Journal: Cancer Science

Article Title: SCD1 inhibition enhances the effector functions of CD8 + T cells via ACAT1 ‐dependent reduction of esterified cholesterol

doi: 10.1111/cas.15999

Figure Lengend Snippet: SCD1 inhibitors enhance the activation of human CD8 + T cells. CD8 + T cells were isolated from human PBMCs using MACS and activated with an anti‐CD3 monoclonal antibody (mAb), anti‐CD28 mAb, and IL‐2. CD8 + T cells were treated with dimethylsulfoxide (DMSO), SCD1 inhibitors (A939572 and CAY10566), SCD1 inhibitors + oleic acid (OA), or SCD1 inhibitors + palmitoleic acid (POA). (A) Intracellular oleic acid levels in human CD8 + T cells. (B) Intracellular palmitoleic acid levels in human CD8 + T cells. (C) IFN‐γ levels in culture supernatants of CD8 + T cells were measured using ELISA. (D) Cell proliferation of CD8 + T cells was evaluated by WST‐1 assay. Data are expressed as means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s., not significant.

Article Snippet: SCD1 inhibitors A939572 and CAY10566 were purchased from APExBIO and Cayman, respectively.

Techniques: Activation Assay, Isolation, Enzyme-linked Immunosorbent Assay, WST-1 Assay

Journal: Cancer Science

Article Title: SCD1 inhibition enhances the effector functions of CD8 + T cells via ACAT1 ‐dependent reduction of esterified cholesterol

doi: 10.1111/cas.15999

Figure Lengend Snippet: Inhibition of SCD1 enhances the effector function of human CD8 + T cells via suppression of esterified cholesterol synthesis by ACAT1. CD8 + T cells were isolated from human PBMCs using MACS and activated by the anti‐CD3 mAb, anti‐CD28 mAb, and IL‐2. (A–C) CD8 + T cells were cultured in AIM‐V medium with DMSO + γCD, 1 μM ACAT1 inhibitors (Avasimibe and CP113818) + γCD, or 1 μM ACAT1 inhibitors + γCD + cholesteryl oleate (ChO). IFN‐γ (A), TNF‐α (B), and GzmB (C) levels in culture supernatants were measured by ELISA. (D) CD8 + T cells were treated with DMSO, SCD1 inhibitors (A939572 and CAY10566), or SCD1 inhibitors + oleic acid (OA). The ratio of esterified cholesterol/cholesterol (left panel) and the amounts of cholesterol and esterified cholesterol (right panel) are shown. (E) CD8 + T cells were cultured in AIM‐V medium with DMSO + γ‐cyclodextrin (γCD), SCD1 inhibitors (A939572 and CAY10566) + γCD, or SCD1 inhibitors + γCD + cholesteryl oleate (ChO). IFN‐γ levels in culture supernatants were measured by ELISA. Data are expressed as means ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s., not significant.

Article Snippet: SCD1 inhibitors A939572 and CAY10566 were purchased from APExBIO and Cayman, respectively.

Techniques: Inhibition, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: SCD1 inhibition enhances the effector functions of CD8 + T cells via ACAT1 ‐dependent reduction of esterified cholesterol

doi: 10.1111/cas.15999

Figure Lengend Snippet: Systemic administration of SCD1 inhibitor enhances IFN‐γ production by CD8 + T cells in vivo. (A–D) C57BL/6 mice bearing MCA205 tumors were treated with an SCD1 inhibitor (A939572; 10 mg/kg), an ACAT1 inhibitor (Avasimibe; 15 mg/kg) or the vehicle (mock). Tumor‐infiltrating CD8 + T cells (CD8 + TILs) were sorted using MACS. (A) Oleic acid levels in tumor tissues and CD8 + TILs. (B) The ratio of esterified cholesterol/cholesterol and the amount of cholesterol and esterified cholesterol in CD8 + TILs are shown. (C, D) CD8 + TILs were cultured ex vivo after SCD1 inhibitor (C) or ACAT1 inhibitor (D) treatment. The next day, IFN‐γ levels in culture supernatants were measured by ELISA. Data are expressed as means ± SD ( n = 5). (E) Mice bearing MCA205 tumors were treated with A939572 or the vehicle and anti‐PD‐1 antibody (200 μg/mouse) or isotype‐matched antibody. (F) Mice bearing MCA205 tumors were treated with an ACAT1 inhibitor (Avasimibe; 15 mg/kg) or the vehicle and anti‐PD‐1 antibody (200 μg/mouse) or isotype‐matched antibody. Tumor growth curves for average tumor volumes are shown (means ± SD; n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s., not significant.

Article Snippet: SCD1 inhibitors A939572 and CAY10566 were purchased from APExBIO and Cayman, respectively.

Techniques: In Vivo, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: SCD1 inhibition enhances the effector functions of CD8 + T cells via ACAT1 ‐dependent reduction of esterified cholesterol

doi: 10.1111/cas.15999

Figure Lengend Snippet: SCD1 is a potential target for enhancing the antitumor effects of CAR‐T cell therapy. GPC1‐specific murine CAR (GPC1‐mCAR)‐T cells and murine control T (mCont‐T) cells were generated as described in Section and then treated with DMSO or A939572. (A) Intracellular oleic acid levels in GPC1‐mCAR‐T cells. (B) The ratio of esterified cholesterol/cholesterol and the amount of cholesterol and esterified cholesterol in GPC1‐mCAR‐T cells are shown. (C, D) GPC1‐mCAR‐T cells or mCont‐T cells and mGPC1‐overexpressing MCA205 tumor cells (MCA205‐mGPC1) were co‐cultured in vitro. (C) IFN‐γ secretion was evaluated by ELISA. (D) Cytotoxic activity was evaluated using a standard Cr 51 ‐releasing assay. Data are expressed as means ± SD ( n = 3). (E) Mice bearing MCA205‐mGPC1 were treated with 5 × 10 6 cells of GPC1‐mCAR‐T cells or mCont‐T cells on day 2 and SCD1 inhibitor was administered by oral gavage twice daily for 16 days starting on day 4. Tumor growth curves for average tumor volumes are shown (means ± SD; n = 5). * p < 0.05, ** p < 0.01, **** p < 0.0001. E/T ratio, effector cell (GPC1‐mCAR‐T cells)/target cell (MCA205‐mGPC1 cell) ratio.

Article Snippet: SCD1 inhibitors A939572 and CAY10566 were purchased from APExBIO and Cayman, respectively.

Techniques: Generated, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Cancer Science

Article Title: SCD1 inhibition enhances the effector functions of CD8 + T cells via ACAT1 ‐dependent reduction of esterified cholesterol

doi: 10.1111/cas.15999

Figure Lengend Snippet: Schematic of the study. Inhibition of SCD1 enhances the antitumor activity of CD8 + T cells by reducing the intracellular oleic acid level, ACAT1‐dependent cholesterol esterification reaction, and esterified cholesterol level.

Article Snippet: SCD1 inhibitors A939572 and CAY10566 were purchased from APExBIO and Cayman, respectively.

Techniques: Inhibition, Activity Assay