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Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets
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Strategies for optimizing CITE-seq for human islets and other tissues.

Sarah J Colpitts1,2, Matthew A Budd3,4, Mahdis Monajemi3,4

  • 1Department of Immunology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada.

Frontiers in Immunology
|March 20, 2023
PubMed
Summary

Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) can be standardized for human tissue studies. This method identifies immune cells in islets, overcoming challenges of tissue digestion and low cell abundance.

Keywords:
CITE-seqflow cytometrypancreassingle cell RNA seqtissue immunity

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Area of Science:

  • Immunology
  • Single-cell biology
  • Genomics

Background:

  • Characterizing the human tissue immune landscape is crucial but hindered by tissue disaggregation effects on cell phenotypes and low immune cell abundance.
  • Standardizing single-cell technologies like CITE-seq for enzymatically digested tissues is essential for reliable immune cell profiling.

Purpose of the Study:

  • To troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for human tissue studies involving enzymatic digestion.
  • To assess antibody epitope susceptibility to enzymatic digestion and optimize CITE-seq protocols for immune cell analysis in human islets.

Main Methods:

  • Tested epitope susceptibility of 92 antibodies on human peripheral blood mononuclear cells after enzymatic digestion.
  • Compared flow cytometry-based CITE-seq antibody titrations with sequencing data to determine optimal antibody concentrations.
  • Utilized CITE-seq to compare immune cells in enzymatically digested islet tissue and enzyme-untreated spleen.

Main Results:

  • Observed significant sensitivity of CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 epitopes to enzymatic treatment.
  • Found flow cytometry accurately predicts optimal antibody concentrations for CITE-seq for most antibodies.
  • Identified distinct immune cell subsets within islets, including T cell subsets, mast cells, and innate lymphoid cells (ILCs), using CITE-seq.

Conclusions:

  • Developed strategies for rational design and testing of CITE-seq antibodies for single-cell immune studies in human islets and other tissues.
  • Demonstrated CITE-seq's utility in identifying immune cells with low abundance or challenging transcriptional signatures in digested tissues.
  • Highlighted potential protective effects of islet structure on resident immune cells against enzymatic digestion.