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 ...

Tumor susceptibility gene 101 (TSG101), an ESCRT-I protein, is implicated in multiple cellular processes and its functional depletion can lead to blocked lysosomal degradation, cell cycle arrest, demyelination and neurodegeneration. Here, we show that loss of TSG101 results in endoplasmic reticulum (ER) stress and this causes ER membrane remodelling (EMR). This correlates with an expansion of ER, increased vacuolation, altered relative distribution of the rough and smooth ER and disruption of three-way junctions. Blocked lysosomal degradation due to TSG101 depletion leads to ER stress and Ca leakage from ER stores, causing destabilization of actin cytoskeleton. Inhibiting Ca release from the ER by blocking ryanodine receptors (RYRs) with Dantrolene partially rescues the ER stress phenotypes. Hence, in this study we have identified the involvement of TSG101 in modulating ER stress mediated remodelling by engaging the actin cytoskeleton. This is significant because functional depletion of TSG101 effectuates ER-stress, perturbs the structure, mobility and function of the ER, all aspects closely associated with neurodegenerative diseases. SUMMARY STATEMENT: We show that tumor susceptibility gene (TSG) 101 regulates endoplasmic reticulum (ER) stress and its membrane remodelling. Loss of TSG101 perturbs structure, mobility and function of the ER as a consequence of actin destabilization.

Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function.

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.

Development of protective immune responses relies on a balance between proinflammatory CD4 T helper (Th) cell populations such as Th17 cells and regulatory CD4 T cells (Tregs) that keep immune activation in check. Evidence that interleukin-2-inducible T cell kinase (Itk) regulates this balance supports therapeutic applications for Itk inhibition.

Article	Year
Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 32422153	2020
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
Balancing immune activation with Itk. Trends in pharmacological sciences\| PubMed ID: 39358175	2024

Zenia Kaul

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