Strategies for optimizing CITE-seq for human islets and other tissues
Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor- matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITEseq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITEseq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues. Overall design: We performed CITE-Seq on enzymatically digested, CD45-enriched, flow-sorted human islets to assess the ability of CITE-Seq oligo-antibodies to bind to target cells. We developed a panel of 61 CITE-Seq oligo-antibodies to select for key lineage and phenotypic immune cell markers, and evaluated how the enzymatic digestion process affected detection of these key cell surface proteins. We repeated this enzymatic digestion on mechanically dissociated human splenocytes and used the same panel of 61 CITE-Seq olig-antibodies to compare immune cell composition to that of islet-resident CD45+ cells.