Dynamic hydrogen peroxide levels reveal a rate-dependent sensitivity in B-cell lymphoma signaling.

Although in vivo extracellular microenvironments are dynamic, most in vitro studies are conducted under static conditions. Here, we exposed diffuse large B-cell lymphoma (DLBCL) cells to gradient increases in the concentration of hydrogen peroxide (H2O2), thereby capturing some of the dynamics of the tumour microenvironment. Subsequently, we measured the phosphorylation response of B-cell receptor (BCR) signalling proteins CD79a, SYK and PLCγ2 at a high temporal resolution via single-cell phospho-specific flow cytometry. We demonstrated that the cells respond bimodally to static extracellular H2O2, where the percentage of cells that respond is mainly determined by the concentration. Computational analysis revealed that the bimodality results from a combination of a steep dose-response relationship and cell-to-cell variability in the response threshold. Dynamic gradient inputs of varying durations indicated that the H2O2 concentration is not the only determinant of the signalling response, as cells exposed to more shallow gradients respond at lower H2O2 levels. A minimal model of the proximal BCR network qualitatively reproduced the experimental findings and uncovered a rate-dependent sensitivity to H2O2, where a lower rate of increase correlates to a higher sensitivity. These findings will bring us closer to understanding how cells process information from their complex and dynamic in vivo environments.

Integrated Single-Cell (Phospho-)Protein and RNA Detection Uncovers Phenotypic Characteristics and Active Signal Transduction of Human Antibody-Secreting Cells.

Single-cell technologies are currently widely applied to obtain a deeper understanding of the phenotype of single-cells in heterogenous mixtures. However, integrated multilayer approaches including simultaneous detection of mRNA, protein expression, and intracellular phospho-proteins are still challenging. Here, we combined an adapted method to in vitro-differentiate peripheral B-cells into antibody-secreting cells (ASCs) (i.e., plasmablasts and plasma cells) with integrated multi-omic single-cell sequencing technologies to detect and quantify immunoglobulin subclass-specific surface markers, transcriptional profiles, and signaling transduction pathway components. Using a common set of surface proteins, we integrated two multimodal datasets to combine mRNA, protein expression, and phospho-protein detection in one integrated dataset. Next, we tested whether ASCs that only seem to differ in its ability to secrete different IgM, IgA, or IgG antibodies exhibit other differences that characterize these different ASCs. Our approach detected differential expression of plasmablast and plasma cell markers, homing receptors, and TNF receptors. In addition, differential sensitivity was observed for the different cytokine stimulations that were applied during in vitro differentiation. For example, IgM ASCs were more sensitive to IL-15, while IgG ASC responded more to IL-6 and IFN addition. Furthermore, tonic BCR activity was detected in IgA and IgM ASCs, while IgG ASC exhibited active BCR-independent SYK activity and NF-κB and mTOR signaling. We confirmed these findings using flow cytometry and small molecules inhibitors, demonstrating the importance of SYK, NF-κB, and mTOR activity for plasmablast/plasma cell differentiation/survival and/or IgG secretion. Taken together, our integrated multi-omics approach allowed high-resolution phenotypic characterization of single cells in a heterogenous sample of in vitro-differentiated human ASCs. Our strategy is expected to further our understanding of human ASCs in healthy and diseased samples and provide a valuable tool to identify novel biomarkers and potential drug targets.

Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics.

To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.

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

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.

BLNCR is a long non-coding RNA adjacent to integrin beta-1 that is rapidly lost during epidermal progenitor cell differentiation.

As our understanding of transcriptional regulation improves so does our appreciation of its complexity. Both coding and (long) non-coding RNAs provide cells with multiple levels of control and thereby flexibility to adapt gene expression to the environment. However, few long non-coding RNAs (lncRNAs) have been studied in human epidermal stem cells. Here, we characterized the expression of 26 lncRNAs in human epidermal keratinocytes, 7 of which we found to be dynamically expressed during differentiation. We performed in depth analysis of a lncRNA located proximal to the epidermal stem cell marker integrin beta-1 (ITGB1) and transcribed in the opposite direction. We dubbed this gene Beta1-adjacent long non-coding RNA, or BLNCR, and found that its expression is regulated by p63 and AP1 transcription factors. Furthermore, BLNCR expression is regulated downstream the integrin and EGF signaling pathways that are key to epidermal stem cell maintenance. Finally, we found that BLNCR expression is rapidly reduced upon induction of differentiation, preceding the down regulation of integrin beta-1 expression. These dynamics closely mirror the loss of proliferative and adhesion capacity of epidermal stem cells in colony formation assays. Together, these results suggest that loss of BLNCR expression marks the switch from a proliferative state towards terminal differentiation in human epidermal stem cells.

Single-Cell ID-seq Reveals Dynamic BMP Pathway Activation Upstream of the MAF/MAFB-Program in Epidermal Differentiation.

Epidermal homeostasis requires balanced and coordinated adult stem cell renewal and differentiation. These processes are controlled by both extracellular signaling and by cell intrinsic transcription regulatory networks, yet how these control mechanisms are integrated to achieve this is unclear. Here, we developed single-cell Immuno-Detection by sequencing (scID-seq) and simultaneously measured 69 proteins (including 34 phosphorylated epitopes) at single-cell resolution to study the activation state of signaling pathways during human epidermal differentiation. Computational pseudo-timing inference revealed dynamic activation of the JAK-STAT, WNT, and BMP pathways along the epidermal differentiation trajectory. We found that during differentiation, cells start producing BMP2-ligands and activate the canonical intracellular effectors SMAD1/5/9. Mechanistically, the BMP pathway is responsible for activating the MAF/MAFB/ZNF750 transcription factor network to drive late-stage epidermal differentiation. Our work indicates that incorporating signaling pathway activation into this transcription regulatory network enables coordination of transcription programs during epidermal differentiation.

Immuno-detection by sequencing enables large-scale high-dimensional phenotyping in cells.

Cell-based small molecule screening is an effective strategy leading to new medicines. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Here we present the immuno-detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. We use ID-seq to simultaneously measure 70 (phospho-)proteins in primary human epidermal stem cells to screen the effects of ~300 kinase inhibitor probes to characterise the role of 225 kinases. The results show an association between decreased mTOR signalling and increased differentiation and uncover 13 kinases potentially regulating epidermal renewal through distinct mechanisms. Taken together, our work establishes ID-seq as a flexible solution for large-scale high-dimensional phenotyping in fixed cell populations.

A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.

Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.