Oleate-mediated enhancement of human natural killer cell function

Abstract

Natural killer (NK) cells are a critical first line of defense against viral infections. 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 infections. Utilizing a non-viral CRISPR-Cas9 knockout screen targeting 31 transcription factors differentially expressed during human NK cell development, we identified myocyte enhancer factor 2C (MEF2C) as a master regulator of human NK cell functionality ex vivo. MEF2C haploinsufficient (MCHS) patients and mice displayed profound defects in NK cell development and effector function, with an increased susceptibility to viral infection. Mechanistically, MEF2C was required for IL-2 and IL-15-mediated increase in lipid content through regulation of the SREBP pathways. Supplementation with oleic acid restored MEF2C-deficient and MCHS patient NK cell cytotoxic function.

Description

[0001] OLEATE-MEDIATED ENHANCEMENT OF HUMAN NATURAL KILLER CELL FUNCTION CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application No.63 / 557,785, filed February 26, 2024, entitled “ OLEATE-MEDIATED ENHANCEMENT OF HUMAN NATURAL KILLER CELL FUNCTION”, which application is incorporated by reference herein. TECHNICAL FIELD The invention is in the fields of immunology and medicine. BACKGROUND OF THE INVENTION Natural killer (NK) cells play a critical role during the early defense against viral infection via direct cytotoxicity against infected cells as well as the production of inflammatory cytokines such as interferon (IFN)-^1. This is highlighted by the increased susceptibility to viruses displayed by NK cell-deficient individuals, often leading to premature death due to disseminated viral infection2-13. In the initial stages of viral infection, NK cells are exposed to a rich milieu of activating signals including proinflammatory cytokines secreted by local myeloid cells as well as direct ligation of activating receptors1. These signals result in transcriptional and epigenetic changes driving a dramatic burst of proliferation, production of effector molecules, and heightened metabolic activity, enabling activated NK cells to deliver a rapid and potent antiviral response. In humans, peripheral NK cells are developmentally stratified by differential expression of the markers CD56, CD16, and CD5714-16. Developmental subsets of human NK cells are functionally distinct, as CD56briCD16- cells produce higher amounts of IFN-^ and display lower cytotoxicity than CD56dimCD16+cells. CD56dimCD16+CD57+cells present the most mature receptor repertoire, the greatest cytolytic activity, and display the greatest sensitivity to CD16 receptor activation14,17. Significant transcriptional and epigenetic reprogramming occurs for NK cells to transition between these functionally distinct developmental stages18. Transcription factors such as EOMES, ETS1, T-BET, NFIL3, and ID2, as well as regulatory proteins like CISH, are well characterized as regulators of NK cell development and effector function after activation19-25. For example, ETS1 expression drives expression of T-BET, ID2, and activating receptors to promote effector function19. Understanding the transcriptional regulation of human NK cell function is clinically significant, as multiple human NK cell deficiencies are caused by mutations in transcription factors (BCL11B, GATA2, IRF8) owing to their pleiotropic roles in gene regulation2,6,26. While mechanistic studies identifying transcription factors important for NK cell antiviral function are largely performed in mice, recent studies suggest that the transcriptional changes that are induced in mouse and human NK cells upon cytokine activation are largely species-specific27. There is a need in the art for methods and material that can enhance human NK cell function. In this context, a direct examination of transcription factor functions in primary human NK cells can facilitate the discovery of gene regulatory networks important for enhancing NK cell function in the clinic. SUMMARY OF THE INVENTION To identify novel transcription factors that control human NK cell function, we developed a targeted non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) based screening approach in primary human peripheral blood mononuclear cell (PBMC)- derived NK cells to evaluate the role of developmentally regulated transcription factors in positive or negative regulation of effector function28,29. Out of 31 genes screened, we found that myocyte enhancer factor 2C (MEF2C) was the sole transcription factor broadly required for human NK cell homeostasis, cytokine production, and cytotoxicity. MEF2C Haploinsufficiency Syndrome (MCHS) is a neurodevelopmental disorder caused by mutations or deletions of one allele of the MEF2C gene. MCHS is characterized by hypotonia, epilepsy, autism, absent speech, intellectual disability, and developmental delay. Mutations of this gene have resulted in severe psychomotor retardation, periodic tremor and an abnormal motor pattern with mirror movement of the upper limbs observed during infancy, hypotonia, abnormal EEG, epilepsy, absence of speech, autistic behavior, bruxism, and mild dysmorphic features, mild thinning of the corpus callosum and delay of white matter myelination in the occipital lobes. Currently, there is no treatment for MEF2C deficiency and care is individualized based on symptoms. As discussed below, we have discovered that MEF2C haploinsufficiency in both human patients and mice results in defective peripheral NK cell development, effector function. Our studies show that loss of MEF2C lowers intracellular lipid content and uptake, with decreased LDLR levels. Unexpectedly, we found that supplementation of NK cells with oleic acid can restore the cytotoxic function of MEF2C-deficient NK cells. Thus, we identify MEF2C as a novel transcriptional regulator of NK cell effector function via the regulation of cytokine-activated lipid metabolic reprogramming and characterize a functional NK cell defect associated with MEF2C haploinsufficiency in humans. Building upon our discoveries, we have developed a number of methods and materials for the modulation of NK cell effector function, for example with NK cells obtained from or present in individuals having MEF2C Haploinsufficiency Syndrome. The invention disclosed herein has a number of embodiments. Embodiments of the invention include methods of modulating NK cell physiology, the methods comprising combining a NK cell with a composition comprising oleic acid, wherein the composition comprises amounts of oleic acid sufficient to increase lipid content in the NK cell (e.g., a CD56dimCD16+NK cell). Typically, the composition also includes a pharmaceutically acceptable carrier. Optionally, the composition comprises a plurality of agents, for example oleic acid and also a cytokine such as IL- 15. In typical embodiments of the invention, the composition comprises amounts of oleic acid sufficient to enhance NK cell cytotoxicity, for example amounts of oleic acid sufficient to enhance antiviral NK cell effector function. In some embodiments of the invention, the NK cell comprises a mutation in the myocyte enhancer factor 2C (MEF2C) gene. In illustrative embodiments, the NK cell is obtained from or present in a patient diagnosed with MEF2C Haploinsufficiency Syndrome (MCHS). In certain embodiments of the invention, the NK cell is one is present in a patient diagnosed with MEF2C Haploinsufficiency Syndrome, and the composition is administered to the patient nonparenterally. Related embodiments of the invention include methods of making a composition, the methods comprising combining oleate with an NK cell, wherein amounts of oleate present in the composition are selected to be sufficient to increase lipid content in the NK cell. Typically in such methods, amounts of oleic acid are selected to be sufficient to enhance NK cell cytotoxicity. In certain embodiments of the invention, the methods further comprise combining the NK cell and oleate with a cytokine such as IL-15. Embodiments of the invention further comprise compositions made by these methods. Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. MEF2C is required for human NK cell proliferation and effector function. (a) Overview of transcription factor knockouts regulating NK cell function. (b) Density of viable TRACcRNPor MEF2CcRNPNK cells 6 days after cRNP editing expanded with IL-2 and IL-15. (c) Left, representative histograms showing dilution of CellTrace Violet (CTV) in TRACcRNPor MEF2CcRNPNK cells on day 6 post cRNP edit. Right, frequency of cells undergone >2 cell divisions. (d) Frequency of Ki67+cells on day 6 post cRNP contour plots (left) and quantification of percent IFN-^+(center) and IFN-^ MFI of cytokine-producing cells (right) of TRACcRNPor MEF2CcRNPNK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and / or IL-18. (f) TNF-^ MFI of NK cells stimulated for 16 h with IL-2, IL-15, K562 cells, IL-12, and IL-18. (g) Specific lysis of K562 cells by edited NK cells after 16 h coculture with IL-2 and IL-15 at indicated effector:target ratios. (h) Representative histograms (left) and quantification (right) of GzmB expression in edited NK cells. (i) Representative histogram (left) and quantification (right) of CD107a expression in edited NK cells cultured for 4 h with IL-2, IL-15, K562 cells, brefeldin A, and monensin in the presence of CD107a-PE antibody. (c-f, h, i) Gated on CD56+CD3- cells. Data are representative of n = 6-11 independent donors presented as individual paired donors. *p < 0.05, **p < 0.01, ***p < 0.001 by paired t test. Figure 2. MEF2C haploinsufficiency syndrome (MCHS) patients present with a functional NK cell deficiency. (a) Representative contour plots and (b) quantification showing NK cell maturation distribution in PBMCs from healthy control or MCHS patients bearing c.90G>T or c.638-2A>G point mutations. PBMCs were isolated from whole blood by Ficoll density separation and immediately stained for NK cell markers. (c) Representative contour plots (left) and quantification of percent IFN-^+(center) and IFN-^ MFI of cytokine-producing cells (right) of CD56dimhealthy 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. (d) Specific lysis of K562 cells by healthy donor control or MCHS patient NK cells cocultured for 16 h with K562 cells and IL-2 / IL-15 after 5 d expansion in IL-2 / 15. (e) Representative histogram of CTV dilution in control or base edited human NK cells 6 days after base editing expanded in IL-2 / 15. (f) Density of viable control or base edited NK cells 6 days after base editing expanded with IL-2 and IL-15. (g) Specific lysis of K562 cells by control or base edited NK cells cocultured for 16 h with K562 cells and IL-2 / IL-15 after 5 d expansion in IL-2 / 15. (h) Representative contour plots (left) and quantification of percent IFN-^+(center) and IFN-^ MFI of cytokine-producing cells (right) of control or base edited NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12. (a,b,e,f,h) Gated on CD56+CD3- cells; (c) Gated on CD56dimCD16+CD56+CD3- cells. Data represent mean ^ SEM or individual paired donors. Data are representative of (b) n = 14, (c,d) n = 6, (e-h) n = 7 independent healthy donors alongside n = 2 MCHS patients each sampled two independent times where applicable. *p < 0.05 by paired t test or Student’s t-test. Figure 3. MEF2C haploinsufficiency disrupts antiviral immunity. (a) Percent IFN-^+(left) and IFN-^ MFI of total NK cells (right) of wild-type or Mef2c+ / -bone marrow-derived splenic NK cells stimulated ex vivo for 4 h with IL-15, IL-12, and / or IL-18 in the presence of brefeldin A / monensin. (b) Percent CD107a+wild-type or Mef2c+ / -bone marrow-derived splenic NK cells stimulated ex vivo with plate-bound anti-Ly49H antibody for 4 h with IL-15, brefeldin A, and monensin. (c) WT or Mef2c+ / -sBMC mice were infected with a sublethal dose of MCMV and weighed daily. Body weight was normalized to starting body weight per mouse before infection on D0. (d) Kaplan-Meier survival curves of wild-type or Mef2c+ / -bone marrow chimeras infected with a sublethal dose of MCMV. (e) Percent IFN-^+(left) and IFN-^ MFI of cytokine-producing cells (right) of wild-type or Mef2c+ / -bone marrow-derived splenic NK cells in wild-type:Mef2c+ / -mBMC mice on D1.5 post MCMV infection. (f) MFI of GzmB of wild-type or Mef2c+ / -bone marrow-derived splenic NK cells in wild-type:Mef2c+ / -mBMC mice on D1.5 post MCMV infection. (g) Representative contour plots showing D0 and D7 splenic NK cell ratios (left) and quantification of D7 ratio (right) in peripheral organs of wild-type or Mef2c+ / -NK cells co-transferred into male Ly49H- / -host mice and infected with MCMV. (a,b) Gated on CD3-TCR^-NK1.1+cells; (e,f,g) Gated on CD3-TCR^- NK1.1+Ly49H+KLRG1+cells. Data are representative of at least 2 independent experiments. Data represent mean ^ SEM or paired wild-type and Mef2c+ / -bone marrow-derived cells from the same mBMC mouse where applicable. Data are representative of (a,b) n = 11 mice, (c,d) n = 23 mice, (e-f) n = 10 mice, and (g) n = 4 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by (a,b, e-g) paired t test, (c) two-tailed Student’s t test, or (d) Mantel-Cox test. Figure 4. MEF2C is required for IL-15 / mTORc1-induced metabolic reprogramming. (a) Immunoblot showing MEF2C and ^-actin loading control protein levels in naive human NK cells or cells stimulated with IL-2 / 15 for 72 h (left) with quantification (right). (b) Immunoblot showing MEF2C and ^-actin loading control protein levels in human NK cells stimulated with IL-2 / 15 for 72 h alone or with MEK inhibitor AZD6244 (50 ^M) or STAT5 inhibitor CAS 285986-31-4 (100 ^M) (left) with quantification (right). (c) Immunoblot showing MEF2C and ^-actin loading control protein levels in human NK cells stimulated with IL-2 / 15 for 72 h alone or with PI3K inhibitor Ly294002 (50 ^M) or mTORc1 inhibitor rapamycin (20 nM) (left) with quantification (right). (d) Representative histograms (left) and MFI of pAKT (center) and pS6 (right) of TRACcRNPor MEF2CcRNPhuman NK cells 6 days post CRISPR edit. FMO, fluorescence minus one control. (e) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TRACcRNPor MEF2CcRNPNK cells measured by Seahorse extracellular flux assay. O, oligomycin; F, FCCP; R / A, rotenone / antimycin A. (f) Quantification of basal ECAR and ratio of OCR to ECAR from (e), normalized to rate per 10,000 cells. Data represent (a) n = 6, (b) n = 4 (MEKi) or n = 6 (STAT5i), (c) n = 5, (d) n = 8, or (e,f) n = 5 paired independent donors. *p < 0.05, **p < 0.01 by paired t-test. Figure 5. MEF2C promotes cytokine activated SREBP signaling and increased lipid content in NK cells. (a) Volcano plots displaying significant differentially expressed genes in cRNP-edited human (left) or mouse (right) NK cells compared to nontargeting controls with labeled genes of interest. NK cells were expanded after CRISPR cRNP editing for 6 days in IL-2 / 15 (human) or 3 days in IL- 15 (mouse) before RNA library preparation and sequencing. For human NK cells, RNA samples were from paired TRACcRNPand MEF2CcRNPNK cells from 3 independent donors. For mouse NK cells, RNA was extracted in triplicate from isolated NK cells from two mice pooled and edited with Rosa26cRNPor Mef2ccRNP. (b) Gene set enrichment analysis of MEF2CcRNPhuman NK cells compared to TRACcRNPcontrol. (c) RNA-seq on human and mouse control and MEF2C knockout NK cells identified 184 conserved differentially expressed genes (DEGs). (d) Heat map showing changes in gene expression of canonical SREBP pathway genes with hierarchical clustering of genes. (e) Representative histograms (left) and MFI of BODIPY 493 / 503 staining (right) in TRACcRNPor MEF2CcRNPNK cells on day 6 post cRNP editing and expanded in IL-2 / 15. FMO, fluorescence minus one control. (f) MFI of BODIPY 493 / 503 in healthy control or MCHS patient NK cells immediately after isolation or after 5 days expansion in IL-2 / 15. (g) BODIPY 493 / 503 MFI of wild-type or Mef2c+ / -bone marrow-derived splenic NK cells in uninfected (left) or D1.5 post MCMV infection (right) wild-type:Mef2c+ / -mBMC mice. (e,f) Gated on CD3-CD56+cells; (g) Gated on naive CD3-TCR^-NK1.1+or D1.5 CD3-TCR^- NK1.1+Ly49H+KLRG1+cells. Data are representative of at least 2 independent experiments. Data represent mean ^ SEM or individual paired donors where applicable. Data are representative of (a-d) n = 6 mice and n = 3 independent donors, (e) n = 7 independent donors, (f) n = 3-8 healthy donors and n = 2 MCHS patients each sampled two independent times, and (g) n = 11 naive and n = 10 D1.5 MCMV mice. *p < 0.05, **p < 0.01 by (e,g) paired t test or (f) two-tailed Student’s t test. Figure 6. Fatty acid supplementation restores MEF2C-deficient NK cell cytotoxicity. (a) Representative histograms (left) and MFI (right) of BODIPY C12 uptake in TRACcRNPor MEF2CcRNPNK cells on day 6 post cRNP editing and expanded in IL-2 / 15. (b) LDLR transcript expression of naive or 3 h IL-2 / 15 activated mouse (left) or human (right) NK cells. (c) Ldlr transcript expression of splenic NK cells at the indicated timepoints following MCMV infection. (d-e) Representative histograms (d) and percent LDLR+and MFI of LDLR (e) of TRACcRNPor MEF2CcRNPhuman NK cells 6 days post CRISPR edit. (f) MFI of LDLR of healthy donor or MCHS patient human NK cells immediately after isolation from peripheral blood or after 5 days IL-2 / 15 stimulation. (g) Quantification of percent IFN-^+(left) and IFN-^ MFI of cytokine-producing cells (right) of TRACcRNPor LDLRcRNPNK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 or IL-18. (h) Specific lysis of K562 cells by edited NK cells after 16 h coculture with IL-2 and IL-15 at indicated effector:target ratios. (i) Specific lysis of K562 cells by untreated healthy donor human NK cells (NT) or cells incubated with 7.5 ug / mL M^CD cholesterol for 1 h or 200 uM BSA-conjugated palmitate or oleate for 24 h after 14 days expansion in IL- 2 / 15. Cells were cocultured for 16 h at a 1:2 E:T ratio. (j) Quantification of percent IFN-^+of untreated or NK cells pretreated with 200 uM BSA-conjugated oleate for 24 h after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12. (k) Left, viable cell density after 24 h pretreatment with 200 uM BSA-conjugated oleate and IL-2 / 15. Right, side scatter (SSC-A) of untreated or NK cells pretreated with 200 uM BSA- conjugated oleate for 24 h. (l) MFI of BODIPY 493 / 503 in healthy control or MCHS patient NK cells immediately after isolation or after 5 days expansion in IL-2 / 15. (m) % fatty acid oxidation (FAO) / amino acid oxidation (AAO) capacity of untreated or NK cells pretreated with 200 uM BSA-conjugated oleate for 24 h measured by SCENITH. (n) Specific lysis of K562 cells by TRACcRNP, MEF2CcRNPNK cells, or MEF2CcRNPNK cells pretreated with 200 uM BSA-conjugated oleate for 24 h at a 1:2 E:T ratio. (o) Specific lysis of K562 cells at the indicated E:T ratios by healthy control, MCHS patient NK cells, or MCHS patient NK cells pretreated with 200 uM BSA-conjugated oleate for 24 h after 5 d IL-2 / 15 expansion. (a,d-g) Gated on CD3- CD56+cells. Data are representative of at least 2 independent experiments. Data represent mean ^ SEM or individual paired donors where applicable. Data are representative of (a) n = 7 independent donors, (b) n = 3 mice and n = 6 independent donors, (c) n = 3 mice, (d, e) n = 6 independent donors, (f, o) n = 3-8 healthy donors and n = 2 MCHS patients each sampled two independent times, (g, j) n = 6, (h) n = 7, (i, m) n = 5, (k-l) n = 3-6, (n) n = 6 independent donors. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by paired t test or Student’s t-test. DETAILED DESCRIPTION OF THE INVENTION Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. In the description of the preferred embodiment, reference may be made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and reagent substitutions may be made without departing from the scope of the present invention. All publications mentioned herein are incorporated by reference to disclose and describe aspects, methods and / or materials in connection with the cited publications. Aspects of the invention disclosed herein are discussed in Li et al., Nat Immunol. 2024 May;25(5):778-789. doi: 10.1038 / s41590-024-01811-2. Epub 2024 Apr 8 (hereinafter “Li et al.”), the contents of which are incorporated by reference. As discussed below, we have discovered that MEF2C haploinsufficiency in both human patients and mice results in defective peripheral NK cell development, effector function, and protection against viral infection. In our studies, mechanistically, MEF2C was induced by IL-15 signaling to increase neutral lipid content through SREBP signaling pathways. We discovered that loss of MEF2C lowered intracellular lipid content and uptake with decreased LDLR levels. Unexpectedly, supplementation of NK cells with oleic acid restores the cytotoxic function of MEF2C-deficient NK cells. Thus, we identify MEF2C as a novel transcriptional regulator of NK cell effector function via the regulation of cytokine- activated lipid metabolic reprogramming and characterize a functional NK cell defect associated with MEF2C haploinsufficiency in humans. Further aspects and embodiments of the invention are discussed in the following sections. MEF2C is a critical regulator of human NK cell effector function Previous studies indicate that the transcriptional responses of activated mouse and human NK cells are distinct between species, suggesting that gene regulatory networks identified in mice may not translate to human immunity27. We therefore sought to identify transcription factors required for primary human NK cell effector function. We referenced previously published multi-omic datasets of FACS-sorted peripheral human NK subsets (CD56bri, CD56dimCD57-, and CD56dimCD57+), which combined ATAC-seq motif analysis, H3K27 monoacetylation ChIP-seq, and RNA- seq to identify 36 transcription factors whose binding sites were enriched in subset- specific putative enhancer regions and were differentially expressed between NK cell subsets30. For each of the 36 transcription factors, we tested 4 distinct sgRNAs to examine which genes were efficiently edited by cRNP editing. This yielded a final set of 31 transcription factors which could be edited with high efficiency in primary human NK cells (Fig. S1a in Li et. al.)30. As differential expression of CD56 and CD16 distinguishes immature cytokine-producing CD56briCD16- cells from mature cytotoxic CD56dimCD16+cells14,15, we reasoned that these transcription factors may be natural regulators of inflammatory or cytotoxic human NK cell function. To directly test the role of these genes in human NK cell effector function, we performed a targeted knockout screen using cRNP editing of primary NK cells isolated from healthy human donor peripheral blood mononuclear cells (PBMCs)28,29(Fig. S1b in Li et. al.). cRNP-edited NK cells were evaluated for viable cell numbers, IFN-^ production in response to cytokine stimulation, and killing of target cells. Of 31 candidates, we identified 11 positive regulators of NK cell function and 10 negative or mixed regulators (Fig. 1a). These included transcription factors previously reported in the literature to positively regulate mouse NK cell function such as TCF7, MYC, and ZEB231-33(Fig. 1a, Fig. S1c-e in Li et. al.). We also identified transcription factor functions consistent with reported immune cell deficiency in patients such as the requirement for RORC for NK cell IFN-^ production34(Fig. S1f in Li et. al.). Multiple positive regulators were novel and had no prior recognized role in human NK cell function. Out of 31 genes screened, we identified myocyte enhancer factor 2C (MEF2C) as the sole gene required for all tested NK cell functions (Fig. 1a). CRISPR cRNP- mediated MEF2C loss was verified by immunoblot and Sanger sequencing (Fig. S2a, b in Li et. al.). Compared to TRAC cRNP-edited (TRACcRNP) NK cells, we recovered lower numbers of viable MEF2C-edited (MEF2CcRNP) cells from the same donor after cRNP editing (Fig. 1b). While we observed a slight increase in apoptotic MEF2CcRNPNK cells, the absolute change was insufficient to account for the difference in total viable cells (Fig. S2c in Li et. al.). Furthermore, loss of MEF2C did not significantly alter the ratio of pro-survival BCL2 versus pro-apoptotic BIM protein (Fig. S2d in Li et. al.). Rather, MEF2CcRNPNK cells displayed a marked proliferative defect evidenced by CellTrace Violet (CTV) dilution and decreased Ki67 expression (Fig. 1c, d). When stimulated with IL-12, IL-18, or a combination of both cytokines, MEF2CcRNPNK cells produced less IFN-^ and TNF-^ (Fig. 1e, f). MEF2CcRNPNK cells were also less cytotoxic against K562 or A375 target cells (Fig. 1g, Fig. S2e in Li et. al.). This killing defect was accompanied by decreased degranulation and lower expression of granzyme B (GzmB) though perforin expression was unaffected (Fig. 1h, i, Fig. S2f in Li et. al.). Together, these results indicate that MEF2C is a critical regulator of IL-2 / 15-activated human NK cell effector function and proliferation. MEF2C haploinsufficiency syndrome is associated with defective NK cell maturation and function To confirm whether disruption of MEF2C expression in humans impacts NK cell function, we studied peripheral blood NK cells from patients with MEF2C haploinsufficiency syndrome (MCHS), a recently described neurological syndrome caused by point mutations or microdeletions involving MEF2C35-42. We identified two MCHS patients with clinical characteristics consistent with previous reports (Fig. S3a in Li et. al., Table 1). Patient 1 initially presented with developmental delay and was identified to have a de novo pathogenic c.638-2A>G intronic mutation upstream of exon 7 of MEF2C by whole exome sequencing, thought to disrupt the splice acceptor site of intron 7 (Fig. S3a in Li et. al.). Patient 2 was found to bear a de novo pathogenic c.90G>T (p.K30N) mutation localized to the DNA-binding MADS domain in MEF2C identified by epilepsy gene panel (Fig. S3a in Li et. al.). Neither patient had notable neutropenia on the most recent complete blood count with differential (Table 1). Patient 1 displayed normal lymphocyte frequencies and counts while the second presented with normal lymphocyte frequencies but decreased absolute lymphocytes (Table 1). Because MEF2C is predominantly expressed in B cells, monocytes, and NK cells, we examined the B cell, T cell, and monocyte compartments of both patients by flow cytometry and found no major changes in frequencies compared to control healthy donor peripheral blood samples (Fig. S3b, c in Li et. al.). However, patient 1 bearing an intronic point mutation (c.638-2A>G) displayed decreased frequencies of cytotoxic CD56dimCD16+NK cells compared to healthy controls accompanied by increased proportions of immature CD56briCD16- / intcells (Fig. 2a, b). Following 5 days of IL-2 / 15 activation ex vivo, CD56dimCD16+patient- derived NK cells produced lower levels of IFN-^ when stimulated with IL-12 and displayed moderately decreased cytotoxicity against K562 target cells compared to healthy controls (Fig. 2c, d). Defects in cytokine production were restricted to the CD56dimpopulation, as MCHS patient total or CD56briNK cells produced similar amounts of IFN-^ as healthy controls, consistent with MEF2C being most highly expressed