Gw3965

Human hepatoma HepG2 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Human primary hepatocytes (HPH) were obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). 6E-CDCA was purchased from Abcam (Cambridge, MA, USA). GW4064 and GW3965 were purchased from Tocris Bioscience (Bristol, UK). OCA was provided by Intercept Pharmaceuticals, Inc. (New York, NY, USA). The expression plasmid for human FXRα was provided by Dr Timothy F. Osborne from Sanford Burnham Prebys Medical Discovery Institute (La Jolla, CA, USA). Expression plasmids for human RXRα and LXRα were obtained from GenScript Corporation (Piscataway, NJ, USA).

CD4⁺ T cells from the spleen and peripheral LNs were positively selected with CD4 microbeads (L3T4; Miltenyi Biotec), and naive CD4⁺ T cells were further sorted as CD4⁺CD25⁻CD62L^highCD44^low cells with the FACSAria III cell sorter (Becton, Dickinson and Company (BD) Biosciences) and stimulated with plate-coated anti-CD3 (1 μg/mL, 145-2C11: BioXCell) and soluble anti-CD28 (1 μg/mL, 37.51; BioXCell) for 3 d in RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich). To measure TCR-mediate protein phosphorylation, flow-sorted naïve CD4⁺ T cells were stimulated with plate-coated anti-CD3 and anti-CD28 (2 μg/mL) before being analyzed at indicated time points. In some experiments, cells were treated with either DMSO vehicle or 1 µM GW3965 (Tocris), 1 µM BIO (Sigma-Aldrich), or mevalonic acid (Sigma-Aldrich).

For immunization studies, mice were subcutaneously immunized with 50 µg KLH (Sigma-Aldrich) or 100 µg NP-OVA (Biosearch Technologies) emulsified in CFA (Sigma-Aldrich), and lymphoid cells from the draining LNs were stained and analyzed by flow cytometry on day 8 to 10. For viral infection experiments, mice were intraperitoneally injected with LCMV-Armstrong (2 × 10⁵ pfu), and lymphoid cells in the spleen were analyzed on day 8. In some experiments, mice were intraperitoneally treated with either Dimethyl sulfoxide (DMSO) vehicle or 20 mg/kg GW3965 (Tocris).
For BM chimera studies, 8- to 10-wk-old sublethally-irradiated Rag1^−/− mice (9 Gy; X-RAD IR160, Precision X-Ray, USA) were i.v. injected 1:1 mixture of WT and Nr1h2^−/− BM cells before 6 wk of reconstitution. The recipients were s.c. immunized with KLH in CFA or infected with LCMV, and lymphoid cells from the draining LNs or spleen were analyzed as indicated (46 (link)).
For OT-II T cell cotransfer studies, 1:1 mixture of flow-sorted WT and Nr1h2^−/− naïve OT-II T cells (2 × 10⁶ cells) were i.v. transferred into sex-matched B6.SJL congenic recipient mice. One day later, the recipients were s.c. injected with 100 µg OVA (Sigma-Aldrich) emulsified in CFA and lymphoid cells from the draining LNs were analyzed on day 8.