Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer1,2,3,4,5. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4+ but not CD8+ T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4+ T lymphocytes have greater mitochondrial mass than CD8+ T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4+ T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4+ T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.
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We would like to thank J. Berzofsky, W. Stoffel, E. M. Shevach and S. Thorgeirsson for helpful discussion. A.M.T. was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases. J.L. was supported by the Intramural Program Grant ZIABC011303, NIH, National Cancer Institute (NCI). M.H. was supported by a European Research Council starting grant (LiverCancerMechanism), the Stiftung Experimentelle Biomedizin (Hofschneider Stiftung), the Pre-clinical Comprehensive Center (PCCC) and the Helmholtz foundation. A.W. was supported by grants from the Krebsliga Schweiz (Oncosuisse) and the Promedica Stiftung, Switzerland. D.E.K., A.H.K., D.W.M. and T.F.G. were supported by the Intramural Research Program of the NIH, NCI.
The authors declare no competing financial interests.
Extended data figures and tables
a, Representative imagines of Oil Red O staining of MYC-ON mice fed MCD or CTR. Scale bar, 100 μm. b, Serum ALT levels analysis. Data are mean ± s.e.m.; n = 4, *P < 0.05, one-way ANOVA. c–e, The effect of the CDAA diet on tumour development in MYC transgenic mice. Experimental setup, representative liver images and liver surface tumour counts are shown. Scale bar, 10 mm. Data are mean ± s.e.m.; n = 6 for CDAA and n = 5 mice for CTR, P = 0.0345, Student’s t-test. f–i, The effect of the CDAA and HF diet on liver carcinogenesis in diethylnitrosoamine (DEN)-injected C57BL/6 mice. Experimental setup, representative tumour-free H&E stainings, macroscopic liver images and surface tumour counts are shown. Scale bar, 100 μm. Data are mean ± s.e.m.; n = 13 for CTR, n = 9 for HF, n = 10 for CDAA, *P < 0.05, one-way ANOVA.
a–j, MYC mice were fed with an MCD diet or CTR diet. a, b, Intrahepatic immune cells were determined by flow cytometry. Composition (a) and absolute numbers (b) of different intrahepatic immune cell subsets in MYC-ON mice, which were kept for 4 weeks on an MCD diet or CTR diet. Data are mean ± s.e.m.; n ≥ 6, *P < 0.05, one-way ANOVA. c, Representative contour plots of intrahepatic CD4+ T lymphocytes. d, Representative dot plots of CD1d-tetramer staining in CD3loCD4+ population. e–g, Absolute number of intrahepatic CD4+ T lymphocytes, frequencies of NK T cells and splenic CD4+ T lymphocytes were measured by flow cytometry. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. h–j, Intrahepatic CD4+ and CD8+ T lymphocyte levels in MYC-ON mice fed with a CDAA diet for 16 weeks. Data are mean ± s.e.m.; n = 6 for CDAA and n = 5 for CTR, *P < 0.05, Student’s t-test. k, l, Intrahepatic CD4+ T lymphocyte levels in DEN-injected BL/6 male mice treated with a CDAA diet, HF diet or CTR for 7 months. Data are mean ± s.e.m.; n = 13 for CTR, n = 9 for HF, n = 10 for CDAA, *P < 0.05, one-way ANOVA. m–p, Intrahepatic CD4+ and CD8+ T lymphocytes in tumour-free C57BL/6 mice treated with a CDAA diet for 16 weeks. TF, tumour free. Data are mean ± s.e.m.; n = 3 for CTR, n = 5 for CDAA, *P < 0.05, Student’s t-test. q–t, Intrahepatic CD4+ and CD8+ T lymphocytes in tumour-free C57BL/6 mice treated with an HF or low-fat (LF) diet for 6 months. Data are mean ± s.e.m.; n = 2 for CTR, n = 5 for LF, n = 5 for HF, *P < 0.05, one-way ANOVA. u–x, CD4+ and CD8+ T lymphocytes in 12-week-old male ob/ob or wild-type lean mice. Data are mean ± s.e.m., n = 5, *P < 0.05, Student’s t-test w, x, MYC mice were fed with MCD or CTR. Macrophage and CD11b+Gr1+ populations were measured. Data are mean ± s.e.m.; n ≥ 4, *P < 0.05, two-way ANOVA.
Extended Data Figure 3 Intrahepatic CD4+ lymphocytes are activated in NAFLD, and CD4 depletion enhances HCC.
a–k, MYC-ON mice were fed with MCD or CTR for 4 weeks. a–d, CD69 and CD44hiCD62Llo subsets in intrahepatic CD4+ T lymphocytes were measured. Data are mean ± s.e.m.; n = 8 for MCD and n = 6 for CTR, *P < 0.05, Student’s t-test. e–g. Ex vivo IFN-γ, IL-4 production in intrahepatic CD4+ T lymphocytes were determined. Data are mean ± s.e.m.; n = 8, *P < 0.05, Student’s t-test. h, Ex vivo staining of T-bet, GATA3, ROR-γt and Foxp3 levels in intrahepatic and splenic CD4+ T lymphocytes. Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. i, Ex vivo IL-17 production by intrahepatic CD4+ T lymphocytes. Data are mean ± s.e.m.; n = 5, *P < 0.05, Student’s t-test. j, Representative dot plots of ROR-γt/IL-17 staining in intrahepatic CD4+ T lymphocytes. k, Absolute number of intrahepatic CD4+ lymphocyte subsets. Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. l, Suppressive function assay of isolated hepatic Treg cells from Foxp3–GFP mice kept on MCD or CTR for 4 weeks. m, Detection of AFP-specific CD4+ T lymphocytes in spleen from MYC-MCD mice. n, Selective depletion of intrahepatic CD4+ T lymphocytes but not NK T cells by i.p. injection of 50 μg anti-CD4 antibody (clone GK1.5). o, p, MYC-ON mice on CTR received 50 μg of GK1.5 antibody or isotype control i.p. once per week for 8 weeks. Representative liver imagines and surface tumour counts are shown. Scale bar, 10 mm. Data are mean ± s.e.m., n = 3.