in CD56dimrather than CD56briNK cells (Fig. S3d, e in Li et. al.). Clinically, this patient reported a history of recurrent otitis media (Table 1). As MEF2C relies on DNA binding for transcriptional activator activity, we next examined NK cells from patient 2 bearing a c.90G>T (p.K30N) disruption to the DNA-binding MADS domain (Fig. S3a in Li et. al.). This patient displayed an even more striking overrepresentation of CD56briCD16- / intcells and decreased CD56dimCD16+NK cells (Fig. 2a, b). Functional testing of patient-derived NK cells revealed profound defects in inflammatory cytokine production from the CD56dimpopulation as well as cytotoxicity, though cytokine production from CD56briNK cells again remained intact (Fig. 2c, d, Fig. S3d in Li et. al.). Clinical findings revealed a history of severe and recurrent viral infections, suggestive of significant immune dysfunction (Table 1). While we observed no major disruptions to perforin expression in MCHS patient NK cells after 5 days of expansion with IL-2 / 15, MCHS patient NK cells displayed defective GzmB expression specifically in CD16intand CD56dimCD16+cells (Fig. S3f, g in Li et. al.). Consistent with the more pronounced defect in cytotoxicity seen in patient 2 bearing a MADS domain point mutation, GzmB production in CD56dimCD16+cells from patient 2 was greatly reduced compared to healthy donor cells or patient 1 (Fig. S3h in Li et. al.). These results indicate that germline MEF2C haploinsufficiency in humans is associated with developmental and functional defects preferentially impacting the mature CD56dimCD16+NK cell subset. As MCHS patients bear germline MEF2C mutations in all somatic cells, we examined whether a cell-intrinsic point mutation in MEF2C in healthy donor-derived NK cells was sufficient to recapitulate the functional defects seen in MCHS patient- derived NK cells. We generated primary human NK cells bearing an intronic point mutation (c.1472+3A>G) in MEF2C via a single guide RNA-targeted Cas9 adenine base editor (ABE8e) (Fig. S3i in Li et. al.). Efficient base editing was confirmed by Sanger sequencing (Fig. S3j in Li et. al.). Compared to electroporation of ABE mRNA alone, the introduction of a MEF2C point mutation was sufficient to impair proliferation, cytotoxicity, and cytokine production in healthy donor-derived human NK cells (Fig. 2e-h). Thus, NK cell-intrinsic point mutations in MEF2C are sufficient to impair healthy mature human NK cell effector function. Mef2c is required for antiviral immunity Mef2c+ / -mouse models have been shown to faithfully recapitulate the neurological aspects of MCHS, as homozygous deficiency in Mef2c is embryonic lethal42,43. We generated mixed bone marrow chimeric (mBMC) mice engrafted with a 1:1 mixture of congenically distinct CD45.1 wild-type and CD45.2 Mef2c+ / -bone marrow to examine the effects of a clinically relevant degree of Mef2c deficiency on NK cell development and effector function (Fig. S4a in Li et. al.). After 4 weeks of bone marrow engraftment, Mef2c+ / -bone marrow-derived NK cells made up a smaller proportion of peripheral NK cells than wild-type bone marrow-derived cells after engraftment from a 1:1 WT:Mef2c+ / -starting ratio (Fig. S4b in Li et. al.). We observed an increase in immature CD27+CD11b+NK cells accompanied by a significant decrease in mature CD27-CD11b+NK cells derived from Mef2c+ / -bone marrow in the periphery, mirroring the developmental block observed in MCHS patient NK cells (Fig. S4c in Li et. al., Fig. 2a, b). Functionally, Mef2c+ / -NK cells produced less IFN-^ when stimulated ex vivo with IL-12 or IL-18 and displayed impaired degranulation upon anti-Ly49H activating receptor ligation ex vivo (Fig. 3a, b). Defective IFN-^ production was restricted to the mature CD27-CD11b+subset of peripheral NK cells (Fig. S4d, e in Li et. al.), like the CD56dim-restricted IFN-^ production defect observed in MCHS patients (Fig. S3d in Li et. al.). To determine whether functional defects in Mef2c+ / -NK cells were sufficient to confer increased susceptibility to viral infection in vivo, we generated single bone marrow chimeric (sBMC) mice engrafted with either wild-type or Mef2c+ / -bone marrow (Fig. S4a in Li et. al.). Upon viral challenge with a sublethal dose of mouse cytomegalovirus (MCMV), Mef2c+ / -sBMC mice lost more weight and succumbed more quickly to infection (Fig. 3c,d). Notably, mice died within the first 5 days of infection. While T cells are initially dispensable against MCMV, NK cells are required for immune protection early on, suggesting that the early mortality of Mef2c+ / -mice was likely attributable to a defect in the initial NK cell response44,45. We next infected WT: Mef2c+ / -mBMC mice to evaluate NK cell effector function in an internally controlled environment (Fig. S4a in Li et. al.). On D1.5 post-MCMV infection, Mef2c+ / -bone marrow-derived NK cells produced lower amounts of IFN-^ and GzmB compared to wild-type bone marrow-derived NK cells in the same host (Fig. 3e, f). As seen ex vivo, IFN-^ production defects were most significant in the mature CD27-CD11b+subset of peripheral NK cells responding to MCMV infection (Fig. S4f in Li et. al.). To examine whether Mef2c haploinsufficiency impacted clonal expansion during MCMV infection, we co-adoptively transferred wild-type and Mef2c+ / -Ly49H+NK cells into Ly49H- / -hosts to clonally proliferate in response to MCMV infection (Fig. S4g in Li et. al.). At the peak of NK cell expansion on D7 post-infection, Mef2c+ / -NK cells displayed markedly impaired expansion compared to co-transferred wild-type cells in multiple organs (Fig.3g). To test whether Mef2c was required for mature NK cell proliferation independent of the role of Mef2c in NK cell development in vivo, we ablated Mef2c expression in mature splenic mouse NK cells using cRNP electroporation (Mef2ccRNP) and adoptively transferred a mixture of congenically distinct Mef2ccRNPand control Rosa26cRNPLy49H+NK cells at a 1:1 ratio to Ly49H- / -hosts and infected with MCMV immediately post transfer (Fig. S4h in Li et. al.). On D7 post-infection, the proportion of Ly49H+KLRG1+Mef2ccRNPNK cells was decreased compared to co-adoptively transferred controls in the peripheral blood of recipient mice (Fig. S4i in Li et. al.). Like human NK cells, Mef2ccRNPNK cells displayed deficient production of IFN-^ in response to activating cytokines as well as impaired cytotoxicity against ^2- microglobulin (^2M)-deficient MC38 tumor targets ex vivo (Fig. S4j, k in Li et. al.). These findings demonstrate that Mef2c is required for both ex vivo and in vivo antiviral activity of mouse NK cells, and Mef2c haploinsufficiency in the hematopoietic compartment of mice is sufficient to increase morbidity and mortality during viral infection. MEF2C is required for IL-15 / mTOR-induced metabolic reprogramming Early during viral infection, activating cytokines in the local environment such as IL-2 and IL-15 stimulate increased NK cell proliferation and effector function1. We therefore hypothesized that MEF2C may be induced by cytokine stimulation to support enhanced effector function. We assessed MEF2C protein expression in healthy donor human NK cells stimulated with IL-2 and IL-15 for 72 hours ex vivo compared to freshly isolated cells and observed marked induction of MEF2C protein levels (Fig.4a). As IL-2 and IL-15 signal through the shared IL-2 / 15R^ and common ^c receptor components46, we pharmacologically inhibited signaling pathways downstream of these shared receptors. While inhibition of the mitogen-activated protein kinase (MAPK) pathway did not impact MEF2C expression, STAT5 was required for IL-2 / 15 dependent increase in MEF2C protein (Fig. 4b). MEF2C induction by IL-2 / 15 stimulation was also dependent on phosphoinositol-3-kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORc1) activity, as inhibition of these pathways decreased MEF2C expression to unstimulated levels (Fig. 4c). In contrast, PI3K and mTORc1 signaling remained intact in MEF2C- deficient NK cells as we observed no consistent changes to phosphorylation of AKT or ribosomal protein S6 across donors between TRACcRNPand MEF2CcRNPcells (Fig. 4d), indicating that MEF2C was not upstream of these pathways. Furthermore, expression of the IL-2 and IL-15 receptor components CD25 / IL-2R^ and CD122 / IL- 2R^ was unaffected by Mef2c heterozygosity in both naïve and D1.5 post-MCMV mouse NK cells as well as MEF2C loss in cRNP-edited human NK cells (Fig. S5a, b in Li et. al.). Indeed, levels of phosphorylated STAT5 and STAT1 remained unchanged in Mef2c+ / -NK cells after MCMV infection, while IL-2 / 15 activated TRACcRNPand MEF2CcRNPhuman NK cells displayed similar phosphorylated STAT5 expression (Fig. S5c, d in Li et. al.). These results suggested that the impaired effector function of NK cells after MEF2C loss is unlikely due to defective cytokine receptor signaling. Previous studies indicate that metabolic reprogramming is required for high levels of effector function in cytokine-treated NK cells47-49. IL-15 potently induces metabolic changes in NK cells via mTOR activation50. Considering the profound functional