Volume 27, Issue 10, Pages 2948-2961.e7 (June 2019) Activation of Mevalonate Pathway via LKB1 Is Essential for Stability of Treg Cells  Maheshwor Timilshina, Zhiwei You, Sonja M. Lacher, Suman Acharya, Liyuan Jiang, Youra Kang, Jung-Ae Kim, Hyeun Wook Chang, Keuk-Jun Kim, Byoungduck Park, Jae-Hyoung Song, Hyun-Jeong Ko, Yun-Yong Park, Min-Jung Ma, Mahesh Raj Nepal, Tae Cheon Jeong, Yeonseok Chung, Ari Waisman, Jae-Hoon Chang  Cell Reports  Volume 27, Issue 10, Pages 2948-2961.e7 (June 2019) DOI: 10.1016/j.celrep.2019.05.020 Copyright © 2019 The Authors Terms and Conditions

Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 1 LKB1 Is Indispensable for Treg Cell Proliferation and Function (A) Immunoblot showing phosphorylated and total proteins in resting and anti-CD3/CD28-stimulated Treg cells, naive CD4+ T cells, and CD8+ T cells from C57BL/6J mice. (B) Frequency of splenic Treg cells from 1- to 4-week-old wild-type and LKB1fl/flFoxp3-Cre mice. Data shown are mean ± SD. (C) Mean fluorescence intensity (MFI) of bromodeoxyuridine (BrdU) and Ki-67 expression in YFP−CD25+CD4+ and YFP+CD25+CD4+ Treg cells from LKB1fl/flFoxp3-Crehet mice. Data shown are mean ± SD. (D) Treg signature molecule expression in YFP+CD25+CD4+ and YFP−CD25+CD4+ Treg cells from LKB1fl/flFoxp3-Crehet mice. Data shown are mean ± SD. (E) Amount of IL-10 in sorted YFP+CD25+CD4+ and YFP−CD25+CD4+ Treg cells from LKB1fl/flFoxp3-Crehet were quantified by cytokine binding assay. Data shown are mean ± SEM. (F) In vitro suppressive activity of Treg cells, assessed as the proliferation of naive CD4+ T cells activated by anti-CD3 plus irradiated antigen-presenting cells (APCs) in the presence of various ratios of Treg cells from wild-type or LKB1fl/flFoxp3-Cre mice, measured as dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE). Data shown are mean ± SEM. (G) CD45RBhiCD4+ T cells from B6.SJL mice expressing the CD45.1 congenic marker were transferred to Rag1−/− mice with PBS or in combination with wild-type or LKB1-deficient Treg cells in a T cell transfer colitis model. Changes in body weight of the Rag1−/− recipients are shown (n = 5). Data shown are mean ± SEM. (H) Histological analysis of H&E staining of the colon section with the histology score were shown from recipient mice at 8 weeks after adoptive transfer. Original magnification, 10× (up; scale bar, 500 μm) or 20× (down; scale bar, 200 μm). Data shown are mean ± SEM. Results represent three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 2 Loss of LKB1 in Treg Cells Induces the Production of Inflammatory Cytokines (A) Amounts of IFN-γ, IL-4, IL-5, IL-13, and IL-17A in purified splenic Treg cells from wild-type and LKB1fl/flFoxp3-Cre mice were quantified by cytokine binding assay. Data shown are mean ± SEM. (B) Amounts of IFN-γ, IL-4, IL-5, IL-13, and IL-17A in purified YFP+CD25+CD4+ and YFP−CD25+CD4+ Treg cells from LKB1fl/flFoxp3-Crehet were quantified by cytokine binding assay. Data shown are mean ± SEM. (C) Disease phenotype of 6-week-old Rag1−/− mice given adoptive transfer of CD45.1+ wild-type Treg cells and/or CD45.2+Foxp3+ LKB1-deficient Treg cells at 8 weeks after transfer. Changes in body weight of Rag1−/− recipients are shown (n = 5). Data shown are mean ± SD. (D) H&E staining of sections of colons from recipient mice at 8 weeks after adoptive