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

Abstract

The transcriptomic and proteomic characterisation of CD4 + T cells at the single-cell level has been performed traditionally by two largely exclusive types of technologies: single cell RNAsequencing (scRNA-seq) technologies and antibody-based cytometry. Here we demonstrate that the simultaneous targeted quantification of mRNA and protein expression in single-cells provides a high-resolution map of human primary CD4 + T cells, and reveals precise trajectories of Th1, Th17 and regulatory T-cell differentiation in blood and tissue. We report modest correlation between mRNA and protein in primary CD4 + T cells at the single-cell level, highlighting the value of protein expression data to characterise cell effector function. This transcriptomic and proteomic hybrid technology provides a massively-parallel, cost-effective, solution to dissect the heterogeneity of immune cell populations and to immunophenotype rare and potentially pathogenic immune subsets, and could have important clinical applications to redefine differentiation and activation of circulating and tissue-resident human immune cells.

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines

“…TCM, TEM and TEMRA) are better described as stages in a continuous trajectory rather than as separate cell populations, as they have been traditionally described based on protein expression and surface markers 24 . Our results are in line with a similar trajectory, which was reported using simultaneous targeted quantification of mRNA and protein expression in single T cells 45 . Interestingly, a similar gradient is also present in innate T cells, as shown by a previous study where higher expression levels of effector molecules were negatively associated with ribosome synthesis and proliferative capacity 46 .…”

Chronic Immune Activation in Systemic Lupus Erythematosus and the Autoimmune PTPN22 Trp620 Risk Allele Drive the Expansion of FOXP3+ Regulatory T Cells and PD-1 Expression

“…This is particularly relevant in SLE patients with active disease, where the disease-specific environment could affect cell function. Therefore, to further investigate the nature of CD25 low FOXP3 + T cells, we employed a recently developed targeted single-cell RNA-sequencing approach combining mRNA and protein quantification (29), to characterize the FOXP3 + cells within both CD127 low CD25 low and CD127 low CD25 hi populations from one SLE patient displaying an expanded Treg compartment, as well as two control donors (one type 1 diabetic and one healthy donor). Clustering analysis of the combined mRNA and protein expression from all ex vivo isolated CD127 low CD25 low ( n = 7,115) and CD127 low CD25 hi ( n = 7,711) cells, revealed diverse functional T-cell subsets, clustering along a naïve-memory differentiation axis, including distinct Treg clusters marked by the expression of FOXP3 and IKZF2 (encoding HELIOS; Figures 4A–C and Supplementary Figure 2A).…”

“…Single-cell RNA sequencing data, was obtained from one SLE patient with an expanded CD25 low FOXP3 + Treg population, one type 1 diabetes (T1D) patient and one healthy control (HC), presented in Trzupek et al (29). Briefly, simultaneous mRNA expression of 397 genes (Human Response Panel; BD Biosciences) and protein expression of 24 T-cell expressed targets (see Supplementary Table 1), were assessed at the single-cell level using the BD Rhapsody and AbSeq system (BD Biosciences), as previously described (29). Flow sorted CD4 + T-cell subsets from each donor were barcoded using oligo-conjugated antibodies (single-cell multiplexing kit; BD Biosciences).…”

“…For this study, we extracted and reanalyzed the data from sorted CD127 low CD25 low ( N = 7,115) and CD127 low CD25 hi ( N = 7,711) T cells passing QC. Detailed description of sorting strategy, library preparation and sequencing is presented in Trzupek et al (29). Data analysis and QC was performed following the BD Biosciences Rhapsody pipeline (BD Biosciences) and the R package Seurat 3.0 (30).…”

Structural Analysis of SARS-CoV-2 ORF8 Protein: Pathogenic and Therapeutic Implications

“…Other effect of the structural mimicry of ORF8 is its ability to activate the immune response by itself due to its similarity with the soluble IL-1β receptor and IL-1RA agonists, stimulating the inflammation process. ORF8 also mimics CD79B (3KG5-A) and CD80 (1DR9-A), which are antigens required to activate B and T cell effector functions, respectively (Vasile et al, 1994;Trzupek et al, 2019). However, ORF8 is not precisely equal to such antigens, and can produce an incomplete stimulation of the receptors.…”