This repository contains scripts for the CD4⁺ T-cell division analysis project (aka Cell Divider). It contains all analysis scripts used for generating the manuscript. All absolute paths up to the project root have been replaced by <ROOT_PATH>/
Immune disease variants point to the causal role of T-cell activation in disease pathogenesis. Activation of CD4+ T-cells triggers a cascade of dynamic changes, propelling cells towards cell division. Yet, T-cells exhibit distinct proliferation rates despite receiving homogenous in vitro stimulation. Activation is typically studied along a time course following stimulation rather than focusing on the extent of cellular response. To address this gap, we generated a high-resolution single-cell map of CD4+ T-cell proliferation.
In the first part of the project, T-cell proliferation was characterised using Cell Trace Violet (CTV) assays. Secondly, we generated a small-scale single-cell transcriptomic and proteomic dataset (Pilot Data) profiling CD4+ memory T-cells from one donor. Cells were activated with 2:1 anti-CD28 and anti-CD3 Dynabeads. In this pilot dataset, we evaluate the experimental design and identify initial division-dependent gene and protein expression. Thirdly, we generate a large-scale single-cell dataset from 4 healthy female donors stimulated with anti-CD3, anti-CD2 and anti-CD28 Immunocult. We activated three CD4+ T-cell subtypes: naive, memory, and regulatory cells. We profiled cells across five generations of division using single-cell transcriptomics and expression of 130 surface proteins. Our multimodal map encompasses 115,013 single cells, with 15,971 - 24,402 cells at individual generations.
Data for the pilot and NMT is available at: https://www.ebi.ac.uk/biostudies/studies/S-BSST2659?query=celldivider
- Data: "<ROOT_PATH>/CellDivider/data/seurat/pilot_seu_memory.rds"
- Calculated using the QC parameters described in paper
- Pilot data from one healthy female donor, using memory isolation kits stimulated with Dynabeads (2:1 ratio, anti-CD3, anti-CD28)
- Time points: Resting, 16 hour, 3 days and 5 days
- RNA data was not corrected for ambient RNA
- Includes SCT_pca, SCT_umap, ADT_pca, ADT_umap
- Data: "<ROOT_PATH>/CellDivider/data/seurat/NMT_seu.rds"
- Using the QC seurat object above, subset to only cells isolated from their respective kits (e.g. transcriptomically-naive-cells-isolated-by-naive-kits, transcriptomically-memory-cells-isolated-by-memory-kits and transcriptomically-treg-cells-isolated-by-treg-kits). This ensures that the different cultures do not influence interpretation as Treg culture was supplemented with IL2, naive and memory cultures were not. However naive cells are suspected to produce more IL2.
- Includes SCT_pca, SCT_umap, ADT_pca, ADT_umap