method-to-study-metabolism-lymphoid-cells-using-chemistry-to-measure

Method to Study Metabolism in Lymphoid Cells using Chemistry to Measure Puromycin Incorporation by Flow Cytometry

Upon antigen stimulation, na&#239;ve T cells undergo rapid proliferation and expansion to effector T cells. Metabolism plays an important role in the ...

Kyoto University Graduate School of Medicine

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cd mice), we demonstrate that, upon activation, Pik3cd CD8 T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cd CD8 cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cd CD8 cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8 T cell effector differentiation, providing insight into phenotypes of patients with APDS.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8 T cells at the expense of central memory.

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

Article	Year
PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8 T cells at the expense of central memory. Cell reports\| PubMed ID: 34644563	2021
In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells. Nature communications\| PubMed ID: 35145086	2022

Hirofumi Shibata

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