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 findings delineate a multi-tiered regulatory scheme linking immune cell lipid flux to nutrient absorption and systemic physiology.

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 μM Biotin was added to the culture media for the last 30 minutes to facilitate labeling. Isolation of biotinylated proteins were performed based on a modified protocol (41). Cells were collected and washed extensively with cold PBS and incubated with lysis buffer (RIPA buffer containing 1X Halt protease and phosphatase inhibitor and 1 mM PMSF) with gentle agitation for 30 minutes on ice. Lysate were cleared by centrifugation at 12,000 xg for 15 minutes and protein concentration was determined by BCA assay. Equal quantities (900 μg) of cleared lysates in lysis buffer were incubated with 75 μl Streptavidin magnetic beads (Thermo Fisher Pierce) overnight at 4 °C with gentle rotation. The beads were then washed at room temperature twice in lysis buffer for 5 minutes each, once with 1 mL 1 M KCl for 2 minutes, once with 1 mL 0.1 M Na2CO3 for ~10 seconds, once with 1 mL 2 M urea in 10 mM Tris-HCl (pH 8.0) for ~10 seconds, and again twice with 1 mL lysis buffer for 2 minutes. 

In solution tryptic digestion for mass spectrometry

The samples were heated at 95 °C in elution buffer (12 mM sodium lauroyl sarcosine, 0.5% sodium deoxycholate, 50 mM triethylammonium bicarbonate (TEAB) containing tris(2-carboxyethyl)phosphine (10 mM) and chloroacetamide (40 mM) for 20 minutes. The samples were then diluted 5-fold with 50 mM TEAB and digested with trypsin (1 μg) for 16 h at 37 °C. A 1:1 (volume/volume) ratio of ethyl acetate plus 1% trifluoroacetic acid (TFA) was added to the samples and samples were vortexed for 5 minutes. Samples were centrifuged at 16000 xg for 5 minutes at room temperature and the supernatant was discarded. The samples were desalted using a modified version of Rappsilber's protocol (91) in which the dried samples were reconstituted in acetonitrile/water/TFA (solvent A, 100 μL, 2/98/0.1, v/v/v) and then loaded onto a small portion of a C18-silica disk (3M, Maplewood, MN) placed in a 200 μL pipette tip. Prior to sample loading the C18 disk was prepared by sequential treatment with methanol (20 μL), acetonitrile/water/TFA (solvent B, 20 μL, 80/20/0.1, v/v/v) and finally with solvent A (20 μL). After loading the sample, the disc was washed with solvent A (20 μL, eluent discarded) and eluted with solvent B (40 μL). The collected eluent was dried in a centrifugal vacuum concentrator and reconstituted in 0.1% Formic Acid (10 μL) for LC-MS/MS analysis.

LC-MS/MS analysis

Aliquots of each sample (5 μL) were injected onto a reverse phase nanobore HPLC column (AcuTech Scientific, C18, 1.8μm particle size, 360 μm x 20 cm, 150 μm ID), equilibrated in solvent A (water/acetonitrile/FA, 98/2/0.1, v/v/v) and eluted (300 nL/minute) with an increasing concentration of solvent B (acetonitrile/water/FA, 98/2/0.1, v/v/v: min/% F; 0/0, 5/3, 18/7, 74/12, 144/24, 153/27, 162/40, 164/80, 174/80, 176/0, 180/0) using an EASY-nLC II (Thermo Fisher Scientific). The effluent from the column was directed to a nanospray ionization source connected to a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive Plus, Thermo Fisher Scientific) acquiring mass spectra in a data-dependent mode alternating between a full scan (m/z 350-1700, automated gain control (AGC) target 3 × 106, 50 ms maximum injection time, FWHM resolution 70,000 at m/z 200) and up to 15 MS/MS scans (quadrupole isolation of charge states 2-7, isolation window 0.7 m/z) with previously optimized fragmentation conditions (normalized collision energy of 32, dynamic exclusion of 30 s, AGC target 1 × 105, 100 ms maximum injection time, FWHM resolution 35,000 at m/z 200).