Extended Data Figure 4 Lipid-laden hepatocytes release C18:2 and induce CD4+ T lymphocyte death via apoptosis.
a, Representative contour plots of ex vivo 7AAD/annexin V staining of intrahepatic CD4+ T lymphocytes from MYC-ON mice fed with MCD or CTR. b, Representative phase-contrast images of primary hepatocytes from MYC-ON mice after MCD or CTR treatment. c–e, Isolated primary hepatocytes from MYC-ON mice on MCD or CTR were cocultured with isolated CD4+ T lymphocytes or splenocytes. Cell death levels were measured by flow cytometry. Data are mean ± s.e.m.; n = 4, one-way or two-way ANOVA. f, g, BODIPY 493/503 staining of CD4+ T lymphocytes in liver, spleen or blood from MYC-ON mice with MCD or CTR. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. h, i, Identification of FFAs in hepatocyte conditioned medium by gas chromatography/mass spectrometry (GC/MS). Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. j, Anti-CD3/28 bead-activated splenocytes were treated with different FFAs, and cell death level in CD4+ or CD8+ T lymphocytes was determined. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. k–m, Dose–response curve and time course of C18:2-induced cell death in CD4+ or CD8+ T lymphocytes. n, Caspase3/7 activity in CD4+ lymphocytes after C18:2 treatment. Data are mean ± s.e.m.; n = 9, *P < 0.05, Student’s t-test. o, Dose–response curve of H2O2-induced cell death in CD4+ or CD8+ T lymphocytes. p, Uptake of C18:2 by CD4+ and CD8+ T lymphocytes after incubation with 50 μM C18:2 for 2 h. Data are mean ± s.e.m.; n = 6, *P < 0.05, two-way ANOVA.
CD4+ and CD8+ T lymphocytes sorted from C18:2-treated splenocytes were subjected to microarray analysis. Pathway analysis was done by ingenuity pathway analysis (IPA). n = 3. Ratio is the number of changed genes divided by total genes in the pathway.
Extended Data Figure 6 Mitochondrial ROS mediates C18:2-induced CD4+ T lymphocyte death in vitro and in vivo.
a, Real-time polymerase chain reaction (PCR) confirmed the gene changes from microarray. Data are mean ± s.e.m.; n = 3, *P < 0.05, two-way ANOVA. b, Cpt1a mRNA level in Jurkat cells after FFA treatment. Data are mean ± SEM; n = 6, *P < 0.05, one-way ANOVA. c, Expression of CPT1a in wild-type and two knockdown Jurkat cells. NT, none-targeting control. d, e, OCR analysis of activated CD4+ and CD8+ T lymphocytes upon C18:2 or C16:0 incubation. f, ROS levels of CD4+ or CD8+ T lymphocytes in splenocytes treated with C18:2 or C16:0. Data are mean ± s.e.m.; n = 8, *P < 0.05, two-way ANOVA. g, Mitochondrial ROS in wild-type and two CPT1 knockdown Jurkat cells. i, Cell death of CD4+ or CD8+ T lymphocytes in splenocytes treated with C18:2 in the presence of NAC or catalase. Data are mean ± s.e.m.; n = 4, *P < 0.05, two-way ANOVA. i, j, In vivo blocking ROS with NAC in MYC-ON mice treated with MCD. Some mice also received CD4 antibody depletion. Experimental setup and representative H&E liver sections are shown. Scale bar, 200 μm.
Extended Data Figure 7 C18:2 induces cell death in human CD4+ T lymphocytes, and NASH patients have lower intrahepatic CD4+ T lymphocytes.
a, Cell death levels of sorted human CD4+ T lymphocytes treated with different FFAs. Data are mean ± s.e.m.; n = 4, *P < 0.05, one-way ANOVA. b, ROS level of CD4+ or CD8+ T lymphocyte in peripheral blood mononuclear cells (PBMCs) treated with C18:2 or C16:0. Data are mean ± s.e.m.; n = 6, *P < 0.05, two-way ANOVA. c, d, Serum ALT and AST concentration in different patients. e, Intrahepatic CD4+ T lymphocyte count in biopsies. CD4+ T lymphocytes were identified by immunohistochemistry. Data are mean ± s.e.m.; normal = 6, NASH = 16, ASH = 8, HBV/HCV = 16, *P < 0.05, one-way ANOVA.
Extended Data Figure 8 Immunohistochemistry staining of intrahepatic CD4+ or CD8+ T lymphocytes in patient biopsies.
Representative CD4 or CD8 immunohistochemistry images of liver biopsies from healthy individuals, NASH, ASH patients or patients with HBV or HCV. For each condition, two different magnifications are shown. Scale bar, 100 μm.
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Ma, C., Kesarwala, A., Eggert, T. et al. NAFLD causes selective CD4+ T lymphocyte loss and promotes hepatocarcinogenesis. Nature 531, 253–257 (2016). https://doi.org/10.1038/nature16969
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