transfer. Original magnification, 10×; scale bar, 100 μm. (E) Images of spleens from Rag1−/− recipient mice at 8 weeks after transfer. (F) Total cell count of splenocytes from Rag1−/− recipient mice at 8 weeks after transfer. Data shown are mean ± SEM. Results represent three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 3 Mevalonate Pathway Is Impaired in LKB1-Deficient Treg Cells (A) mRNA expression of different metabolic pathway genes of Treg cells from wild-type and LKB1fl/flFoxp3-Cre mice was determined by RT-PCR. Data shown are mean ± SD. (B and C) mRNA expression of lipid metabolic pathway genes (B) and HMGCS and HMGCR protein expression (C) of Treg cells from YFP+CD25+CD4+ and YFP−CD25+CD4+ Treg cells from LKB1fl/flFoxp3-Crehet mice were determined after 1 h of resting and stimulation with anti-CD3/CD28. Data shown are mean ± SD. (D and E) mRNA (D) and protein expression (E) of HMGCS and HMGCR in CD4+ T cells from wild-type and LKB1fl/flCd4-Cre mice were determined by RT-PCR after 1 h of resting and stimulation with anti-CD3/CD28. Data shown are mean ± SEM. (F) 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) enzyme activity of CD4+ T cells was evaluated from WT and LKB1fl/flCd4-Cre mice with or without anti-CD3/CD28 stimulation. Data shown are mean ± SEM. (G) Immunoblot analysis of SREBP1 in resting and anti-CD3/CD28-stimulated CD4+ T cells from WT and LKB1fl/flCd4-Cre mice is shown. The precursor form of SREBP-1 p125 and the mature active form SREBP-1p68 are shown. β-actin serves as a loading control. Results represent three independent experiments. ∗p < 0.05; ∗∗p < 0.01. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 4 Loss of HMGCR in Treg Cells Induces Inflammatory Cytokine Production and Onset of Fatal Inflammatory Disorder (A) IFN-γ and IL-17 production in splenic Treg cells from in vivo simvastatin-treated LKB1+/+Foxp3-Cre mice. (B) Quantification of cytokines in purified Treg cells from PBS- or simvastatin-treated LKB1+/+Foxp3-Cre mice measured by cytokine binding assay. Data shown are mean ± SEM. (C) Immunoblot analysis of HMGCS1 in Treg cells after transfection with scramble shRNA and/or HMGCS1 shRNA. (D) Expression of IFN-γ from scramble and/or HMGCS1 shRNA-transfected Foxp3+ Treg cells. (E) Comparative evaluation of the morbid percentage in WT, HMGCRfl/flFoxp3-Cre, and LKB1fl/flFoxp3-Cre mice (n = 5–7 mice per group). (F) Amounts of different cytokines secreted by Treg cells from controls containing Foxp3-GFP reporter or HMGCRfl/+Foxp3-Cre mice containing YFP reporter were quantified by cytokine binding assay. Data shown are mean ± SEM. Results represent three independent experiments. ∗p < 0.05; ∗∗∗p < 0.001. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 5 Mevalonate Restores Foxp3 Expression, Stability, and Function in LKB1-Deficient Treg Cells (A) Mevalonate quantification from purified WT and LKB1-deficient Treg cells (1 × 106 cells) by LC-MS/MS. Data shown are mean ± SEM. (B) Frequency of Foxp3+CD25+ Treg cells in spleen and PLN from PBS-treated wild-type and LKB1fl/flFoxp3-Cre mice and/or mevalonate-treated wild-type and LKB1fl/flFoxp3-Cre mice. Data shown are mean ± SD (n = 4–5 mice per group). (C) Amounts of different cytokines in Treg cells from PBS- or mevalonate-treated wild-type and LKB1fl/flFoxp3-Cre mice were quantified by cytokine binding