Multispectral fluorescence-activated cell sorting of B and T cell subpopulations from equine peripheral blood

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 CVS21⁺CD3^- 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.