Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation
- University of Dundee, United Kingdom
Abstract
T cell expansion and differentiation are critically dependent on the transcription factor c-Myc (Myc). Herein we use quantitative mass-spectrometry to reveal how Myc controls antigen receptor driven cell growth and proteome restructuring in murine T cells. Analysis of copy numbers per cell of >7000 proteins provides new understanding of the selective role of Myc in controlling the protein machinery that govern T cell fate. The data identify both Myc dependent and independent metabolic processes in immune activated T cells. We uncover that a primary function of Myc is to control expression of multiple amino acid transporters and that loss of a single Myc-controlled amino acid transporter effectively phenocopies the impact of Myc deletion. This study provides a comprehensive map of how Myc selectively shapes T cell phenotypes, revealing that Myc induction of amino acid transport is pivotal for subsequent bioenergetic and biosynthetic programs and licences T cell receptor driven proteome reprogramming.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Raw mass spec data files and MaxQuant analysis files for naïve WT, and TCR activated MycWT, MyccKO, Slc7a5WT and Slc7a5cKO T cells are available on the ProteomeXchange data repository (https://www.ebi.ac.uk/pride/archive/login) and can be accessed with identifier PXD016105. Raw mass spec data files and MaxQuant analysis files for OT-1 TCR time-course data are available on the ProteomeXchange data repository (https://www.ebi.ac.uk/pride/archive/login) and can be accessed with identifier PXD016443.
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OT1 T cell activation time courseProteomeXchange, PXD016443.
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Single-cell RNA sequencing of OT-I CD8+ T cells after stimulation with different affinity ligandsArrayExpress identifier: E-MTAB-6051.
Article and author information
Author details
Funding
Wellcome (097418/Z/11/Z)
- Doreen A Cantrell
Wellcome (205023/Z/16/Z)
- Doreen A Cantrell
Wellcome (202950/Z/16/Z)
- Doreen A Cantrell
European Molecular Biology Organization (ALTF 1543-2015)
- Julia M Marchingo
European Commission (705984)
- Julia M Marchingo
- Doreen A Cantrell
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were performed under Project License PPL 60/4488 and P4BD0CE74. The University of Dundee Welfare and Ethical Use of Animals Committee accepted the project license for submission to the Home Office. Mice were bred and maintained in the WTB/RUTG, University of Dundee in compliance with UK Home Office Animals (Scientific Procedures) Act 1986 guidelines.
Copyright
© 2020, Marchingo et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Quantitative analysis of how Myc controls T cell proteomes and metabolic pathways during T cell activation
eLife 9:e53725.
https://doi.org/10.7554/eLife.53725
Further reading
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- Immunology and Inflammation
- Medicine
Background:
Individuals with Down syndrome (DS), the genetic condition caused by trisomy 21 (T21), display clear signs of immune dysregulation, including high rates of autoimmunity and severe complications from infections. Although it is well established that T21 causes increased interferon responses and JAK/STAT signaling, elevated autoantibodies, global immune remodeling, and hypercytokinemia, the interplay between these processes, the clinical manifestations of DS, and potential therapeutic interventions remain ill defined.
Methods:
We report a comprehensive analysis of immune dysregulation at the clinical, cellular, and molecular level in hundreds of individuals with DS, including autoantibody profiling, cytokine analysis, and deep immune mapping. We also report the interim analysis of a Phase II clinical trial investigating the safety and efficacy of the JAK inhibitor tofacitinib through multiple clinical and molecular endpoints.
Results:
We demonstrate multi-organ autoimmunity of pediatric onset concurrent with unexpected autoantibody-phenotype associations in DS. Importantly, constitutive immune remodeling and hypercytokinemia occur from an early age prior to autoimmune diagnoses or autoantibody production. Analysis of the first 10 participants to complete 16 weeks of tofacitinib treatment shows a good safety profile and no serious adverse events. Treatment reduced skin pathology in alopecia areata, psoriasis, and atopic dermatitis, while decreasing interferon scores, cytokine scores, and levels of pathogenic autoantibodies without overt immune suppression.
Conclusions:
JAK inhibition is a valid strategy to treat autoimmune conditions in DS. Additional research is needed to define the effects of JAK inhibition on the broader developmental and clinical hallmarks of DS.
Funding:
NIAMS, Global Down Syndrome Foundation.
Clinical trial number:
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- Immunology and Inflammation
Since the precursor frequency of naive T cells is extremely low, investigating the early steps of antigen-specific T cell activation is challenging. To overcome this detection problem, adoptive transfer of a cohort of T cells purified from T cell receptor (TCR) transgenic donors has been extensively used but is not readily available for emerging pathogens. Constructing TCR transgenic mice from T cell hybridomas is a labor-intensive and sometimes erratic process, since the best clones are selected based on antigen-induced CD69 upregulation or IL-2 production in vitro, and TCR chains are polymerase chain reaction (PCR)-cloned into expression vectors. Here, we exploited the rapid advances in single-cell sequencing and TCR repertoire analysis to select the best clones without hybridoma selection, and generated CORSET8 mice (CORona Spike Epitope specific CD8 T cell), carrying a TCR specific for the Spike protein of SARS-CoV-2. Implementing newly created DALI software for TCR repertoire analysis in single-cell analysis enabled the rapid selection of the ideal responder CD8 T cell clone, based on antigen reactivity, proliferation, and immunophenotype in vivo. Identified TCR sequences were inserted as synthetic DNA into an expression vector and transgenic CORSET8 donor mice were created. After immunization with Spike/CpG-motifs, mRNA vaccination or SARS-CoV-2 infection, CORSET8 T cells strongly proliferated and showed signs of T cell activation. Thus, a combination of TCR repertoire analysis and scRNA immunophenotyping allowed rapid selection of antigen-specific TCR sequences that can be used to generate TCR transgenic mice.