defects observed in our initial screen (Fig. 1), we hypothesized that MEF2C induction may broadly promote NK cell metabolic changes in response to cytokine stimulation and mTORc1 activation. Seahorse extracellular flux analysis of IL-2 / 15 activated TRACcRNPand MEF2CcRNPNK cells revealed decreased extracellular acidification rate (ECAR) and an increased ratio of oxygen consumption rate (OCR) to ECAR in the absence of MEF2C (Fig. 4e, f). There was no significant difference in maximal OCR, suggesting that this metabolic shift was specific to adaptations resulting from cytokine activation (Fig. S5e in Li et. al.). These metabolic changes were confirmed across mature NK cell subsets by single-cell metabolic profiling using SCENITH, which revealed robust inhibition of translation by oligomycin treatment as well as decreased glycolytic capacity and increased dependency on oxidative phosphorylation for ATP production upon MEF2C loss (Fig. S5f in Li et. al.). While these trends were consistent across all NK cell developmental subsets, altered metabolism was seen most significantly in the CD16+CD57- subset, suggesting that this subset is most dependent on MEF2C for maintaining metabolic fitness after cytokine activation (Fig. S5g in Li et. al.). Thus, MEF2C is required for mTORc1-mediated metabolic reprogramming upon IL-15 stimulation in mature peripheral human NK cells. MEF2C mediates NK cell SREBP signaling and lipid metabolism To interrogate the genes regulated by MEF2C that may control NK cell metabolism, we performed bulk transcriptomics in human and mouse CRISPR-edited NK cells. Upon MEF2C loss, most differentially expressed genes (DEGs) were decreased in expression in both human and mouse NK cells, suggesting that MEF2C primarily acts as a transcriptional activator in NK cells (Fig. 5a). We observed a notable decrease in expression of genes associated with SREBP-mediated lipid metabolism in both species, where SCD / Scd1 encoding stearoyl-CoA desaturase was one of the most significantly downregulated genes in both mouse and human cRNP- edited cells (Fig. 5a). Indeed, gene set enrichment analysis (GSEA) performed on all DEGs in MEF2CcRNPhuman NK cells compared to TRACcRNPcontrols revealed decreased expression of genes associated with fatty acid metabolism, cholesterol homeostasis, mTORc1 signaling, and glycolysis pathways (Fig. 5b). We identified 184 DEGs conserved between species regulated by MEF2C (Fig. 5c) and performed gene ontology analysis on this gene set to confirm GSEA findings. Gene ontology analysis identified most pathways altered by MEF2C loss were related to mitosis and cell division, consistent with the defective proliferative phenotype we observed in multiple settings (Fig. S6b in Li et. al.). Exclusion of mitosis-associated pathways revealed enrichment in SREBP signaling, lipid metabolism, and glycolysis in agreement with GSEA analysis, as well as hippocampal and substantia nigra brain development reflective of the neurodevelopmental delays seen in MCHS patients (Fig. S6c in Li et. al.). Indeed, genes associated with canonical SREBP signaling were downregulated in both MEF2C-deficient human and mouse NK cells (Fig. 5d, Fig. S6d in Li et. al.). Decreased SREBF1 and SCD transcript expression were confirmed by qRT-PCR (Fig. S6e in Li et. al.). Consistent with dysregulated lipid homeostasis upon MEF2C loss, we observed significantly decreased total neutral lipid content in IL-2 / 15 stimulated MEF2CcRNPNK cells by BODIPY 493 / 503 staining (Fig. 5e). While unstimulated healthy donor and MCHS patient-derived NK cells initially had similar lipid content, MCHS patient-derived NK cells displayed lower lipid levels after 5 days of IL-2 / 15 stimulation compared to healthy donor controls, suggesting that MEF2C is necessary for cytokine-induced increase in lipid content in human NK cells (Fig. 5f). In vivo, while MCMV infection increased lipid content in both wild- type and Mef2c+ / -mouse NK cells in mBMC mice (Fig. S6f in Li et. al.), Mef2c+ / -cells increased lipid stores less than wild-type cells on D1.5 post infection despite starting with similar lipid levels before infection (Fig. 5g). These findings highlight the key role of MEF2C in promoting SREBP-mediated lipid metabolism in cytokine stimulated NK cells both in vitro and during in vivo viral infection. Lipid supplementation restores cytotoxicity in MEF2C-deficient NK cells Intracellular lipid content can be modulated through lipid synthesis pathway activity or uptake of environmental lipids via receptors like CD36 or low-density lipoprotein receptor (LDLR)51,52. IL-2 / 15 stimulated MEF2CcRNPNK cells displayed impaired lipid uptake evidenced by decreased BODIPY C12 uptake (Fig. 6a). Indeed, analysis of ex vivo cytokine-stimulated mouse and human NK cells revealed increased LDLR transcript expression upon IL-2 / 15 stimulation (Fig. 6b). Splenic NK cells similarly increased Ldlr expression throughout the expansion phase of MCMV infection, reaching a peak in expression at D7 post-infection (Fig.6c). Consistent with bulk transcriptomic findings in cRNP-edited cells (Fig. 5a), we observed markedly decreased LDLR protein levels upon cRNP-mediated MEF2C loss in healthy donor human NK cells (Fig. 6d, e). After IL-2 / 15 stimulation, MCHS patient-derived NK cells likewise increased LDLR levels less than healthy donor NK cells (Fig. 6f). To evaluate the role of LDLR in maintaining human NK cell effector function, we ablated LDLR expression in healthy donor primary NK cells by cRNP electroporation and evaluated cytotoxicity and cytokine production of cRNP-edited cells. Loss of LDLR protein expression and decreased lipid content after cRNP editing was confirmed by flow cytometry (Fig. S6g, h in Li et. al.). LDLRcRNPNK cells phenocopied MEF2CcRNPNK cells, displaying decreased production of IFN-^ as well as lower cytolytic activity against K562 targets compared to paired TRACcRNPcontrols (Fig. 6g, h), suggesting that LDLR-mediated lipid uptake is required for optimal activated human NK cell function ex vivo. SREBP proteins are key drivers of lipid metabolism in mammalian cells53. SREBP1 is a master activator of fatty acid synthesis, driving the elongation and desaturation of the C16:0 fatty acid palmitate into C18:1 oleate via a C18:0 intermediate, stearate (Fig. S6i in Li et. al.). This process relies on the activity of fatty acid elongase 6 (ELOVL6) and stearoyl-CoA desaturase (SCD). Likewise, SREBP2 activation increases activity of sterol synthetic and uptake pathways. As we observed profoundly decreased expression of ELOVL6, SCD, HMGCR, and SQLE upon MEF2C loss in both human and mouse NK cells, we hypothesized that supplementation of NK cells with the lipid products of SREBP-mediated lipid synthesis may rescue MEF2C-deficient NK cell function. In cytokine-stimulated human NK cells, oleate but not cholesterol or palmitate supplementation increased cytotoxicity against K562 targets (Fig. 6i). However, oleate treatment decreased IFN- ^ production in response to IL-12 (Fig. 6j). 24 hours of oleate treatment did not significantly impact NK cell number but resulted in increased granularity, as measured by side scatter (Fig. 6k). Furthermore, oleate-treated wild type NK cells displayed significantly increased lipid content and trended toward increased mitochondrial oxidative capacity (Fig. 6l, m). In a lipid-deficient setting, oleate pretreatment was sufficient to restore cytotoxicity of MEF2CcRNPNK cells to the levels of corresponding paired donor TRACcRNPcells, consistent with SCD as one of the most significantly downregulated genes upon MEF2C loss (Fig. 6n, Fig. 5a). Notably, oleate treatment of IL-2 / 15 stimulated MCHS patient NK cells was sufficient to increase cytotoxicity to varying degrees. Lipid treatment of MEF2C c.638-2A>G patient NK cells markedly restored killing activity to near healthy adult donor levels, while supplementation of MEF2C c.90G>T patient NK cells only modestly improved cytotoxicity (Fig. 6o). Thus, we show that upon cytokine activation, MEF2C links PI3K / Akt-induced mTORc1 activation and downstream lipid metabolism programs under the control of SREBPs to promote antiviral NK cell effector function (Fig. S6i in Li et. al.). Germline disruption of these programs controlled by MEF2C results in NK cell defects in MCHS patients and Mef2c+ / -mice, resulting in increased susceptibility to viral infection. While prior studies have applied pooled CRISPR cRNP screens to link genes to immune cell function in T cells, monocytes, and monocyte-derived dendritic cells, our findings represent the first use of a non-viral targeted CRISPR cRNP screen in primary human NK cells. Our study highlights the utility of this approach to directly study gene function in human cells and reveal targets with both novel cellular function and direct clinical relevance. Furthermore, we show that Cas9 base editing can also be used to validate cell-intrinsic functional effects of patient gene variants in healthy donor primary NK cells. Cas9 base editing has been used to study hematopoiesis and attribute pathogenicity to variants of unknown significance (VUS) in