Abstract
BackgroundTraditionally, 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.MethodsWe 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.ResultsWe 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.ConclusionsThe 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.
Highlights
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 and antibody-based cytometry
Simultaneous protein quantification increases the power of scRNA-seq to dissect the functional heterogeneity of human CD4+ T cells In this study, we wanted to investigate the power of a unified high-throughput experimental workflow combining targeted scRNA-seq and the quantification of protein expression at the single-cell level, to dissect the heterogeneity of human primary CD4+ T cells in the blood
We initially profiled the expression of 397 genes at the mRNA level, coupled with 37 protein targets (Additional file 1: Table S1) using the BD AbSeq technology, in CD4+ T cells isolated from the blood of an systemic lupus erythematosus (SLE) patient
Summary
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. In contrast to cytometry-based technologies, which are limited to the concurrent detection of up to a few tens of protein markers, scRNA-seq technologies allow to profile the entire transcriptome, with a recent explosion in different platforms becoming available to immunologists [2, 3]. These fundamentally differ in the cell capture methods and resulting sensitivity, ranging from a few hundreds of cells profiled with high sensitivity using plate-based capture methods such as SMART-seq2 [4], to tens of thousands of cells profiled with lower sensitivity using wholetranscriptome scRNA-seq platforms, such as 10× Genomics [5], Seq-Well [6] or Drop-seq [7]
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