Systemic hypoxia inhibits T cell response by limiting mitobiogenesis via matrix substrate-level phosphorylation arrest

  1. Amijai Saragovi
  2. Ifat Abramovich
  3. Ibrahim Omar
  4. Eliran Arbib
  5. Ori Toker
  6. Eyal Gottlieb
  7. Michael Berger  Is a corresponding author
  1. The Lautenberg center for Immunology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University Medical School, Israel
  2. The Ruth and Bruce Rappaport, Faculty of Medicine, Technion - Israel Institute of Technology, Israel
  3. Faculty of Medicine, Hebrew University of Jerusalem; The Allergy and Immunology Unit, Shaare Zedek Medical Center, Israel
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Systemic chronic oxygen restriction inhibits CD8+ T cell activation and response.

(A) Schematic of the experiment presented in panels B-H. C57BL6 mice were primed intradermally in the ear pinna with 5 × 106 transduction units (TU) of Lv-OVA or left untreated. Twenty-four hours …

Figure 1—figure supplement 1
Systemic chronic oxygen restriction inhibits CD8T cell activation and response.

(A–C) C57BL6 mice were primed intradermally in the ear pinna with 5 × 106 TU of Lv-OVA or left untreated. Twenty-four hours following the viral challenge, mice were transferred to chambers and …

Figure 2 with 2 supplements
Following mitochondrial remodeling, activated CD8+ T cell become tolerant to inhibition of OXPHOS.

(A) Schematic of experiment presented in panels B-E. CellTrace-labeled splenocytes were activated with anti-CD3/28 and transferred to chambers containing 1% O2 for 5 (middle/red), or 18 hr …

Figure 2—figure supplement 1
Oligomycin inhibits CD8+ T cell respiration, activation, and proliferation.

(A) Oxygen Consumption Rate (OCR) of isolated naïve CD8+ T cells treated with the indicated oligomycin, FCCP concentrations or left untreated. (n = 5 biological replicates). (B) Bar graph …

Figure 2—figure supplement 2
Acquisition of respiratory restriction tolerance is not correlated with increased glycolytic activity.

(A) Extra Cellular Acidification Rate (ECAR), measured by seahorse, of Naïve CD8+ T cells (gray), T-Early (red) and T-Late (pink) under basal conditions (BASAL) or oligomycin treatment (Max). Dashed …

Figure 3 with 1 supplement
Respiratory restriction has only marginal effect on cytoplasmic function during early activation.

(A) Heatmap showing relative amounts of key energy-related metabolites (as indicated in the figure) extracted from CD8+ T cells activated for 5 hr using anti-CD3/28 (T-Early) oligomycin treated or …

Figure 3—figure supplement 1
Inhibition of ANT2 leads to increased mitochondrial membrane polarization in CD8+ T cells.

(A) Label-free quantification (LFQ) of ANT1 relatively to the LFQ of ANT2 in naïve CD8+ T cells measured by MS analysis.(n = 5 mice grouped into six replicates, P value ****<0.0001). (B) Flow …

Figure 4 with 1 supplement
Respiratory restriction leads to energetic crisis within the matrix compartment in early activated CD8+ T cell.

(A-D) Splenocytes from mito-Dendra2 mice were activated with anti-CD3/28 for 5 hr and then transferred to a chamber containing 1% O2 or left in atmospheric oxygen pressure (21% O2). Twenty-four …

Figure 4—figure supplement 1
Respiratory restriction induces an energetic crisis within the matrix compartment in early activated CD8+ T Cell.

A) Splenocytes were stimulated using anti CD3/CD28 for 9, 12 and 24 hr. Cells were then treated with oligomycin for one hour or left untreated. Isolated CD8+ T cells were then analyzed using qRT-PCR …

Figure 5 with 1 supplement
FCCP treatment rescues respiratory-restricted CD8+ T cells by stimulating matrix-localized substrate-level phosphorylation, elevating ATP, and reducing AMP/GMP concentrations.

