L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity
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L-arginine modulates T cell metabolism and enhances survival and anti-tumor Activity How metabolic reprogramming of T cells impact T cells fate and function? Indeed, these cells enhance their metabolic throughput upon antigenic stimulation to ensure the production of sufficient biomass and energy for their proliferation, differentiation and effector functions. The naïve T cells, which utilize oxidative phosphorylation for energy supply but upon activation, these cells switch their metabolic pathways toward glycolysis. An important question arises here that whether differential metabolic reprogramming (i.e., use of oxidative phosphorylation versus glycolytic activity) directs the fate and function of a given cell? And whether this process shapes the immune response and individual cell survival? The researchers from Institute for Research in Biomedicine took advantage of high-resolution Mass Spectrometry to investigate dynamics of proteome and metabolome following activation of naïve T cells.
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L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity
- 1. L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity Roger Geiger, Jan C. Rieckmann, Tobias Wolf, Camilla Basso, Yuehan Feng, Tobias Fuhrer, Maria Kogadeeva, Paola Picotti, Felix Meissner, Matthias Mann, Nicola Zamboni, Federica Sallusto and Antonio Lanzavecchia Institute for Research in Biomedicine, Universita` della Svizzera italiana, Bellinzona 6500, Switzerland Institute of Microbiology, ETH Zurich, Zurich 8093, Switzerland Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried 82152, Germany Institute of Biochemistry, ETH Zurich, Zurich 8093, Switzerland Institute of Molecular Systems Biology, ETH Zurich, Zurich 8093, Switzerland Center of Medical Immunology, Institute for Research in Biomedicine, Universita` della Svizzera italiana, Bellinzona 6500, Switzerland Coach Professor: Dr. Chen, Yu-Ju Sit-in Professor: Prof. Yu-Ling, Shih Course Coordinator: Dr. Shie, Jiun-Jie Dated: 20180516 Presenter: Gul Muneer Cell 13 October, 2016 DOI: 10.1016/j.cell.2016.09.031 1
- 2. L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor Activity Roger Geiger†, Jan C. Rieckmann, Tobias Wolf, Camilla Basso, Yuehan Feng, Tobias Fuhrer, Maria Kogadeeva, Paola Picotti, Felix Meissner, Matthias Mann‡, Nicola Zamboni, Federica Sallusto and Antonio Lanzavecchia† 2 †Institute for Research in Biomedicine, Universita` della Svizzera italiana, Bellinzona 6500, Switzerland †Institute of Microbiology, ETH Zurich, Zurich 8093, Switzerland ‡Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried 82152, Germany
- 3. T Cell from Bone Marrow Activated T CellNaïve T cell Thymus Antigen Presenting Cell Differentiation Central Memory T cells (Tcm) Express CCR7⁺, CD62L & IL-2 Release less or no IFN-γ Th1 Th2 TCM TEM Re-stimulation/ Re-activation Effector Memory T cells (Tem) Express No CCR7‾ and IL-2 Release high IFN-γ Differentiating Cytokines: IL-12 IL-4 IFN-γ IL-4 T Cells Proliferation & Differentiation
- 4. Activated T Cell APC Antigenic Stimulation Naïve T cell Secondary Lymphoid Organs Nutrients Oxidative Phosphorylation Nutrients Glycolytic Activity Apoptotic Cell Memory Cell After Immune Response ~ 95% ~ 5% Metabolic Pathways of T cells
- 5. Activated T Cell Tumor Microenvironment Antigen Presenting Cell T cell Dysfunction/Exhausted Apoptotic Cell Glucose Interferon-γ L-Arginine CD3ξ Chain Degradation Expression Insufficient Nutrient Myeloid-Derived Suppressor Cells Tumor Cell Tumor Cell Tumor Cell Tumor Cell Tumor Cell T cell Metabolic Fitness in Antitumor Immunity
- 6. APC METABOLOME? Activated T Cell PROTEOME? Activation Induced Changes Questions of the Study? What are the changes in proteome & metabolome when a naïve T cells undergoes activation? How change in proteome & Metabolome determines the fate of activated T cell? T CELL FATE?
