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MEF2C regulates NK cell effector functions through control of lipid metabolism

Nature Immunology (2024)Cite this article

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An Author Correction to this article was published on 19 April 2024

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Abstract

Natural killer (NK) cells are a critical first line of defense against viral infection. Rare mutations in a small subset of transcription factors can result in decreased NK cell numbers and function in humans, with an associated increased susceptibility to viral infection. However, our understanding of the specific transcription factors governing mature human NK cell function is limited. Here we use a non-viral CRISPR–Cas9 knockout screen targeting genes encoding 31 transcription factors differentially expressed during human NK cell development. We identify myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C-haploinsufficient patients and mice displayed defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for an interleukin (IL)-2- and IL-15-mediated increase in lipid content through regulation of sterol regulatory element-binding protein (SREBP) pathways. Supplementation with oleic acid restored MEF2C-deficient and MEF2C-haploinsufficient patient NK cell cytotoxic function. Therefore, MEF2C is a critical orchestrator of NK cell antiviral immunity by regulating SREBP-mediated lipid metabolism.

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Fig. 1: MEF2C is required for human NK cell proliferation and effector function.
Fig. 2: Patients with MCHS present with a functional NK cell deficiency.
Fig. 3: MEF2C haploinsufficiency disrupts antiviral immunity.
Fig. 4: MEF2C is required for IL-15- and mTORC1-induced metabolic reprogramming.
Fig. 5: MEF2C promotes cytokine-activated SREBP signaling and increased lipid content in NK cells.
Fig. 6: Fatty acid supplementation restores MEF2C-deficient NK cell cytotoxicity.

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Data availability

Sequence data have been deposited in the GEO database under the accession code GSE245463. Gene expression data in sorted human immune cells were provided by the DICE project. Publicly available RNA-seq datasets for human peripheral NK cells sorted by flow cytometry were accessed at GSE112813. Publicly available RNA-seq datasets for cytokine-stimulated NK cells were accessed at GSE140035. RNA-seq data were aligned using the reference mouse genome mm10 or the reference human genome hg38. All other data are available in the main text or Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We deeply thank the patients with MCHS and their families who participated in this study. We thank the nurses, staff and physicians at Greenwood Genetic Center for their outstanding evaluation and care for children with genetic disorders and inborn errors of immunity. We thank the blood donors, E. Faure, A. Catapang and the UCLA CFAR Virology Core for providing healthy donor peripheral blood samples. We thank S. Feng and the UCLA Broad Stem Cell Research Center High-Throughput Sequencing Core for assistance with RNA-seq. We thank the B. Moriarity laboratory at the University of Minnesota for providing the plasmid encoding ABE8e and associated protocols. We thank M. Lechner at UCLA for sharing the β2M−/− MC38 cell line. We thank the O’Sullivan, Bensinger, Cowan, Covarrubias, Su, Divakaruni, Hoffman and Christofk laboratories for helpful discussion. We acknowledge the rich literature of inborn errors of immunity and NK cell deficiency and regret that not all studies could be discussed. T.E.O. is supported by the National Institutes of Health (R01AI145997, R01AI174519) and the Hypothesis Fund. J.H.L. is supported by the National Institutes of Health (T32GM008042, T32AR071307, T32AI007323, 1F30AI181449-01) and a UCLA Molecular Biology Institute Whitcome Fellowship. C.D.L. is supported by the National Institutes of Health (T32GM008042). C.G.B. is supported by a UCLA Eugene Cota V. Robles Fellowship. C.W.C. is supported by the National Institutes of Health (R01MH111464) and a SFARI Pilot Award (649452). A.S.D. is supported by the National Institutes of Health (R35GM138003), the W.M. Keck Foundation (995337) and an Agilent Early Career Professor Award. A.B.B. is supported by the National Institutes of Health (T32GM136614). The UCLA CFAR Virology Core is supported by the National Institutes of Health (5P30AI028697).