(A-D) CellTrace-labeled splenocytes were stimulated using anti-CD3/28. Nine hours post activation cells were left untreated or treated with 1 μM FCCP, 60 nM oligomycin or with a combination of FCCP …

Figure 5—figure supplement 1
Schematic of the suggested model; respiratory restriction through inhibition of complex V by oligomycin leads to increase in mitochondrial membrane potential and decreased electron flow.

(Top) Decreased electron flow leads to inhibition of TCA cycle and substrate-level phosphorylation since that these processes are coupled. (Bottom) Schematic of the suggested model for rescuing …

Figure 6 with 1 supplement
Short oxygen exposure rescues CD8+ T cells activated under hypoxia in vivo.

(A) Schematic describing the experiment in B-E. CellTrace-labeled mouse splenocytes were activated with anti-CD3/28 under 1% or atmospheric oxygen pressure (21% O2). Twenty-four hours post …

Figure 6—figure supplement 1
Short oxygen exposure rescues CD8+ T cells activated under hypoxia in vivo.

C57BL6 mice were primed intradermally in the ear pinna with 5 × 106 TU of Lv-OVA or left uninfected. Twenty-four hours after the viral challenge, mice were divided into four groups: (1) Lv-OVA …

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyAnti-mouse-CD8α (Rat Monoclonal) clone 53–6.7BiolegendCat# 10071
Cat# 10072
Cat# 10072
FACS 1:500
AntibodyAnti-mouse-CD44 (Rat Monoclonal) clone IM7BiolegendCat# 10452FACS 1:1000
AntibodyAnti-mouse-CD69 (Armenian Hamster Monoclonal) clone H1.2F3BiolegendCat# 10451FACS 1:500
AntibodyAnti-mouse-CD25 (Rat Monoclonal) clone 3C7BiolegendCat# 10190FACS 1:500
AntibodyAnti-mouse-CD62L (Rat Monoclonal) clone MEL-14BiolegendCat# 10441FACS 1:500
AntibodyAnti-mouse TCR Vα2 (Rat Monoclonal) clone B20.1BiolegendCat# 12780FACS 1:1000
AntibodyAnti-mouse-Ki67 (Rat Monoclonal) clone 16A8BiolegendCat# 652423FACS 1:100
AntibodyPurified anti mouse-C3ϵ (Armenian Hamster monoclonal) clone 145–2 C11BiolegendCat# 100340Activation 0.1 μg/ml
AntibodyPurified anti mouse-CD28 (Syrian Hamster monoclonal) clone 37.51BiolegendCat# 102116Activation 0.1 μg/ml
AntibodyPurified anti-human-CD3ϵ (Mouse monoclonal) clone OKT3BiolegendCat# 317326Activation 0.1 μg /ml
AntibodyPurified anti-human-CD28 (Mouse monoclonal) clone CD28.2BiolegendCat# 302934Activation 0.1 μg/ml
AntibodyAnti-human-CD8α (Mouse monoclonal) clone HIT8aBiolegendCat# 30090FACS 1:400
AntibodyAnti-human-CD25 (Mouse monoclonal) clone M-A251BiolegendCat# 35610FACS 1:500
AntibodyAnti-mouse AMPKα (Rabbit monoclonal)Cell SignallingCat# 2532WB 1:1000
AntibodyAnti-mouse phospho-AMPKα (Rabbit monoclonal)Cell SignallingCat#: 2531WB 1:1000
AntibodyDonkey Anti-Rabbit IgG H and L (HRP) (Donkey polyclonal)abcamCat# ab97085WB 1:10000
AntibodyAnti-Ubiquitin (Mouse monoclonal) clone FK2Merck-MilliporeCat# ST1200IP 2 μg
Chemical compound, drugOligomycin ACayman ChemicalsCat# 113421 nM - 1 μM
Chemical compound, drugRotenoneCayman ChemicalsCat# 139951 μM
Chemical compound, drugAntimycin ACayman ChemicalsCat# 194331 μM
Chemical compound, drugFCCPCayman ChemicalsCat# 152181 μM
Chemical compound, drugBongkrekic Acid (ammonium salt)Cayman ChemicalsCat# 190791 μM - 2 μM
