Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells

Gerlach, Jan P.; Buggenum, Jessie A.G.L. van; Tanis, Sabine E.J.; Hogeweg, Mark; Heuts, Branco M. H.; Muraro, Mauro J.; Elze, Lisa; Rivello, Francesca; Rakszewska, Agata; Oudenaarden, Alexander van; Huck, Wilhelm T. S.; Stunnenberg, Hendrik G.; Mulder, Klaas W.

doi:10.1038/s41598-018-37977-7

Cited by 82 publications

(74 citation statements)

References 34 publications

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“…Recently, two similar approaches, CITE-seq [9] and REAP-seq [10], have been described to measure protein expression using oligo-conjugated antibodies in parallel with scRNA-seq data. Furthermore, other applications are currently being developed to integrate the growing portfolio of single-cell omics technologies [38,39]. A fundamental difference with the approach described in this study is that these technologies all rely on wholetranscriptome data, providing a high-level crosssectional representation of all polyA mRNA transcripts in the cell.…”

Section: Discussionmentioning

confidence: 99%

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

et al. 2020

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Background: Traditionally, the transcriptomic and proteomic characterisation of CD4 + T cells at the single-cell level has been performed by two largely exclusive types of technologies: single-cell RNA sequencing (scRNA-seq) and antibody-based cytometry. Here, we present a multi-omics approach allowing the simultaneous targeted quantification of mRNA and protein expression in single cells and investigate its performance to dissect the heterogeneity of human immune cell populations. Methods: We have quantified the single-cell expression of 397 genes at the mRNA level and up to 68 proteins using oligo-conjugated antibodies (AbSeq) in 43,656 primary CD4 + T cells isolated from the blood and 31,907 CD45 + cells isolated from the blood and matched duodenal biopsies. We explored the sensitivity of this targeted scRNA-seq approach to dissect the heterogeneity of human immune cell populations and identify trajectories of functional T cell differentiation. Results: We provide a high-resolution map of human primary CD4 + T cells and identify precise trajectories of Th1, Th17 and regulatory T cell (Treg) differentiation in the blood and tissue. The sensitivity provided by this multi-omics approach identified the expression of the B7 molecules CD80 and CD86 on the surface of CD4 + Tregs, and we further demonstrated that B7 expression has the potential to identify recently activated T cells in circulation. Moreover, we identified a rare subset of CCR9 + T cells in the blood with tissue-homing properties and expression of several immune checkpoint molecules, suggestive of a regulatory function. Conclusions: The transcriptomic and proteomic hybrid technology described in this study provides a cost-effective solution to dissect the heterogeneity of immune cell populations at extremely high resolution. Unexpectedly, CD80 and CD86, normally expressed on antigen-presenting cells, were detected on a subset of activated Tregs, indicating a role for these co-stimulatory molecules in regulating the dynamics of CD4 + T cell responses.

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Section: Discussionmentioning

confidence: 99%

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

et al. 2020

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“…Unfortunately, alcohol-based fixation and permeabilization is not compatible with many antibody clones and/or fluorophores that are necessary for identification of immune cell subsets of interest. More recently, the RAID protocol was introduced for the combined analysis of intracellular phosphoprotein concentrations and transcriptomics in single cells ( 80 ). This protocol utilizes fixation with chemically-reversible fixatives (DSP, SPDP), permeabilization with a detergent supplemented with RNase inhibitor, and cross-linking reversal with DTT.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells

2020

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Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.

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“…Microfluidic devices were fabricated by using photo-and soft lithography based on the AutoCAD files with channel designs that were kindly provided by Dr. Linas Mazutisd 5 . Silicon wafers were spin-coated with a uniform layer of SU8-2050 photoresist (MicroChem Co., USA), soft-baked, UV exposed through transparency mask (JD Phototools, UK), baked again post-exposure, developed and hard-baked according to manufacturer's protocol (MicroChem Co., USA).…”

Section: Microfluidic Device Fabrication For Single-cell Barcodingmentioning

confidence: 99%

Single-cell intracellular epitope and transcript detection revealing signal transduction dynamics

Rivello

Buijtenen

Matuła

et al. 2020

Preprint

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Current high-throughput single-cell multi-omics methods cannot concurrently map changes in (phospho)protein levels and the associated gene expression profiles. We present QuRIE-seq (Quantification of RNA and Intracellular Epitopes by sequencing) and use multi-factor omics analysis (MOFA+) to map signal transduction over multiple timescales. We demonstrate that QuRIE-seq can trace the activation of the B-cell receptor pathway at the minute and hour time-scale and provide insight into the mechanism of action of an inhibitory drug, Ibrutinib.

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Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells

Cited by 82 publications

References 34 publications

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

Discovery of CD80 and CD86 as recent activation markers on regulatory T cells by protein-RNA single-cell analysis

Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells

Single-cell intracellular epitope and transcript detection revealing signal transduction dynamics

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