- 7. EDTA Blood Ficoll RBCs Granulocytes Ficoll PBMCs (Interphase) Plasma CFG CD4⁺ CD8⁺ APC B Cell Peripheral Blood Mononucleated Cells (PBMCs) + + + + + + + FACS Cell Sorter Sorting Isolation of Human T Cells 1. Isolation 2. Magnetic Cell Sorting 3. Fluorescence Activated Cell Sorting
- 8. MS MS/MSLC …………...…………... Data Analysis & Integration Proteins Metabolites Activationtimecourse MS/MSLC Schematic View of the Experimental Approach Differential proteins/ metabolites Expression Analysis of proteins/ Metabolites of Pathways
- 9. Proteomic & Metabolomic Changes Following Activation of Human Naïve CD4⁺ Cells TCR/CD3 α-CD3α-CD28 CD28 CD4⁺ Beads/Plate bound Antibodies Collectively, these data reveals different changes occurring in the Proteome and Metabolome of activated Human T cells Total Proteins: 7,816 Differently Expressed Proteins: 2,824 Total Metabolites: 429 Up regulated: 49 Down regulated: 14
- 10. L-Arginine Is Rapidly Metabolized Upon Activation
- 11. Ornithine CAT-1 Arginine Arginine N-Acetyl ornithine OAT ACY1SRM ALDH18A1 Citrulline Fumarate Arginosucinate N-Acetyl Citrulline PutrescineSpermidine Guanidino Butanamide ALDH18A1 Proline Pyrroline-5 Carboxylate Glutamate Glutamate-5 Semialdehyde Glutamate-5 Phosphate Urea NO ASL GAMT AGMAT ARG2 PYCR2 PYCR1 PYCRL Metabolic Fate of L-arginine -6-4-2-4-22424600 Log2FoldChange PROTEINSMETABOLITES norNOHA Collectively, these data show that L-arginine is taken up by activated T cells in amounts exceeding the requirements for protein synthesis and can be rapidly converted by into downstream metabolites. Creatine Agmatine
- 12. L-Arginine Globally Influences Metabolism of Activated Human T Cells: Metabolome Analysis Naïve T cells Activation Plate bound Antibodies + 3 mM L-Arginine/L- ornithine MS Metabolites
- 13. HK1 HK2 SLC2A1SLC2A3 GlucoseGlucose Glucose Glucose-6-P Fructose-6-P PFKM PFBP1 ALDOCALDOA Fructose-1,6-bis-P Gly-3-P DHAPTPI1 GAPDH PGK1 1,3 BPG 3-P-Glycerate PGAM1 2-P-Glycerate P-enol-pyruvate ENO1 ENO2 ENO3 PKM Pyruvate Acetyl-CoA PCK2 PC Lactate LDHA LDHB Citrate Cis-Aconitase Isocitrate OxaloacetateMalate Fumarate Succinate α-Ketoglutarate ACO₂ ACO₂ IDH3A IDH3B IDH3AOGDH SDHA SDHB SDHC SDHD FH MDH2 TCA CYCLE PHGDH PSAT1 PSPH Glutamate P-Serine Serine GPI PFKP PFKL -1-0.5-2-1140.511.500 Log2FoldChange METABOLITESPROTEINS -1.5 Differential Analysis of Glycolysis & TCA
- 14. Glucose & O₂ Consumption in Activated Human T Cells After 72 hours Fresh Medium After 24 hours Glucose Assay Kit Naïve T cells Activation Plate bound Antibodies L-Arginine CD4⁺ Naïve T cells Activation Plate bound Antibodies L-Arginine CD4⁺ 7 X 10⁶ Cells Seahorse XF-24 Extracellular Flux Analyzer 4 days later Collectively, these data demonstrate that an increase in intracellular L-arginine levels tilted the metabolism in activated T cells from glycolysis toward mitochondrial OXPHOS. 56 96 136
- 15. Effect of L-Arginine on Human T Cell Proliferation & Differentiation 10⁵ Naïve T cells Activation & Th1 Polarization Plate bound Antibodies + IL-12 + L-Arginine IFN- γ Quantification Phorbol 12-myristate 13 acetate & Ionomycin Re-stimulation Cell Trace Violet (CTV) 3rd Generation Brightness NumberofCells 2nd Generation Flow Cytometry Labelling with Proliferative dye (CTV) Activation Plate bound Antibodies+ L-Arginine Flow Cytometry 1st Generation
