Single cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse immune cell subtypes including T-bet⁺B cells

Abstract

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. Immunology research in non-traditional model organisms can be advantageous or even necessary (e.g. for host-pathogen interaction studies), but 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. Here, 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 (PBMCs) 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 + PRF1lymphocytes, 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). Unexpectedly, 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.Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in nontraditional model organisms, and form the basis for an immune cell atlas of horse peripheral blood.

Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet+ B cells

“…scRNA-Seq data processing scRNA-Seq data are available in the NCBI GEO repository, accession number GSE148416 [79]. Analysis R code is available on GitHub, BradRosenbergLab/equinepbmc [80].…”

“…The datasets generated and analyzed during the current study are available in the NCBI GEO repository, https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE148416 [79]. The analysis R code generated during the current study is available on GitHub https://github.com/BradRosenbergLab/ equinepbmc [80].…”

“…cgi?acc=GSE148416 [79]. The analysis R code generated during the current study is available on GitHub https://github.com/BradRosenbergLab/ equinepbmc [80].…”

Single-cell RNA sequencing of equine mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility

“…Reads were assigned to cell barcodes, mapped and quantified per gene using CellRanger (v 3.0.1, 10X Genomics) with default parameters ("standard workflow"). Raw BAM files were extracted and processed with the End Sequence Analysis Toolkit [33] and a workflow optimized for equine single cell RNA-seq analysis as described in Patel et al 2020 [34]. Briefly, extracted BAM files were modified such that cell barcode and UMI information were appended to the corresponding read name entry for processing by ESAT.…”

Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture

“…In contrast, we observed the most consistent expression of CD90 in MSC cultured with 10% ePL media and expression of CD45 was almost undetectable in all media. Furthermore, we made an interesting observation of CD14 detection on the majority of MSC when using an equine-specific antibody for CD14 (Kabithe et al, 2010 ; Wagner et al, 2013 ; Bonelli et al, 2017 ; Schnabel et al, 2019 ; Larson et al, 2020 ; Patel et al, 2020 ). According to the ISCT, CD14 should not be expressed on human MSC (Dominici et al, 2006 ) as it is a glycolipid anchored membrane glycoprotein expressed on monocytes and macrophages (Tesfaigzi, 2006 ; Braun et al, 2010 ) and a leukocyte-secreted molecule in response to LPS stimulation and inflammation, as demonstrated in horses (Wagner et al, 2013 ).…”