30-Color Longitudinal Full-Spectrum Immunophenotyping and Sorting of Human Circulating Immune Cell Subsets Implicated in Systemic Autoimmune Rheumatic Diseases

Abstract

Purpose and Appropriate Sample TypesThis 30-color panel was developed to enable the enumeration and purification of distinct circulating immune cell subsets implicated in the pathogenesis of systemic autoimmune diseases including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc; scleroderma), Sjogrens disease (SjD), idiopathic inflammatory myopathy (IIM), and others. While designed for application to peripheral blood mononuclear cells, the inclusion of CD45 coupled with the ability to extract cellular autofluorescence spectral signatures enables the application of this panel to other tissue types. Of the 30 total markers, this panel employs 18 markers to profile T cell subsets consisting of different memory subsets and T helper polarities, > 10 markers to profile B cell subsets including double-negative B cells, and a total of 8 lineage markers to identify immune lineages including monocyte and natural killer cell subsets, conventional dendritic cells, plasmacytoid dendritic cells, and basophils. This panel reproducibly identifies target populations with excellent resolution over several months of data acquisition with minimal batch effects, offering investigators a practical approach to sort immune cell subsets of interest for downstream applications while simultaneously collecting high parameter immunophenotypic information using a limited sample quantity.