Data from: Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells
Data files
Feb 16, 2024 version files 6.48 MB
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count_matrix.txt
6.47 MB
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QC_alignment_overview.txt
10.31 KB
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README.md
3.21 KB
Abstract
CD8+ T cells rely on actin cytoskeleton remodeling to search for target cells and assemble the immunological synapse (IS) for lethal hit delivery. We here investigated how the energy expenditure related to actin remodeling might influence the fitness of human cytotoxic T cells. We first established that the spreading ability of CD8+ T cells in conditions of LFA-1 and TCR engagement mirrored the cytotoxic potential of these cells. Morphological and functional fitness were both potentiated by IL-2, which co-stimulated the transcription of glycolytic enzymes, actin isoforms and the subunits of the ARP2/3 complex. This molecular program scaled with F-actin content and cell spreading. Blockade of glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility and synaptic adhesion, while blockade of mitochondrial OXPHOS affected F-actin less severely and selectively reduced cell elongation during confined migration. Although T cells deficient for the ARP2/3 subunit ARPC1B increased their ATP content upon IL-2 exposure, their morphological and functional defects were only partially corrected, pointing to the pivotal position of ARP2/3 mediated actin polymerization as integrator of T cell energetic state. Our study therefore highlights that the ability of effector T cells to migrate, form IS and ultimately kill target cells depends on a tight coordination of their metabolic and actin remodeling activities.
README: Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells
Abstract:
CD8+ T cells rely on actin cytoskeleton remodeling to search for target cells and assemble the immunological synapse (IS) for lethal hit delivery. We here investigated how the energy expenditure related to actin remodeling might influence the fitness of human cytotoxic T cells. We first established that the spreading ability of CD8+ T cells in conditions of LFA-1 and TCR engagement mirrored the cytotoxic potential of these cells. Morphological and functional fitness were both potentiated by IL-2, which co-stimulated the transcription of glycolytic enzymes, actin isoforms and the subunits of the ARP2/3 complex. This molecular program scaled with F-actin content and cell spreading. Blockade of glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility and synaptic adhesion, while blockade of mitochondrial OXPHOS affected F-actin less severely and selectively reduced cell elongation during confined migration. Although T cells deficient for the ARP2/3 subunit ARPC1B increased their ATP content upon IL-2 exposure, their morphological and functional defects were only partially corrected, pointing to the pivotal position of ARP2/3 mediated actin polymerization as integrator of T cell energetic state. Our study therefore highlights that the ability of effector T cells to migrate, form IS and ultimately kill target cells depends on a tight coordination of their metabolic and actin remodeling activities.
Method Summary:
Library preparation was conducted on 1 ng of cDNA using the Nextera XT library preparation kit (Illumina). Sequencing was performed using the 50 bp single-read setup on the Illumina HiSeq 3000/4000 platform. Illumina adaptors were trimmed from raw reads using Picard tools (GATK). Basic quality control of 50 bp single end reads was performed with fastQC. Additional, qualimap and fastqc-screen reports were screened for outlying samples in terms of multi-mapping reads, possible contaminations (rRNA, intronic RNA, E. coli, etc.) and distribution of read coverage. Reads were then aligned to human genome build GRCh38 using STAR aligner with standard settings. Aligned reads were counted using featureCounts function of Rsubread package. The resulting count matrix was then analyzed using DESeq2 pipeline.
Description of the data and file structure
count_matrix.txt: This file contains the count matrix generated by Rsubread. The column names represent sample IDs, while the row names correspond to Entrez IDs (i.e., NCBI Gene Identifiers).
QC_alignment_overview.txt: The row names in this file match the column names in count_matrix.txt. The column names are derived from NGS quality control tools. The column average_mapped_READ_length is expressed in base pairs. All other numeric columns represent percentage values.
Sharing/Access information
This data were specifically produced for this project.
Code/Software
Code to reproduce the basic analysis described in the paper can be accessed via GitHub.
Methods
Five hundred cells were lysed in 4 µl of lysis buffer and cDNA synthesis and enrichment were performed following the Smart-seq2 protocol. Briefly, library preparation was conducted on 1 ng of cDNA using the Nextera XT library preparation kit (Illumina). Sequencing was performed using the 50 bp single-read setup on the Illumina HiSeq 3000/4000 platform. Illumina adaptors were trimmed from raw reads using Picard tools (GATK). Basic quality control of 50 bp single end reads was performed with fastQC. Additionally, qualimap and fastqc-screen reports were screened for outlying samples in terms of multimapping reads, possible contaminations (rRNA, intronic RNA, E. coli, etc.) and distribution of read coverage. Reads were then aligned to human genome build GRCh38 using STAR aligner with standard settings. Aligned reads were counted using featureCounts function of Rsubread package. The resulting count matrix was then analyzed using DESeq2 pipeline.