Comparative Profiling of the Immune System in Sarcoidosis via CITE-Seq and Flow Cytometry

Abstract

CITE-Seq enables simultaneous single cell transcriptome and proteome analysis via combining single cell RNA-seq with oligo-labeled antibodies. Conventional techniques such as flow or mass cytometry have caveats including the number of epitopes accurately detected or an inability to recover samples for transcriptome analysis. These limitations are prohibitive for multivariate analysis of limited clinical samples. We developed a CITE-seq assay that enables comprehensive immune cell profiling of Sarcoidosis. Sarcoidosis is a granulomatous lung disease characterized by abnormal CD4+ T cell Th1 activity. However, the disease etiology and course are variable and the underlying molecular drivers remain unknown. The long-term goal of this study is to utilize CITE-seq to identify immune molecular pathways of Sarcoidosis pathogenesis. In our initial studies, we analyzed PBMC’s by CITE-seq vs. flow cytometry and observed similar cell profiles. However, the synergy of protein detection coupled with transcriptome analysis via CITE-seq enhanced cell subset identification vs. flow or scRNA-seq alone. We utilized CITE-seq in an ongoing longitudinal study of Sarcoidosis subjects to enhance resolution of the immune components contributing to disease. We compared CITE-seq to a flow cytometry panel analyzing the differential contributions of various CD4+ T cell lineages. The enhanced granularity provided by CITE-seq elucidated molecular pathways associated with disease pathogenesis. Thus, moving forward CITE-seq can provide the resolution and multivariate data collection required to identify the inflammatory drivers of Sarcoidosis.

From Karl Wurm and Guy Scadding's staging to 18F-FDG PET/CT scan phenotyping and far beyond: perspective in the evading history of phenotyping in sarcoidosis

“…Another method that combines single-cell transcriptomics with proteomics, which is called cellular indexing of transcriptomes and epitopes (CITE-Seq), is a sequencing-based method that allows simultaneous quantification of cell surface protein and single-cell sequencing data, enabling both surface marker and transcriptomic phenotyping of immune cells that are dysregulated in various diseases ( 97 ). Since cell surface proteins are indicators of cell phenotype and functional status, CITE-seq may provide a superior method for profiling immune cells in sarcoidosis when compared with scRNA-seq or proteomic analysis alone ( 98 ). While “omics” cannot be directly applied to everyday clinical practice, they could provide significant, distinct, and exclusive insights into the described phenotypes of sarcoidosis by combining clinical, laboratory, imaging, and histologic characteristics with molecular signatures in a sort of “reverse phenotyping” approach.…”