CD34+hematopoietic stem progenitor cells (HSPCs) in patients with clinically diagnosed disorders of hematopoiesis54. Therapeutically, adenine base editors have been used to correct pathogenic CD3^ severe combined immunodeficiency (SCID) mutations in HPSCs and restore T cell development in a preclinical study55. Future applications of base editor-based functional validation of patient variants may be enhanced by computational prediction of variants most likely to disrupt protein structure, narrowing the range of variants requiring functional testing and potentially screening for new immune deficiencies56. Our results suggest that MCHS, primarily a neurodevelopmental disorder, unexpectedly presents with NK cell defects. Case reports suggest MCHS patients may display increased susceptibility to infection, but the cause of these recurrent infections is unknown35,38. Interestingly, pathogenic MCHS mutations tend to arise de novo as seen in our patients, in contrast to most characterized NK cell deficiencies which tend to be inherited in an autosomal recessive manner (Table 1)57. Other scenarios of combined neurodevelopmental and NK cell defects suggest that the central nervous system and immune compartment may share key developmental programs despite originating from distinct embryonic germ layers. Patients with heterozygous mutations in BCL11B present with autism-like intellectual disability like MCHS as well as varying degrees of NK cell developmental arrest and defects in other immune compartments26. Studies suggest that children with autism spectrum disorders have functionally defective NK cells with decreased cytotoxic capacity58. A subset of Rett syndrome driven by MECP2 overexpression known as MeCP2 duplication / triplication syndrome can similarly present with associated NK cell and T cell defects59. Mutations in multiple positive regulators identified by the present study (KLF3, NR4A2, SETBP1, ZEB2 (Mowat-Wilson syndrome), RORA) are also associated with reported intellectual disability or neurodevelopmental disorders in ClinVar yet lack formally reported immune defects. Mechanistically, emerging studies suggest that type 1 cytokines like IFN-^ produced by meningeal T and NK cells can modulate brain circuitry to affect behavior60,61. Ifng deficiency or neuronal deletion of Ifngr1 in mice disrupted inhibitory neuronal circuit activity in the prefrontal cortex (PFC) resulting in defective social behavior as well as increased seizure susceptibility60. Similarly, loss of meningeal mouse NK cell-derived IFN-^ decreased homeostatic inhibitory GABA-ergic signaling in the PFC61. Immune deficiencies associated with pediatric neurological disorders may be particularly undertreated, as frequent infections in these patients may be attributed to increased oral and pharyngeal secretions and impaired clearance rather than innate immune defects62. Therefore, further study will be crucial to better identify and care for these potentially immunodeficient patients. MEF2C deficiency predominantly affects mature peripheral NK cell populations, displaying functional effects in CD56dimCD16+MCHS patient NK cells and the corresponding CD27-CD11b+mouse NK cell population. As this mature subset represents most peripheral NK cells in both species, defects in this population result in an overall decrease in NK cell function and increased susceptibility to viral infection observed in both mouse models and MCHS patients even with normal CD56briNK cell activity. These findings are consistent with the differential expression of MEF2C in NK cell subsets, highest in the CD56dimCD16+population. As previous studies have found unique metabolic demands between the more inflammatory CD56briversus cytotoxic CD56dimhuman NK cells, our results suggest that differential requirements for lipid metabolism may also exist between these subsets and deserve further investigation63. Our study also identifies MEF2C as a previously unidentified master transcription factor linking extracellular cytokine stimulation and mTORc1 activation to SREBP-mediated lipid homeostasis in NK cells. SREBP signaling is required for NK cell metabolic reprogramming in Scap-deficient mouse models, which lack activation of both SREBP1 and SREBP2, though these studies focus on SREBP- mediated regulation of the citrate malate shuttle rather than lipid homeostasis64. Adaptive B and T cell responses similarly require SREBPs for full function65-67. Thus, our results support the critical role of SREBPs as key metabolic mediators in a broad array of immune populations. While kinases such as S6 kinase downstream of mTORc1 have been suggested to be required for induction of glycolysis and SREBP activation in response to mitogenic signals in other cell types68-70, we observed reduced glycolysis in human NK cells following MEF2C loss despite normal S6 kinase phosphorylation, suggesting that S6 kinase activity alone is not sufficient to activate SREBPs in NK cells. Rather, the transcriptional activator activity of MEF2C is likely required in addition to S6 kinase activation for IL-15-mediated metabolic reprogramming in NK cells. These findings may also point to MEF2C as an alternate mechanism for mTORc1 to activate SREBP signaling in NK cells, supported by studies in other cell types demonstrating that deletion of S6 kinase fails to abrogate SREBP1 activation71. Nuclear mTORc1 has been suggested to enhance histone acetylation and suppress histone deacetylase (HDAC) activity72-74. As MEF2C is repressed by class II HDACs75-78, mTORc1-mediated suppression of HDACs may increase MEF2C activity upon NK cell cytokine stimulation. Additionally, we find that STAT5 is also required for cytokine-dependent induction of MEF2C, suggesting that cooperation between STAT5 and mTOR activity may stimulate metabolic rewiring of activated NK cells similar to reported STAT-mTOR crosstalk in T cells79. Similarly, the mechanisms by which MEF2C activates downstream metabolic pathways are likely complex. As a MEF2 family member, MEF2C relies on a family- conserved MADS DNA-binding domain and associated MEF2 protein-binding domain to mediate transcriptional regulatory activity80. MEF2C may regulate lipid metabolism gene activity either through direct transactivation or the recruitment of additional transcription factors. Sterol species produced by increased SREBP activity can serve as both metabolites and signaling molecules through liver X receptor (LXR) signaling81. Thus, the downstream mechanisms of MEF2C-mediated lipid and glucose homeostasis in NK cells are likely multifactorial. Prior studies have suggested divergent effects of lipid metabolism on NK cell function. Examination of NK cells from obese patients indicated that elevated intracellular lipid levels from heightened amounts of circulating fats impair NK cell cytotoxicity via PPAR^ / ^-mediated mTOR inhibition82. Diffuse large B-cell lymphoma-associated NK cells similarly displayed increased lipid levels and corresponding decreased production of IFN-^ which was recapitulated by ex vivo fatty acid treatment83. Conversely, high cholesterol environments have been proposed to augment the antitumor activity of NK cells in hepatocellular carcinoma by increasing lipid raft formation84. While supraphysiologic cellular lipid levels may have beneficial or detrimental effects on NK cell function during obesity or cancer, our findings propose that there is also a minimum requirement for lipid metabolism to adequately support NK cell effector functions after cytokine activation. Our results suggest that the lipid transporter LDLR is required for optimal human NK cell effector function, which is in agreement with studies showing CD8+T cells derived from Ldlr-deficient mice are similarly impaired in proliferation, cytokine production, and cytotoxicity51. While we confirm previous results demonstrating that palmitate exposure inhibits cytotoxic NK cell function82, we find that specific treatment with oleate alone uniquely boosts NK cell cytotoxicity in both wild-type and MEF2C-deficient NK cells. As SREBP activity is highly sensitive to negative feedback inhibition via INSIG / SCAP-mediated repression, lipid supplementation with intermediates such as cholesterol or palmitate rather than end products of lipid metabolic pathways have inhibitory effects on SREBP function in other non-immune cell types, similar to our observations in NK cells53,81. Indeed, previous studies suggest that NK cells treated ex vivo with statins or peripheral NK cells from patients on statin therapy both display defective effector function85,86. Because statins inhibit cholesterol synthesis through the inhibition of HMG-CoA reductase, these findings may reflect an inhibitory buildup of SREBP2 pathway intermediates in statin-treated NK cells that may similarly inhibit NK cell function. Thus, metabolic supplementation with a panel of rationally selected lipids may represent a promising avenue for augmenting MCHS patient NK cells and NK cell adoptive therapy for cancer. Materials and Methods Mice. Mice were bred at UCLA according to animal welfare guidelines of the UCLA Institutional Animal Care and Use