DOI: 10.5061/dryad.69p8cz98t
title: Data from: Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells

description: 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.
description: 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, <em>E. coli</em>, 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: # 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.
 [https://github.com/Mwielscher/RNAseq
 ](https://github.com/Mwielscher/RNAseq)