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ABSTRACT: Effects of Asparagine Deprivation on CD8+ T Cell Activation

PROVIDER: PRJNA1219369 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32235745_1.fastq.gz	Fastqsanger.gz
	SRR32235745_2.fastq.gz	Fastqsanger.gz
	SRR32235746_1.fastq.gz	Fastqsanger.gz
	SRR32235746_2.fastq.gz	Fastqsanger.gz
	SRR32235747_1.fastq.gz	Fastqsanger.gz

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Project description:The Effect of Asparagine Deprivation on CD8+ T Cell Activation

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Project description:Study of the effects of asparagine deprivation on germinal centre B cells.

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Asparagine availability controlsgerminal centre B cell homeostasis

Project description:Germinal centre (GC) B cells proliferate at some of the highest rates of any mammalian cell. Yet the metabolic processes which enable this are poorly understood. We performed integrated metabolomic and transcriptomic profiling of GC B cells, and found that asparagine metabolism is highly upregulated. Asparagine is conditionally essential to B cells, and its synthetic enzyme, asparagine synthetase (ASNS) is markedly upregulated following their activation, through the integrated stress response sensor general control non-derepressible 2 (GCN2). When Asns is deleted, B cell survival in low asparagine conditions is severely impaired. Using stable isotope tracing, we found that metabolic adaptation to the absence of asparagine requires ASNS, and that the synthesis of nucleotides is particularly sensitive to asparagine deprivation. Conditional deletion of Asns in B cells selectively impairs GC formation, associated with a reduction in RNA synthesis rates. Finally, removal of environmental asparagine by asparaginase was found to also severely compromise the GC reaction.

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Project description:IL2 deprivation time course for day 8 human CD8 T cells activated for 3 days with TransAct T-cell activation beads. PBMC (Cambridge Bio) were defrosted and CD8+ T-cells isolated using a human CD8+ T-cell isolation kit. CD8+ T-cells were activated at 1 x 106 cells/mL with 1 in 100 TransAct T-cell activation beads (Miltenyi) and 20 ng/mL IL-2 in complete RMPI (RMPI 1640, 10% FBS, penicillin/streptomycin) for three days. Cells were then washed out of activation and then maintained in complete RMPI with 20 ng/mL IL-2 for five days. To remove IL-2 for IL-2 deprivation, cells were washed twice in pre-warmed media, then replated in complete media without IL-2. Cells were deprived of IL-2 for 1hr, 3hrs, 5hrs, 8hrs, 24hrs or 48hrs.

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Project description:Microarray analysis of WT and Tfap4-KO CD8 T cells during early activation

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Oncogenic KRAS mediates amino acid metabolism during nutrient stress via regulation of NRF2 and ATF4 in NSCLC [shKRAS low Q]

Project description:Glutamine is a key nutrient for tumor cells that supports nucleotide and amino acid biosynthesis, replenishes the TCA cycle intermediates and contributes to redox metabolism. We identified oncogenic KRAS as a critical regulator of the response to glutamine deprivation in NSCLC. Full activation of the ATF4 stress response pathway is dependent on expression of NRF2 downstream of oncogenic KRAS in NSCLC. Through this mechanism, KRAS alters amino acid uptake and metabolism and sustains mTORC1 signaling during nutrient stress. Furthermore, we identified regulation of asparagine synthetase (ASNS) as a key effect of oncogenic KRAS signaling via ATF4 during glutamine deprivation, and a potential therapeutic target in KRAS mutant NSCLC.

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Oncogenic KRAS mediates amino acid metabolism during nutrient stress via regulation of NRF2 and ATF4 in NSCLC [shKRAS high Q]

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Asparagine regulates RIG-I stability to suppress anti-tumor immunity in bladder cancer

Project description:Tumor cells often employ many ways to restrain type I interferon (IFN-I) signaling to evade immune surveillance. However, whether cellular amino acid metabolism regulate this process remains unclear and its effects on antitumor immunity are relatively unexplored. Here, our study reports that asparagine generated by asparagine synthetase (ASNS) inhibits IFN-I signaling and promotes immune escape in bladder cancer. We further show that depletion of ASNS strongly limits in vivo tumor growth in a CD8+ T cell-dependent manner, thus boosting the immunotherapy efficacy. Moreover, clinically approved ASNase synergizes with anti-PD-1 therapy in suppressing tumor growth in mouse models of bladder cancer. Mechanistically, asparagine intensifies the interaction of E3 ligase CBL and RIG-I, promoting K48-linked polyubiquitination and degradation of RIG-I, thus suppressing RIG-I mediated IFN signaling and anti-tumor immune response. Clinically, ASNS is overexpressed in muscle-invasive bladder cancer and correlated with poor response of immunotherapy. Together, our findings uncover asparagine as a natural metabolite to modulate RIG-I-mediated IFN-I signaling, providing the basis for developing the combinatorial use of ASNase and anti-PD-1 for bladder cancer.

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