Committee. For all mouse experiments, 8-10- week-old age- and sex-matched littermates were used according to approved institutional protocols. Bone marrow chimeric mice were generated as previously described87. Briefly, host mice were lymphodepleted by intraperitoneal (i.p.) injection of busulfan (1 mg / mL) 25 mg per kg for 3 consecutive days (75 mg / kg total dose).24 hours following the final busulfan injection, isolated bone marrow from wild-type, Mef2c+ / -, or a 1:1 mixture of wild-type:Mef2c+ / -bone marrow resuspended in anti- NK1.1 (1 mg / mL, clone PK136) was transferred by intravenous (i.v.) injection. Bone marrow chimeric mice were allowed to engraft for at least 4 weeks before experimental studies. MCMV infection. MCMV (Smith strain) was serially passaged through BALB / c mice. On third passage, MCMV viral stocks were prepared by dissociation of salivary glands via dounce homogenization as previously described88. In vivo MCMV infection studies were performed by infecting mice with 7.5 x 103plaque-forming units in 0.5 mL of PBS i.p. Mice were monitored and weighed daily and euthanized if body weight decreased >20% of initial body weight. Mouse NK cell culture. Mouse spleens were collected and dissociated as previously published88. NK cells were isolated by negative immunomagnetic selection using the EasySep Mouse NK cell isolation kit (Stem Cell). Cytokine and plate-bound antibody stimulation experiments were performed as previously described87. For cytokine stimulation, 2 x 104purified mouse NK cells were cultured for 4 h at 37^C in CR-10 medium (RPMI 1640 + 25 mM HEPES + 10% FBS, 1% L-glutamine, 1% 200 mM sodium pyruvate, 1% MEM-NEAA, 1% penicillin-streptomycin, 0.5% sodium bicarbonate, and 0.01% 55 mM 2-mercaptoethanol) supplemented with brefeldin A (1:1000, Biolegend), monensin (2 uM, Biolegend), recombinant mouse IL-15 (50 ng / mL, PeproTech), mouse IL-12 (20 ng / mL, PeproTech), and / or mouse IL-18 (10 ng / mL, Biolegend). For plate-bound antibody stimulation, 2 x 104purified mouse NK cells were stimulated with 4 mg / mL precoated antibody against Ly49H (clone RM4- 5) for 4 h at 37^C with recombinant mouse IL-15 (50 ng / mL, PeproTech) and brefeldin A and monensin. For all stimulation experiments, control cells were cultured in CR-10 medium with brefeldin A and monensin alone (no treatment / NT). Adoptive transfer of NK cells. For whole splenocyte transfer, 5 x 107splenocytes were resuspended in PBS and injected i.v. into recipient mice. For transfer of CRISPR cRNP edited NK cells, NK cells were rested in complete CR-10 media with mouse IL-15 (50 ng / mL, PeproTech) for 10 min at 37^C immediately after CRISPR cRNP electroporation. After resting, congenically distinct cRNP-edited NK cells were mixed at a 1:1 ratio of CD3-NK1.1+Ly49H-KLRG1- cells, resuspended in PBS, and injected i.v. into recipient mice. Recipient mice were immediately infected with MCMV by i.p. injection. Human subjects. 14 milliliters of peripheral blood were obtained in Vacutainer CPT tubes (BD) from pediatric patients with MEF2C haploinsufficiency syndrome at the Greenwood Genetic Center, Greenwood, South Carolina. All blood samples were obtained from patients providing written informed consent according to Greenwood Genetic Center and UCLA approved IRB protocols. All human patient studies were approved by the ethics committees at Greenwood Genetic Center and UCLA. Human NK cell culture. Healthy primary human NK cells were isolated and cultured as previously described87. Briefly, human PBMCs were obtained either from healthy anonymous donors provided by the UCLA CFAR Virology Core or in Vacutainer CPT tubes (BD) for MCHS patient and healthy control peripheral blood. NK cells were isolated via negative immunoselection using the EasySep Human NK Cell Isolation Kit (Stem Cell Technologies). Isolated NK cells were expanded in NK MACS medium (Miltenyi Biotech) supplemented with recombinant human IL-2 (100 IU / mL, PeproTech) and recombinant human IL-15 (20 ng / mL, Peprotech) and cultured in 24-well G-Rex plates (Wilson Wolf). Cytokine stimulation assays were performed on cells activated for 15 d with IL-2 / IL-15 or CRISPR cRNP-edited cells on D6 post CRISPR edit. To stimulate cytokine production, NK cells were cultured in CR-10 media supplemented with human IL-2 (100 IU / mL, PeproTech), human IL-15 (20 ng / mL, Peprotech), K562 human leukemia cells, human IL-12, and / or human IL- 18 for 16 h then analyzed by intracellular flow cytometry. For lipid supplementation studies, NK cells were cultured in NK MACS medium (Miltenyi Biotech) with recombinant human IL-2 (100 IU / mL, PeproTech), recombinant human IL-15 (20 ng / mL, Peprotech), and either M^CD-cholesterol (7.5 ug / mL, Sigma), BSA- conjugated palmitate (200 uM, Cayman Chemical), or BSA-conjugated oleate (200 uM, Cayman Chemical). Guide RNA design. Synthetic sgRNAs were purchased from SYNTHEGO. For CRISPR cRNP editing, guide sequences were identified from previously published mouse and human whole genome CRISPR libraries described previously89. For Cas9 base editing, guide sequences were designed based on specific patient point mutations or using SpliceR v1.2.090. Top 2-4 high efficiency sgRNAs per target were validated by Sanger sequencing and immunoblotting. Gene targets with indel scores below 20 for all 4 tested sgRNAs were removed from the final screen. Negative control guides targeting TRAC were designed by and purchased directly from SYNTHEGO. CRISPR cRNP editing. cRNP editing of isolated NK cells was performed as previously published29,88. cRNP complexes were generated as follows. Synthetic sgRNA (120 pmol, Synthego), Alt-R electroporation enhancer (9 pmol, IDT), and water were added to a 1.5 mL tube per sample. In a separate tube, recombinant SpCas9 (20 pmol, SYNTHEGO) was diluted 1:5 with water, then diluted SpCas9 was added to sgRNA-enhancer mixture and incubated for 10 minutes at room temperature to make cRNP complexes. NK cells were electroporated with cRNP complexes using a Neon Transfection system (Thermo Fisher) with voltage 1900V, pulse width 20 ms, and 1 pulse. Electroporated cells were either incubated for 90 min at 37^C before collection and in vitro culture in complete medium or incubated for 10 min at 37^C before collection and adoptive transfer in vivo. Gene editing efficiency was evaluated by Sanger sequencing and immunoblot after 3 d of in vitro culture for mouse NK cells or 6 d for human NK cells. Cas9 base editing. Synthetic mRNA encoding adenine base editor ABE8e was produced by TriLink Genomics from plasmids provided by the Moriarity laboratory. Base editing of human NK cells was performed as previously described90. Isolated NK cells were electroporated with 5 ug ABE8e mRNA, Alt-R electroporation enhancer (20 pmol, IDT), Protector RNase inhibitor (diluted 1:5, Roche), and sgRNA (120 pmol, SYNTHEGO). Electoporation was performed using a Neon transfection system (Thermo Fisher) with voltage 1800V, pulse width 10 ms, and 2 pulses. Base editing efficiency was evaluated by Sanger sequencing after 6 d of in vitro culture. PCR and Sanger sequencing. PCR and Sanger sequencing to validate CRISPR gene editing was performed as previously published. DNA from isolated NK cells was isolated using PureLink Genomic Mini kit (Thermo Fisher) and DNA concentration was measured using a Nanodrop Eight Spectrophotometer (Thermo Fisher). 50 ng of isolated DNA per sample was amplified by PCR using custom primers surrounding a 500-1,000 bp region targeted by sgRNAs. Sanger sequencing of PCR samples for cRNP-edited or Cas9 base edited DNA was performed by Azenta and editing efficiency was analyzed using inference of CRISPR editing (ICE) (SYNTHEGO) for cRNP editing or EditR v1.0.10 for base editing. Proliferation assays. CellTrace Violet (CTV) (Thermo Fisher) stock solution was prepared according to manufacturer protocols and as previously described88. Stock CTV solution was diluted 1:10,000 in 37^C PBS and purified human NK cells were labeled for 20 min at 37°C protected from light then quenched with complete media for 5 min at 37°C. Cells were cultured for 6 days in complete NK MACS Media before surface staining and analysis by flow cytometry. Apoptosis assays. Annexin V and PI staining were performed according to manufacturer protocols (Thermo Fisher, V13242). Briefly, working solutions of FITC Annexin V and PI were prepared in 1x annexin-binding buffer. Cells were stained in 1:20 diluted stock FITC Annexin V and 100 ng PI for 15 min at room temperature protected from light before flow cytometry analysis. Tumor killing assays. For patient or CRISPR cRNP-edited human NK cells, isolated NK cells were cultured with GFP-expressing K562 cells at indicated effector to target ratios for 16 h at 37^C in the presence of recombinant human IL-2 (100 IU / mL, PeproTech) and recombinant human IL-15 (20 ng / mL, Peprotech). Frequency of remaining viable GFP+ cells after coculture was quantified by flow cytometry. For mouse CRISPR cRNP-edited NK cells, isolated NK cells were cultured with CTV- labeled ^2M- / -MC38 target cells for 16 h at an effector to target ratio of 2:1 in the presence