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Authors and Affiliations

  1. Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Joey H. Li, Adalia Zhou, Cassidy D. Lee, Siya N. Shah, Jeong Hyun Ji, Vignesh Senthilkumar, Eddie T. Padilla, Qinyan Feng, Christian G. Bustillos, Luke Riggan & Timothy E. O’Sullivan

  2. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Joey H. Li, Cassidy D. Lee, Jeong Hyun Ji, Eddie T. Padilla, Christian G. Bustillos, Luke Riggan & Timothy E. O’Sullivan

  3. David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Joey H. Li & Cassidy D. Lee

  4. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Andréa B. Ball & Ajit S. Divakaruni

  5. Department of Neuroscience, Medical University of South Carolina, Charleston, SC, USA

    Alain Greige & Christopher W. Cowan

  6. Greenwood Genetic Center, Greenwood, SC, USA

    Fran Annese, Jessica A. Cooley Coleman & Steven A. Skinner

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Contributions

J.H.L. designed the project, performed and analyzed all experiments and wrote the paper with input from all authors. A.Z., C.D.L., S.N.S. and Q.F. performed and analyzed experiments. J.H.J. analyzed sequencing data. V.S., S.N.S., E.T.P. and C.G.B. tested sgRNA species and performed the CRISPR cRNP screen. A.B.B. and A.S.D. performed and analyzed Seahorse metabolic experiments. L.R. developed the initial human NK cell culture and CRISPR cRNP system. A.G. and C.W.C. provided Mef2c+/− bone marrow. F.A., J.C.-C. and S.A.S. performed clinical evaluations of patients with MCHS and coordinated and provided patient samples. T.E.O. conceived and designed the project, supervised experiments and wrote the paper.

Corresponding author

Correspondence to Timothy E. O’Sullivan.

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Competing interests

T.E.O. is a scientific advisor for Modulus Therapeutics and Xyphos Biosciences, companies that have a financial interest in human NK cell-based therapeutics. The Regents of the University of California have filed a provisional patent application with the United States Patent and Trademark Office for using oleate supplementation as a method of augmenting adoptive NK cell therapy. J.H.L. and T.E.O. are listed as inventors on this patent application. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Functional CRISPR cRNP screen in primary human NK cells identifies positive regulators of human NK cell function.

(a) Differentially expressed transcription factors between human NK cell developmental subsets. (b) Primary human NK cells are isolated from fresh human PBMCs via negative magnetic bead selection. Following expansion in IL-2/15 for 9 days, cells are electroporated with CRISPR-Cas9 RNP complexes. CRISPR-edited NK cells are further expanded for 6 days before functional and flow cytometric analyses. (c) Left, quantification of percent IFN-γ+ in TRACcRNP or TCF7cRNP NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and/or IL-18. Right, specific lysis of K562 cells after 16 h coculture with IL-2 and IL-15 at indicated effector:target ratios. (d) Left, density of viable TRACcRNP or MYCcRNP NK cells 6 days after cRNP editing expanded with IL-2 and IL-15. Right, quantification of percent IFN-γ+ in TRACcRNP or MYCcRNP NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and/or IL-18. (e) Density of viable TRACcRNP or ZEB2cRNP NK cells 6 days after cRNP editing expanded with IL-2 and IL-15. (f) Quantification of percent IFN-γ+ in TRACcRNP or RORCcRNP NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and/or IL-18. (g) Representative gating strategy for peripheral human NK cells. Data are representative of n = 6-8 independent donors presented as individual paired donors. *p < 0.05, **p < 0.01 by two-sided paired t test. Specific p-values are as follows: c percent IFN-γ+ NT = 0.1686, IL-18 = 0.0200, IL-12 = 0.0136, IL-12/18 = 0.8445, % specific lysis 1:8 = 0.0399, 1:4 = 0.0366; d cells/mL = 0.0040, percent IFN-γ+ NT = 0.0769, IL-18 = 0.0128, IL-12 = 0.0244, IL-12/18 = 0.4241; e = 0.0216, f = 0.0456.