Chemical compound, drugEZview Red Protein G
Affinity Gel
Sigma-AldrichCat# E3403
Chemical compound, drugProtease Inhibitor CocktailSigma-Aldrich IsraelP8340WB and IP 1:100
Sequence-based reagentUbc FThis paperPCR primersGCCCAGTGTTACCACCAAGA
Sequence-based reagentUbc RThis paperPCR primersCCCATCACACCCAAGAACA
Sequence-based reagentRpl13 FThis paperPCR primersATGACAAGAAAAAGCGGATG
Sequence-based reagentRpl13 RThis paperPCR primersCTTTCCTGCCTGTTTCCGTA
Sequence-based reagentmt-Rnr FThis paperPCR primersCATACTGGAAAGTGTGCTTGGA
Sequence-based reagentmt-Rnr RThis paperPCR primersGTGTAGGGCTAGGGCTAGGA
Sequence-based reagentmt-Rnr1 FThis paperPCR primersACCGCGGTCATACGATTAAC
Sequence-based reagentmt-Rnr1 RThis paperPCR primersCCCAGTTTGG
GTCTTAGCTG
Sequence-based reagentmt-Rnr2 FThis paperPCR primersGGGATAACAGCGCAATCCTA
Sequence-based reagentmt-Rnr2 RThis paperPCR primersGATTGCTCCGGTCTGAACTC
Commercial assay, kitMitoProbe TMRM Assay Kit for Flow CytometryThermo Fischer:Cat# M20036FACS 50 nM
Commercial assay, kitProteaseMAX SurfactantPromega CorpCat# V2071
Commercial assay, kitCellTrace Violet Cell Proliferation Kit, for flow cytometryThermo Fischer: Molecular ProbesCat# C34571FACS 1:100
Commercial assay, kitEasySep Mouse CD8+T Cell Isolation KitSTEMCELL TechnologiesCat# 19853A
Commercial assay, kitDirect-zol RNA MiniPrep PlusZymo ResearchCat# R2071
Commercial assay, kitProtoScript First Strand cDNA Synthesis KitNew England BioLabs, IncCat# E6300L
Commercial assay, kitPower SYBR Green PCR Master MixApplied BiosystemsCat# 4367660
Strain, strain background Mus musculusC57BL/6JJackson LaboratoryStock No: 000664Wild type
Strain, strain background Mus musculusSlc25a5tm1.1Nte/JJackson LaboratoryStock No: 029482ANT2flox/lox
Strain, strain background Mus musculusC57BL/6-Tg(TcraTcrb)1100Mjb/JJackson LaboratoryStock No: 003831OT1
Strain, strain background Mus musculusB6.Cg-Tg(Lck-cre)1CwiN9 (Lck-Cre)TaconicModel # 4197Lck-Cre
Strain, strain background Mus musculusGt(ROSA)26Sortm1.1(CAG-Mito-Dendra2) DccDr. Tsvee Lapidot from the Weizmann Institute of Sciencemito-Dendra2
Recombinant DNA reagentLv-OVA-GFPDr. Avihai Hovav from the Hebrew University of JerusalemOvalbumin and GFP expressing lentiviral plasmid
Recombinant DNA reagentpCMV-VSV-Ga gift from Bob Weinberghttps://www.addgene.org/8454/Addgene Plasmid #8454VSV-G envelope expressing plasmid
Recombinant DNA reagentpsPAX2a gift from Didier Trono https://www.addgene.org/12260/Addgene Plasmid #12260Lentiviral packaging plasmid
Software, algorithmKaluza softwareBeckman CoulterFACS acquisition software
Software, algorithmFACS Express 6De Novo SoftwareFACS analysis software
Software, algorithmSeahorse WaveAgilentOCR and EACAR analysis
Software, algorithmPerseusMetabolic and proteomic analysis
Software, algorithmPrism 8GraphPadGraphs and Heatmaps, statistical analysis
Software, algorithmThermo XcaliburThermo Fisher ScientificMetabolomics LC-MS data acquisition
Software, algorithmTraceFinder 4.1Thermo Fisher ScientificMetabolomics LC-MS data analysis

Additional files

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