T cell cholesterol transport links intestinal immune responses to dietary lipid absorption

The intrinsic pathways that control membrane organization in immune cells and the impact of such pathways on cellular functions are not well defined. Here we show that the nonvesicular cholesterol transporter Aster-A links plasma membrane (PM) cholesterol availability in T cells to immune signaling and systemic metabolism. Aster-A is recruited to the PM during T-cell receptor (TCR) activation, where it facilitates the removal of “accessible” cholesterol. Loss of Aster-A leads to PM cholesterol accumulation, resulting in enhanced TCR nano-clustering and signaling, and Th17 cytokine production. Furthermore, Aster-A associates with STIM1 and negatively regulates STIM1-dependent Ca2+ flux during activation of mouse and human T cells. Finally, mucosal Th17 response towards commensals is restrained by PM cholesterol remodeling. Ablation of Aster-A in T cells stimulates IL-22 production, which reduces intestinal fatty acid absorption and confers resistance to diet-induced obesity. These findin..., Biotin affinity purification and sample preparation for proximity labeling
EL-4 cell lines stably transduced with pRetroX-3xHA-TurboID-Aster-A or pRetroX-3HA-TurboID (empty vector) were constructed as described above. Tight TRE promoter has a leakage expression in EL-4 cell line, which resulted in moderate expression of 3x-HA-TurboID-Aster-A and stable cell lines were therefore used without doxycycline induction.
TurboID or TurboID-Aster-A expressing EL-4 cells were cultured in 10% FCS complete culture media or starved with a cholesterol-depletion media containing 1% Lipoprotein-deficient serum, 5 μM Simvastatin, 50 µM Mevalonate for 18-24 h. Depleted cells were either loaded with 100 μΜ MβCD-Cholesterol or maintained in fresh cholesterol-depletion media for 1 h and 15 minutes. Cholesterol-rich samples are maintained in 10% FCS complete media for the same time. 50 Î¼M Biotin was added to the culture media for the last 30 minutes to facilitate labeling. Isolation of biotinylated prot..., # T cell cholesterol transport links intestinal immune responses to dietary lipid absorption

Dataset DOI: [10.5061/dryad.w9ghx3g31](10.5061/dryad.w9ghx3g31)

## Description of the data and file structure

### Files and variables

#### File: wc_QE_080622_Tontonoz_Yajing_5_A-LPDS.raw

**Description:** Raw mass spectrometry peptide identification data for pull-down of biotinylated proteins from EL-4 cells overexpressing TurboID-Aster-A, starved overnight in media containing lipoprotein deficient serum (LPDS), simvastatin and mevalonate. 

#### File: wc_QE_080622_Tontonoz_Yajing_3_Turbo-Chol.raw

**Description:** Raw mass spectrometry peptide identification data for pull-down of biotinylated proteins from EL-4 cells overexpressing only TurboID, starved overnight in media containing lipoprotein deficient serum (LPDS), simvastatin and mevalonate, and reloaded with MbCD-Cholesterol. 

#### File: wc_QE_080622_Tontonoz_Yajing_2_Turbo-LPDS.raw

**Description:** Raw mass spectrometry peptide iden...,