Extended Data Fig. 2 MEF2C is required for human NK cell function without impacting fitness.

(a) Immunoblot showing MEF2C protein expression in TRACcRNP or MEF2CcRNP NK cells 6 days after CRISPR cRNP editing. (b) Indel percentage by CRISPR cRNP editing in TRACcRNP or MEF2CcRNP NK cells 6 days after editing. Sanger sequencing results were analyzed using SYNTHEGO ICE analysis software. (c) Quantification of annexin+ early apoptotic or annexin+PI+ late apoptotic TRACcRNP or MEF2CcRNP NK cells 6 days after editing. (d) MFI of BCL2, BIM, or ratio of BCL2/BIM MFI in TRACcRNP or MEF2CcRNP NK cells 6 days after editing. (e) Specific lysis of A375 human melanoma cells after 16 h coculture with TRACcRNP or MEF2CcRNP NK cells in the presence of IL-2/15. (f) MFI of perforin in TRACcRNP or MEF2CcRNP NK cells 6 days after editing. Data are representative of n = 4−7 independent donors presented as individual paired donors. *p < 0.05, **p < 0.01 by two-sided paired t test. Specific p-values are as follows: c = 0.0361, 0.0204; d BCL2 = 0.0047, BIM = 0.2848, BCL2/BIM = 0.1384; e = 0.0405; f = 0.9390.

Source data

Extended Data Fig. 3 MEF2C haploinsufficiency disrupts CD56dim NK cells without impacting CD56bri or other circulating immune populations.

(a) Schematic of pathogenic point mutations in MCHS patients. (b) Expression of MEF2C in FACS-sorted peripheral human immune cells based on DICE database data. (c) Frequency of non-NK cell immune populations in peripheral blood of healthy donor control or MCHS patients. (d) Percent IFN-γ+ (above) and IFN-γ MFI of cytokine-producing cells (below) of total (left) or CD56bri (right) healthy donor control or MCHS patient NK cells stimulated for 16 h with IL-2, IL-15, K562 cells, and IL-12 after 5 d expansion in IL-2/15. (e) MEF2C transcript expression in NK cell maturation subsets. (f-g) MFI of perforin (f) or GzmB (g) in healthy donor or MCHS patient NK cells by maturation subset. (h) MFI of GzmB in healthy donor or MCHS patient CD56dim NK cells. (i) Schematic showing adenine base editor (ABE8e) mediated generation of MEF2C point mutation. (j) Assessment of point mutation frequency at targeted base using MEF2C-targeting sgRNA in conjunction with electroporation of ABE8e mRNA in healthy primary human NK cells. Point mutation rate was evaluated by Sanger sequencing and analysis using the EditR package on day 6 post ABE8e electroporation in culture with IL-2/15. (c) Left, gated on CD3+CD14- T cells, CD3−CD14+ monocytes, and CD3−CD19+ B cells. Center, gated on CD3+CD14−CD4+ or CD3+CD14−CD8+ cells. Right, gated on CD3−CD14+CD16hi classical, CD3−CD14+CD16int intermediate, or CD3−CD14+CD16lo non-classical monocytes. (d,f-h) Gated on CD3−CD56+ cells or CD3−CD56dimCD16+ cells. Data represent mean ± SEM. Data are representative of (c,d,f-h) n = 5-8 independent healthy donors alongside n = 2 MCHS patients each sampled two independent times, (e) n = 3−4 independent donors, or (j) n = 6 independent donors. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-sided Student’s t test. Specific p-values are as follows: d CD56bri percent IFN-γ+ NT = 0.0010, IL-18 = 0.0029; g = 0.0198; h = 0.0039; j < 0.0001.

Extended Data Fig. 4 Mef2c haploinsufficiency impairs antiviral immunity.

