Abstract
BackgroundTraditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers.ResultsHere, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1− lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse.ConclusionsTaken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.
Highlights
Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species
Single-cell Ribonucleic acid (RNA)-Seq of equine peripheral blood mononuclear cells (PBMC) resolves a diversity of immune cell types We performed scRNA-Seq on fresh PBMC collected from 7 healthy adult horses of different breeds, ages, and sexes (Table 1)
Upon inspection of sequence alignments for select genes, we frequently observed reads mapped immediately downstream of annotated transcript regions (Additional file 1: Fig. S1B). This pattern is consistent with incomplete annotation of transcript 3′ untranslated regions (UTRs; the most frequent transcript region captured by 10X Chromium 3′ scRNASeq [21]), which is common in non-traditional model organisms relative to mouse or human reference transcriptomes [22]
Summary
Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. Many biological phenomena relevant to human health and society involve specific animal species, such as the circulation of emerging zoonotic pathogens in animal reservoirs [2], and the health of domesticated livestock. Studying diverse species can prove challenging due to a dearth of experimental tools available for more commonly investigated laboratory organisms.
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