RT Journal Article
SR Electronic
T1 Porcine intestinal innate lymphoid cells and lymphocyte spatial context revealed through single-cell RNA sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.09.475571
DO 10.1101/2022.01.09.475571
A1 Wiarda, Jayne E.
A1 Trachsel, Julian M.
A1 Sivasankaran, Sathesh K.
A1 Tuggle, Christopher K.
A1 Loving, Crystal L.
YR 2022
UL http://biorxiv.org/content/early/2022/01/11/2022.01.09.475571.abstract
AB Intestinal lymphocytes are crucial members of the mucosal immune system with impact over outcomes of intestinal health versus dysbiosis. Resolving intestinal lymphocyte complexity and function is a challenge, as the intestine provides cellular snapshots of a diverse spectrum of immune states. In pigs, intestinal lymphocytes are poorly described relative to humans or traditional model species. Enhanced understanding of porcine intestinal lymphocytes will promote food security and improve utility of pigs as a biomedical model for intestinal research. Single-cell RNA sequencing (scRNA-seq) was performed to provide transcriptomic profiles of lymphocytes in porcine ileum, with 31,983 cells annotated into 26 cell types. Deeper interrogation revealed previously undescribed cells in porcine intestine, including SELLhi γδ T cells, group 1 and group 3 innate lymphoid cells (ILCs), and four subsets of B cells. Single-cell transcriptomes in ileum were compared to those in porcine blood, and subsets of activated lymphocytes were detected in ileum but not periphery. Comparison to scRNA-seq human and murine ileum data revealed a general consensus of ileal lymphocytes across species. Lymphocyte spatial context in porcine ileum was conferred through differential tissue dissection prior to scRNA-seq. Antibody-secreting cells, B cells, follicular CD4 αβ T cells, and cycling T/ILCs were enriched in ileum with Peyer’s patches, while non-cycling γδ T, CD8 αβ T, and group 1 ILCs were enriched in ileum without Peyer’s patches. scRNA-seq findings were leveraged to develop advanced toolsets for further identification of ILCs in porcine ileum via flow cytometry and in situ staining. Porcine ileal ILCs identified via scRNA-seq did not transcriptionally mirror peripheral porcine ILCs (corresponding to natural killer cells) but instead had gene signatures indicative of tissue- and activation-specific functions, indicating potentially similar roles to intestinal ILCs identified in humans. Overall, the data serve as a highly-resolved transcriptomic atlas of the porcine intestinal immune landscape and will be useful in further understanding intestinal immune cell function.Competing Interest StatementThe authors have declared no competing interest.ACDAdvanced Cell DiagnosticsARSAgricultural Research ServiceASCantibody-secreting cellBCRB cell receptorBSAbovine serum albuminc1cluster 1c2cluster 2CCAcanonical correlation analysiscDCconventional dendritic cellCPTCell Preparation TubeDAdifferential abundanceDCdendritic cellDEdifferentially expressedDEGdifferentially expressed geneDGEdifferential gene expressionDNdouble-negativeDOEDepartment of EnergyDTTdithiothretolDZdark zoneEDTAethylenediamine tetraacetic acidFCSfetal calf serumFFPEformalin-fixed, paraffin-embeddedFMOfluorescence-minus-oneFSC-Aforward scatter areaFSC-Hforward scatter heightGALTgut-associated lymphoid tissueGCgerminal centerGOGene OntologyHBSSHank’s balanced salt solutionHRPhorseradish peroxidaseIACUCinstitutional animal care and use committeeIELintraepithelial lymphocyteIFimmunofluorescenceIHCimmunohistochemistryILCinnate lymphoid cellIQRinterquartile rangeISHin situ hybridizationlogFClog fold-changeLTilymphoid tissue inducerLZlight zoneMDSmultidimensional scalingNBFneutral-buffered formalinNoSig no significancepDCplasmacytoid dendritic cellNhoodneighborhoodNKnatural killerNKTnatural killer TORAUOak Ridge Associated UniversitiesORISEOak Ridge Institute for Science and EducationPBMCperipheral blood mononuclear cellPBSphosphate buffered salinePBSTphosphate buffered saline with TweenPCprinciple componentPCAprinciple component analysisPPPeyer’s patchRNA-seqRNA sequencingRTroom temperaturescRNA-seqsingle-cell RNA sequencingSNNshared nearest neighborSSC-Aside scatter heightT-IELintraepithelial T lymphocytet-SNEt-distributed stochastic neighbor embeddingTAtransit amplifyingTCRT cell receptorTFHT follicular helperTFRT follicular regulatoryTregT regulatoryUMIunique molecular identifierUSDAUnited States Department of Agriculture