- 16. Role of L-Arginine on Human T Cell Survival Naïve T cells Activation Plate bound Antibodies + IL-2 + L-Arginine After 3 days Ctrl Medium No IL & L-Arginine Cell Viability
- 17. Activation Plate bound Antibodies + IL-2 + L-Arginine After 3 days Ctrl Medium + Arginase Inhibitors Cell Viability Supporting the Notion: L-Arginine Regulates T Cell Survival CAT-1 Ornithine Arginine Urea Cycle ASL ARG2 L-Arginine Naïve T cells Collectively, these data indicate that elevated intracellular L-arginine levels directly induced metabolic changes and longevity of human CD4+ and CD8+ T cells, independently of downstream metabolites. norNOHA= Nω-hydroxy-nor-Arginine BEC= Boronoethylcystein
- 18. Host Mice Receiving L- Arginine 1.5 g/Kg APC CD99.1⁺ CD4⁺ Transfer Influenza HA₁₁₀₋₁₁₉ Peptide 2x10⁵ cells Naïve Transgenic TCR T cells Immunization (s.c) CD99.2⁺ Solution of antigen FACS Cell Sorter 15days post Immunization Amount of Transgenic T cells L-Arginine Influences Mouse T cell Survival In Vivo
- 19. CD4⁺CD8⁺ Naïve Arg2-/- cells Activation Plate bound Antibodies + IL-2 + L-Arginine +/- Arginase Inhibitor After 4 days Ctrl Medium Cell Viability After 2 days L-Arginine Influences Mouse T cell Survival In Vitro CAT-1 Ornithine Arginine Urea Cycle ASL ARG2This data shows, inhibition of arginases in T cells or deletion of ARG2 in mouse T cells did not affect cell proliferation, suggesting that the downstream fate of L-arginine is less important in T cells than the levels of free L-arginine.
- 20. WT Mice APC CD99.1⁺ CD4⁺ Immunization CD45.2⁺ CD99.2⁺ Chicken Ovalbumin aFACS Cell Sorter 15days post Immunization Amount of Cells L-Arginine Influences Mouse T cell Survival In Vivo CD8⁺ Naïve WT CD45.1 cells CD8⁺ Naïve Arg2-/- CD45.2⁺, CD90.2⁺ cells Transferred Taken together, these findings provide evidence that intracellular L-arginine concentrations, can be elevated by dietary supplementation and can increase the survival capacity of antigen activated T cells in vivo.
- 21. Protein Structural Changes Across T Cell Proteome CD4⁺ Naïve T cells Homogenization Proteins L-Arginine/ D-Arginine Metabolite Proteinase K Denature & Trypsin Half-tryptic Tryptic
- 22. PTPN6SSB B2M DDX17 PSIP1ACIN1 WIBG XRCC6 TSN ACTR2MTPN CAP1 RPL4 RPS10 RPS3A RPS25 RPL6 DDX54 BAZ1B RPS11SPLICING APOPTOSIS RIBOSOME CYTOSKELETON DNAREPAIR Proteins experience structural changes in response to L-Arginine Thus, global approach revealed several proteins with various functions that structurally respond to elevated intracellular L-arginine levels Total Proteins: 5856 Proteins not affected by Proteinase K: 20
- 23. BAZ1B, PSIP1 & TSN Mediate L-Arginine Dependent Reprogramming of T cells BAZ1B = Bromodomain Adjacent To Zinc Finger Domain 1B (Tyrosine-protein kinase) PSIP1 = PC4 and SFRS1 interacting protein 1 (Epithelium-derived growth factor) TSN = Translin (DNA-binding protein) Knockout of transcriptional regulators affected L-arginine’s beneficial role on T- cell survival.
