Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood.
Abstract: Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3. Clone CVS21 bound non-T CD3 cells in addition to CD8 T cells. Further analysis using co-labeling with CD3 and multiple B cell antibodies revealed that most of these CVS21CD3 cells were B cells. Clone CVS8 and anti-equine CD4 clone CVS4 only labeled CD3 cells and were mutually exclusive. To identify equine B-cell subpopulations, anti-equine Pan-Ig (clone CVS36), IgM (clone 1-22), and IgG1 (clone CVS45), as well as anti-human CD21 (clone B-ly4) were tested. Anti-equine Pan-Ig antibody labeled 88 ± 7.6% of CD3 lymphocytes. Anti-equine IgM often produced a continuum of stain intensity of PBMC from several adult horses. Anti- IgG1 and -CD21 labeled overlapping subsets of CD3 cells. Based on these results, combined with conjugate availability, a final panel of anti-CD4 (CVS4), -CD8 (CVS8), -IgG1 (CVS45), and -CD21 (B-ly4) antibodies was used to sort CD4 T cells, CD8 T cells, and CD21 and/or IgG1 B cells simultaneously. The identity of the sorted populations was confirmed by qRT-PCR for cell lineage markers. The described analysis emphasizes the need for increased availability of reagents and use of multispectral flow for equine immunophenotypic analysis. This new multispectral flow cytometry protocol will facilitate studies on equine lymphocyte responses and will have broad application across studies of infectious and immune-mediated disease.
Copyright © 2018 Elsevier B.V. All rights reserved.
Publication Date: 2018-03-23 PubMed ID: 29678226DOI: 10.1016/j.vetimm.2018.03.010Google Scholar: Lookup
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- Journal Article
Summary
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The researchers developed a method to identify and categorize the lymphocyte (a type of white blood cell) subpopulations in horses using multispectral fluorescence-activated cell sorting. This method will improve analysis of equine immune system responses and contribute to the study of infectious and immune-mediated diseases in horses.
Background
- In previous methods of immune phenotyping, cells are characterised based on single or double markers. This makes intricate phenotypic descriptions and subpopulation identification challenging.
- In this study, the researchers aimed to develop a new multispectral flow cytometry protocol using commercially available, pre-conjugated antibodies to cell surface markers in order to identify and sort equine lymphocyte subpopulations.
Method
- The researchers compared two clones of anti-equine CD8, CVS8 and CVS21, in conjunction with CD3.
- CVS21 was found to bind to non-T CD3 cells and CD8 T cells, while CVS8 and anti-equine CD4 clone CVS4 only labelled CD3 cells.
- To identify equine B-cell subpopulations, they tested various antibodies and determined that anti-equine Pan-Ig antibody labelled 88 ± 7.6% of CD3 lymphocytes.
- Anti-equine IgM, IgG1 and anti-human CD21 were found to label overlapping subsets of CD3 cells.
Results
- Based on the results and availability of conjugate, a final panel of anti-CD4 (CVS4), -CD8 (CVS8), -IgG1 (CVS45), and -CD21 (B-ly4) antibodies was selected.
- The researchers were able to simultaneously sort CD4 T cells, CD8 T cells, and CD21 and/or IgG1 B cells.
- The identity of the sorted populations was confirmed through real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for cell lineage markers.
- This newly formulated method emphasises the need for increased availability of reagents and use of multispectral flow for equine immunophenotypic analysis.
Conclusion
- The newly proposed multispectral flow cytometry protocol will enable more accurate studies on equine lymphocyte responses.
- It has a broad application across studies of infectious and immune-mediated disease in horses.
Cite This Article
APA
Tomlinson JE, Wagner B, Felippe MJB, Van de Walle GR.
(2018).
Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood.
Vet Immunol Immunopathol, 199, 22-31.
https://doi.org/10.1016/j.vetimm.2018.03.010 Publication
Researcher Affiliations
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14850, USA. Electronic address: jet37@cornell.edu.
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14850, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14850, USA.
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14850, USA.
MeSH Terms
- Animals
- B-Lymphocytes / cytology
- Female
- Flow Cytometry / methods
- Flow Cytometry / veterinary
- Horses / blood
- Horses / immunology
- Male
- T-Lymphocyte Subsets / cytology
Citations
This article has been cited 6 times.- Sage SE, Nicholson P, Peters LM, Leeb T, Jagannathan V, Gerber V. Single-cell gene expression analysis of cryopreserved equine bronchoalveolar cells.. Front Immunol 2022;13:929922.
- Cequier A, Romero A, Vázquez FJ, Vitoria A, Bernad E, Fuente S, Zaragoza P, Rodellar C, Barrachina L. Equine Mesenchymal Stem Cells Influence the Proliferative Response of Lymphocytes: Effect of Inflammation, Differentiation and MHC-Compatibility.. Animals (Basel) 2022 Apr 11;12(8).
- Patel RS, Tomlinson JE, Divers TJ, Van de Walle GR, Rosenberg BR. Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet(+) B cells.. BMC Biol 2021 Jan 22;19(1):13.
- Gugliandolo E, Crupi R, Biondi V, Licata P, Cuzzocrea S, Passantino A. Protective Effect of Silibinin on Lipopolysaccharide-Induced Inflammatory Responses in Equine Peripheral Blood Mononuclear Cells, an In Vitro Study.. Animals (Basel) 2020 Nov 3;10(11).
- Saldinger LK, Nelson SG, Bellone RR, Lassaline M, Mack M, Walker NJ, Borjesson DL. Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro.. Vet Ophthalmol 2020 Jan;23(1):160-170.
- Hillmann A, Paebst F, Brehm W, Piehler D, Schubert S, Tárnok A, Burk J. A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells.. PLoS One 2019;14(6):e0218949.
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