the booster. Lung fluid was collected 3 days before and 7 days after infection. A third unvaccinated, uninfected pig (NAÏVE) was lavaged at 28 and 33 days of age.