assay. Data shown are mean ± SEM. (D) In vitro suppressive activity of Treg cells, assessed as the proliferation of naive CD4+ T cells activated by anti-CD3 plus irradiated APCs in the presence of various ratios of Treg from PBS- or mevalonate-treated WT and LKB1fl/flFoxp3-Cre mice, measured as dilution of CFSE. Data shown are mean ± SEM. (E) H&E staining of indicated tissue sections (skin, kidney, lung, and liver) from PBS- and/or mevalonate-treated wild-type and LKB1fl/flFoxp3-Cre mice. Histology scores are shown in the bar diagrams. Original magnification, 10×; scale bar, 100 μm. (F) Survival curve of untreated or mevalonate-treated wild-type and LKB1fl/flFoxp3-Cre mice (n = 6–8 mice per group). Results represent three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 6 GGPP Restores Foxp3 Expression and Autoimmune Phenotype in LKB1fl/flFoxp3-Cre Mice (A) Schematic representation of the mevalonate pathway. (B) GGPP, coenzyme Q10, and cholesterol quantification from purified WT and LKB1-deficient Treg cells (1 × 106 cells) by LC-MS/MS. Data shown are mean ± SD. (C) Frequency of Foxp3+CD25+ Treg cells from PBS-, GGPP-, or cholesterol-treated wild-type and LKB1fl/flFoxp3-Cre mice. (D) Amounts of different cytokines in Treg cells from PBS- or GGPP-treated wild-type and LKB1fl/flFoxp3-Cre mice were quantified by cytokine binding assay. Data shown are mean ± SEM. (E) In vitro suppressive activity of Treg cells, assessed as the proliferation of naive CD4+ T cells activated by anti-CD3 plus irradiated APCs in the presence of various ratios of Treg from PBS- or GGPP-treated WT and LKB1fl/flFoxp3-Cre mice, measured as dilution of CFSE. Data shown are mean ± SD. (F) Representative pictures of spleen and peripheral lymph node (PLN; axillary, superficial cervical, brachial, and inguinal lymph nodes) from PBS- or GGPP-treated wild-type and LKB1fl/flFoxp3-Cre mice. (G) H&E staining of indicated tissue sections (skin, kidney, lung, and liver) from PBS- and/or GGPP-treated wild-type and LKB1fl/flFoxp3-Cre mice. Original magnification, 10×; scale bar, 100 μm. (H) Survival curve of untreated or GGPP-treated wild-type and LKB1fl/flFoxp3-Cre mice (n = 5 mice per group). Results represent three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions

Figure 7 GGPP Enhances STAT5 Activation to Maintain Treg Lineage Stability (A) Purified naive CD4 T cells were differentiated for Th1, Th17, and Treg differentiation conditions with or without GGPP. Percentages of in vitro differentiated Th1, Th17, and Treg cells were shown. Data shown are mean ± SEM. (B) Protein prenylation state in untreated Treg cells and Treg cells treated with geranylgeranyltransferase I inhibitor (GGTi; 10 μM) or GGPP (10 μM) sorted from 3-week-old WT and LKB1fl/flFoxp3-Cre mice. (C and D) Purified Treg cells from 3-week-old WT and LKB1fl/flFoxp3-Cre mice were isolated and stimulated with anti-CD3/CD28 with or without GGPP (10 μM) for 24 h. (C) Immunoblot analysis of STAT5 activation in resting and IL-2-stimulated Treg cells is shown. (D) Immunoblot analysis of Smad2/3 activation in resting and TGF-β-stimulated Treg cells is shown. β-actin serves as a loading control. Results represent three independent experiments. ∗p < 0.05. Cell Reports 2019 27, 2948-2961.e7DOI: (10.1016/j.celrep.2019.05.020) Copyright © 2019 The Authors Terms and Conditions