(a) Schematic of WT:Mef2c+/- mixed bone marrow chimeric (mBMC) mice or WT and Mef2c+/- single bone marrow chimeric (sBMC) mice. (b) Percent WT or Mef2c+/- bone marrow-derived NK cells on D0 or D42 post bone marrow transplant of WT:Mef2c+/- mBMC mice. (c) Representative contour plots (left) and frequencies (right) of peripheral NK cell subsets of wild-type:Mef2c+/- mixed bone marrow chimeric (mBMC) mice after 4 weeks engraftment. (d-e) Percent IFN-γ+ (left) and IFN-γ MFI of cytokine-producing cells (right) of splenic NK cells from WT:Mef2c+/- mBMC mice stimulated ex vivo with IL-15 and IL-12 (d) or IL-18 (e) stratified by maturation subset. (f) Percent IFN-γ+ (left) and IFN-γ MFI of cytokine-producing cells (right) of splenic NK cells from WT:Mef2c+/- mBMC mice on D1.5 post MCMV. (g) Schematic of adoptive transfer of WT:Mef2c+/- mBMC splenocytes. (h) Schematic of adoptive transfer of CRISPR edited Rosa26cRNP or Mef2ccRNP NK cells. (i) Representative contour plots (left) and frequency (right) of Rosa26cRNP or Mef2ccRNP Ly49H+KLRG1+ mouse NK cells on D0 and D7 post MCMV. (j) Percent IFN-γ+ of Rosa26cRNP or Mef2ccRNP NK cells stimulated for 4 h with IL-12 or IL-18. (k) Percent specific lysis of MC38 β2 M-/- by Rosa26cRNP or Mef2ccRNP NK cells at 1:1 E:T. (l) Representative gating strategy for splenic mouse NK cells. (b-e,j) Gated on CD3−TCRβ-NK1.1+ cells. (f,i) Gated on CD3−TCRβ-NK1.1+Ly49H+KLRG1+ cells. Data shown as mean ± SEM, paired WT and Mef2c+/- NK cells from the same mBMC mouse, or paired Rosa26cRNP and Mef2ccRNP NK cells from the same mouse where applicable. Data representative of at least 2 independent experiments. Data are representative of (b,c) n = 23, (d,e) n = 11, (f) n = 10, (i,j) n = 4, and (k) n = 7 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-sided paired t test. Specific p-values are as follows: b < 0.0001; c DN = 0.0020, DP < 0.0001, CD11b SP < 0.0001; d CD11b SP percent IFN-γ+ = 0.0144, CD11b SP IFN-γ MFI = 0.0469; e CD11b SP percent IFN-γ+ = 0.0447; f CD11b SP percent IFN-γ+ = 0.0032; DP IFN-γ MFI = 0.0409, CD11b SP IFN-γ MFI = 0.0016; i < 0.0001; j IL-12 percent IFN-γ+ = 0.0484, IL-18 percent IFN-γ+ = 0.0466; k = 0.0009.

Extended Data Fig. 5 MEF2C is required for cytokine-activated metabolic reprogramming.