- 24. PTPN6SSB B2M DDX17 PSIP1ACIN1 WIBG XRCC6 TSN ACTR2MTPN CAP1 RPL4 RPS10 RPS3A RPS25 RPL6 DDX54 BAZ1B RPS11SPLICING APOPTOSIS RIBOSOME CYTOSKELETON DNAREPAIR Proteins experience structural changes in response to L-Arginine Taken together, these data provide evidence that BAZ1B, PSIP1, and TSN interact with L- arginine and play a role in the reprograming of T cells toward increased survival capacity.
- 25. L-Arginine Improves Anti-Tumor T cell Response In Vitro CD8⁺ Naïve OT-I cells Plate bound Antibodies + IL-2 + L-Arginine After 4 days Ctrl Medium Cell Viability After 2 days Chicken Ovalbumin 257-264 Peptide Activation
- 26. CD8⁺ Naïve CD90.1⁺ CD45.1/2⁺ OT-I T cells CD8⁺ Naïve CD90.1⁺ CD45.1⁺ OT-I T cells Plate bound Antibodies + IL-2 Activation Plate bound Antibodies + IL-2 + L-Arginine Activation Mice FACS Cell Sorter Expansion of OT-I Cells After 4 days L-Arginine Improves Anti-Tumor T cell Response in Vivo
- 27. T Cells Maintained Tcm (Central Memory) Like State Naïve OT-I T cells Activation Plate bound Antibodies + L-Arginine IFN- γ Quantification Phorbol 12-myristate 13 acetate & Ionomycin Re-stimulation Cell Trace Violet (CTV) 3rd Generation Brightness NumberofCells 2nd Generation Flow Cytometry Labelling with Proliferative dye (CTV) Activation Plate bound Antibodies+ L-Arginine Flow Cytometry 1st Generation CD8⁺
- 28. B16-OVA Melanoma Cells WT C57BL/6 Mice s.c. Injected Activated OT-I T cells (in vitro in L-Arginine) CD8⁺ After 10 days WT C57BL/6 Mice L-Arginine Improves Anti-Tumor T cell Response in Vivo
- 29. B16-OVA Melanoma Cells WT C57BL/6 Mice s.c. Injected Naïve OT-I T cells On 5th Day L-Arginine (1.5g/Kg body weight) CD8⁺ On 6th Day On 7th Day Immunization Chicken Ovalbumin L-Arginine Improves Anti-Tumor T cell Response in Vivo Collectively, these data demonstrate that elevated L-arginine levels increased the survival capacity of CD8+ T cells and their antitumor activity in vivo.
- 30. L-Arginine - L-Arginine Glycolysis OXPHOS Glycolysis OXPHOS + + Sensors SURVIVAL Activated T cell ARG2 ARG2 Central-Memory T Cell L-arginine is catabolized into downstream metabolites mainly by Arginase2. Elevated intracellular L-arginine reprograms metabolism & shift metabolism from glycolysis to OXPHOS. 3 nuclear proteins sense elevated L-arginine promotes T cell survival and maintains cell in Tcm like state. All these phenotype changes are responsible for improved anti-tumor activity. CONCLUSION
- 31. Thank you very much For Your Patience! Note: Survival refers to “Lymphocyte survival” not animal or patients survival! Don’t start taking Dietary L-arginine supplements!