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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 T...

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Autores principales: Colpitts, Sarah J., Budd, Matthew A., Monajemi, Mahdis, Reid, Kyle T., Murphy, Julia M., Ivison, Sabine, Verchere, C. Bruce, Levings, Megan K., Crome, Sarah Q.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10014726/
https://www.ncbi.nlm.nih.gov/pubmed/36936943
http://dx.doi.org/10.3389/fimmu.2023.1107582
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author Colpitts, Sarah J.
Budd, Matthew A.
Monajemi, Mahdis
Reid, Kyle T.
Murphy, Julia M.
Ivison, Sabine
Verchere, C. Bruce
Levings, Megan K.
Crome, Sarah Q.
author_facet Colpitts, Sarah J.
Budd, Matthew A.
Monajemi, Mahdis
Reid, Kyle T.
Murphy, Julia M.
Ivison, Sabine
Verchere, C. Bruce
Levings, Megan K.
Crome, Sarah Q.
author_sort Colpitts, Sarah J.
collection PubMed
description 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 CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq 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.
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spelling pubmed-100147262023-03-16 Strategies for optimizing CITE-seq for human islets and other tissues Colpitts, Sarah J. Budd, Matthew A. Monajemi, Mahdis Reid, Kyle T. Murphy, Julia M. Ivison, Sabine Verchere, C. Bruce Levings, Megan K. Crome, Sarah Q. Front Immunol Immunology 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 CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq 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. Frontiers Media S.A. 2023-03-01 /pmc/articles/PMC10014726/ /pubmed/36936943 http://dx.doi.org/10.3389/fimmu.2023.1107582 Text en Copyright © 2023 Colpitts, Budd, Monajemi, Reid, Murphy, Ivison, Verchere, Levings and Crome https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Colpitts, Sarah J.
Budd, Matthew A.
Monajemi, Mahdis
Reid, Kyle T.
Murphy, Julia M.
Ivison, Sabine
Verchere, C. Bruce
Levings, Megan K.
Crome, Sarah Q.
Strategies for optimizing CITE-seq for human islets and other tissues
title Strategies for optimizing CITE-seq for human islets and other tissues
title_full Strategies for optimizing CITE-seq for human islets and other tissues
title_fullStr Strategies for optimizing CITE-seq for human islets and other tissues
title_full_unstemmed Strategies for optimizing CITE-seq for human islets and other tissues
title_short Strategies for optimizing CITE-seq for human islets and other tissues
title_sort strategies for optimizing cite-seq for human islets and other tissues
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10014726/
https://www.ncbi.nlm.nih.gov/pubmed/36936943
http://dx.doi.org/10.3389/fimmu.2023.1107582
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