(a) MFI of CD25/IL-2Rα (left) or CD122/IL-2Rβ (right) of peripheral blood NK cells from WT or Mef2c+/- sBMC mice on D0 or D1.5 of MCMV infection. (b) MFI of CD25/IL-2Rα (left) or CD122/IL-2Rβ (right) of TRACcRNP or MEF2CcRNP human NK cells 6 days post CRISPR edit. (c) MFI of pSTAT5 (left) or pSTAT1 (center) and representative histogram of pSTAT1 expression (right) in splenic NK cells from WT or Mef2c+/- sBMC mice on D1.5 of MCMV infection. FMO, fluorescence minus one control. (d) MFI of pSTAT5 (left) and representative histogram (right) of TRACcRNP or MEF2CcRNP human NK cells 6 days post CRISPR edit. FMO, fluorescence minus one control. (e) Maximal respiration of TRACcRNP or MEF2CcRNP human NK cells 6 days post CRISPR edit evaluated by Seahorse extracellular flux assay. (f) Metabolic dependencies (left) and translation rate (right) of TRACcRNP or MEF2CcRNP human NK cells 6 days post CRISPR edit measured by SCENITH. Gluc dep, glucose dependence; Mito dep, mitochondrial dependence; Glyc cap, glycolytic capacity; FAO/AAO cap, fatty acid oxidation/amino acid oxidation capacity; Co, control; DG, 2-deoxyglucose; O, oligomycin. (g) Metabolic dependencies of TRACcRNP or MEF2CcRNP human NK cells 6 days post CRISPR edit measured by SCENITH, stratified by maturation subset. (a) Gated on CD3−TCRβ-NK1.1+ cells. (b,f,g) Gated on CD3-CD56+ cells. Data represent (a) n = 5-7 mice per group, (b,d) n = 3 paired independent donors, (c) n = 3-4 mice per group, (e) n = 5, or (f,g) n = 4-6 paired independent donors. *p < 0.05, **p < 0.01 by two-sided paired t test. Specific p-values are as follows: f mito dep = 0.0091, glyc cap = 0.0091, Co puro MFI = 0.0140, O puro MFI = 0.0040; g CD16− = 0.0492, 0.0492; CD16+CD57− = 0.0491, 0.0322, 0.0322, 0.0491.

Extended Data Fig. 6 MEF2C maintains SREBP activity in mouse and human NK cells.

(a) Heatmap showing differentially expressed genes from RNA-seq performed on human and mouse control and MEF2C knockout NK cells. (b) Gene Ontology pathway analysis of downregulated genes conserved between human MEF2CcRNP and mouse Mef2ccRNP NK cells compared to control edited cells ranked by FDR. (c) Gene Ontology pathway analysis of downregulated genes conserved between human MEF2CcRNP and mouse Mef2ccRNP NK cells compared to control edited cells ranked by FDR, excluding mitosis and cell division-related pathways. (d) Heat maps showing changes in gene expression of canonical SREBP pathway genes separated by biological replicate with hierarchical clustering of genes. (e) Transcript expression of SREBF1 and SCD1 in TRACcRNP or MEF2CcRNP human NK cells 6 days post CRISPR edit. (f) MFI of BODIPY 493/503 of total splenic NK cells from naive and D1.5 MCMV-infected mice. (g-h) MFI of LDLR (g) or BODIPY 493/503 (h) in TRACcRNP or LDLRcRNP human NK cells 6 days post CRISPR edit. (i) Proposed model of MEF2C-directed lipid metabolic reprogramming driving NK cell effector function in response to cytokine stimulation and mTORc1 activation. Data represent mean ± SEM or individual paired donors where applicable. (f) Gated on naïve CD3−TCRβ-NK1.1+ cells or D1.5 CD3−TCRβ-NK1.1+Ly49H+KLRG1+ cells. (g-h) Gated on CD3−CD56+ cells. Data are representative of (a-d) n = 6 mice and n = 3 independent donors, (e) n = 6−7 independent donors, (f) n = 13 naive and 10 D1.5 mice, or (g-h) n = 6 independent donors. *p < 0.05, **p < 0.01 by two-sided paired t test or Student’s t test. Specific p-values are as follows: e = 0.0108, 0.0421; f = 0.0092; g = 0.0032, h = 0.0485.

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Fig. 4 and Extended Data Fig. 2.

Unprocessed immunoblots for Fig. 4a–c and Extended Data Fig. 2a.

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Li, J.H., Zhou, A., Lee, C.D. et al. MEF2C regulates NK cell effector functions through control of lipid metabolism. Nat Immunol (2024). https://doi.org/10.1038/s41590-024-01811-2

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