of recombinant mouse IL-15 (50 ng / mL, Peprotech). Frequency of remaining viable CTV+cells after coculture was quantified by flow cytometry. Flow cytometry. Single cell suspensions were stained with fluorophore-conjugated antibodies (Biolegend, Thermo Fisher, eBioscience). Intracellular staining for cytokines was performed using the Cytofix / Cytoperm Fixation / Permeabilization kit (BD). Intracellular staining for lipids was performed using the eBioscience Foxp3 / Transcription Factor staining kit (Thermo Fisher). Intracellular staining for phosphorylated proteins was performed using 4% formaldehyde fixation followed by ice cold 100% methanol permeabilization and antibody staining following manufacturer recommendations. Flow cytometry was performed using an Attune NxT Acoustic Focusing cytometer (Thermo Fisher). Data were analyzed using FlowJo v.10.7.2 (TreeStar). The following fluorophore-conjugated antibodies were used: human CD3 (UCHT1), human CD19 (SJ25-C1), human CD14 (TuK4), human CD4 (RPA-T4), human CD8 (RPA-T8), human CD56 (TULY56), human CD16 (CB16), human CD57 (TB01), human IFN-^ (B27), human TNF-a (MAb11), human GzmB (GB11), human PRF (B-D48), human CD107a (H4A3), human LDLR (C7), human CD25 (BC96), human CD122 (TU27), human CD218a (H44), mouse CD45.1 (A20), mouse CD45.2 (104), mouse CD3 (17A2), mouse TCR^ (H57-597), mouse NK1.1 (PK136), mouse CD27 (LG.3A10), mouse CD11b (M1 / 70), mouse Ly49H (3D10), mouse KLRG1 (2F1), mouse IFN-^ (XMG1.2), mouse GzmB (GB11), mouse CD107a (1D4B), mouse CD25 (PC61), mouse CD122 (TM-B1), mouse CD218a (P3TUNYA), mouse / human phospho-S6 Ribosomal Protein (Ser235 / 236) (D57.2.2E), mouse / human phospho-AKT (D9E) (Ser473), mouse / human phospho-STAT1 (Tyr701) (A-2), mouse / human phospho-STAT5 (C71E5) (Tyr694), mouse / human phospho-STAT5 (C11C5) (Tyr694), goat anti-rabbit IgG H&L FITC (polyclonal). Staining for neutral lipids was performed using BODIPY 493 / 503 (500 ng / mL, Thermo Fisher). Lipid uptake was evaluated by uptake of BODIPY C12 (500 ng / mL, Thermo Fisher). Quantitative PCR. For quantitative PCR, RNA was isolated from NK cells using the Quick-RNA Micro-prep kit (Zymo). RNA concentration was measured using a Nanodrop Eight Spectrophotometer (Thermo Fisher) and complementary DNA (cDNA) was synthesized using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher). Undiluted cDNA was directly assessed in a SYBR Green PCR assay (Applied Biosystems) in a 384 well plate using a LightCycler 480. Each sample was plated in 5 technical replicates per PCR primer pair. Expression was normalized to b- actin housekeeping gene for each sample, then each experimental sample was normalized to paired donor control CRISPR cRNP-edited sample for each donor. Immunoblot analysis. Immunoblots were performed as previously published87,88. Cell lysates were prepared in Pierce RIPA buffer (Thermo Fisher) with HALT protease / phosphatase inhibitor (Thermo Fisher). Protein concentration was quantified using the Pierce BCA assay (Thermo Fisher). SDS-PAGE was performed on NuPage Novex 4-12% Bis-Tris protein gels (Thermo Fisher) in an XCell II blot module (Thermo Fisher), transferred to PVDF membranes (Millipore Sigma), and blocked overnight in 5% nonfat dry milk in 1x TBS and 0.1% Tween-20. Proteins of interest were detected using mouse anti-human MEF2C, rabbit anti-human B-actin (1:10,000 dilution; Cell Signaling CST4970), goat anti-mouse horseradish peroxidase (1:2,000 dilution, Thermo Fisher), and goat anti-rabbit horseradish peroxidase (1:10,000 dilution, Thermo Fisher). Proteins were detected with the SuperSignal West Pico PLUS ECL kit (Thermo Fisher) and visualized on an Azure Biosystems c280 imager. Respirometry. Respirometry studies were conducted in a Seahorse XFe96 Analyzer. All experiments were conducted at 37°C, and at pH 7.4. Rates of oxygen consumption and extracellular acidification of NK cells were measured in medium with 8 mM glucose, 2 mM glutamine, 2 mM pyruvate, and 5 mM HEPES. On the day of the experiment, cells were plated at 2.0 x 105or 2.5 x 105cells / well in Seahorse XFe96 microplates coated with Cell-Tak at a final concentration of 0.075 mg / mL. Plates were centrifuged at 500 g for 4 min. Respiration was measured in response to oligomycin (2.25 µM), FCCP (two sequential pulses of 0.5 µM), and rotenone (0.2 µM) with antimycin A (1 µM). Respiratory parameters were calculated as previously described91. Single-cell metabolic profiling (SCENITH). SCENITH was performed as previously published88,92. Briefly, 2.0 x 105purified human NK cells in complete NK MACS media were cultured with control (DMSO), 2-deoxyglucose (DG), oligomycin (O), or both DG and O for 4 hours at 37°C together with puromycin. After treatment, cells were stained for surface markers and intracellular staining for puromycin using a custom anti-puromycin antibody provided by the R. Argüello group. Metabolic dependencies were calculated based on geometric MFI of puromycin from inhibitor conditions as published92. RNA-seq library construction and sequencing. RNA was isolated from purified NK cells using the Quick-RNA Micro-prep kit (Zymo). Ribosomal RNA was depleted from samples using the QIAseq FastSelect -rRNA HMR kit (Qiagen). RNA libraries were prepared according to manufacturer instructions using the Stranded Total RNA Prep kit (Illumina, 20040525). Libraries were barcoded using the IDT for Illumina RNA UD Indexes Set A (Illumina, 20040553) and PCR-amplified libraries were cleaned up using Ampure XP beads (Beckman Coulter). Library quality and concentration were assessed by HSD1000 tape on 2200 Tapestation (Agilent) and Qubit 2.0 Fluorometer, and barcoded libraries were pooled to equimolar ratios for sequencing. Sequencing was performed on a NovaSeq 6000 system (Illumina). RNA-seq analysis. Bulk RNA-seq fastq files were first checked for quality using FastQC, then trimmed to remove low-quality reads and adaptors using Trimmomatic (version 0.40). The parameters used were SLIDINGWINDOW:4:20 MINLEN:40 LEADING:3 TRAILING:3. Then, reads were aligned to either the reference mouse genome (mm10) or the reference human genome (hg38) with STAR (version 2.7.11). STAR was run with default parameters and the –quantMode geneCounts parameter to generate ReadsPerGene.out.tab files, which are tab- delimited files with the raw number of reads mapped to each gene. The unstranded reads (column 2 of ReadsPerGene.out.tab) were combined into one counts matrix using Excel. Using DESeq2 (version 1.24.0) with default parameters, differential expression analysis was performed, and the counts matrix was normalized. All genes were filtered for significantly differentially expressed genes by p < 0.05. Only genes with counts per million (CPM) > 40 were used for differential expression analysis. The normalized counts matrix was used to calculate log2 fold change of differentially expressed genes (DEGs), and heatmaps were generated using the pheatmap package in R based on a |log2 fold change| cutoff of 0.5. Hierarchical clustering was performed using standard parameters in R v4.3.2. Volcano plots were generated using ggplot2 v3.4.4. Gene ontology analysis was performed using Gene Ontology and Panther Database with the DEGs as input. Gene set enrichment analysis was performed using the fgsea package v3.18 with input genes ranked by log2FC*-log10(p-value) and Hallmark human reference pathways h.all.v2023.2 obtained from MSigDB. The Benjamini-Hochberg false discovery rate (FDR) was used for multiple comparison correction of pathway analysis. DICE expression data analysis. Gene expression in FACS-sorted human peripheral immune cells was accessed from the Database of Immune Cell Expression, expression quantitative trait loci and Epigenomics (DICE) project93. Data collection and statistical analyses. For graphs, data are shown as mean ^ SEM, paired control and CRISPR cRNP-edited NK cells from the same human donor or mouse, or paired congenically distinct NK cells within the same host animal. Statistical differences were identified using an unpaired two-tailed Student’s t-test or paired t-test unless otherwise indicated in the figure legend. Kaplan-Meier survival curves were compared using the log rank (Manel-Cox) test with correction for testing multiple hypotheses. P < 0.05 was deemed significant. Graphs and statistical analyses were generated using Graphpad Prism 10. Data availability. Sequencing data are accessible from the GEO database under GSE245463 (reviewer access token: cjadcuiqvlcvhev). All other data are available in the main text, supplementary materials, or by request from the authors.

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[0003] 

[0004] T a e . Summary o c nca ea ures o C S pa ens. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. CONCLUSION This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS:

1. A method of modulating NK cell physiology, the method comprising combining a NK cell with a composition comprising oleic acid, wherein the composition comprises amounts of oleic acid selected to be sufficient to increase lipid content in the NK cell.

2. The method of claim 1, wherein the NK cell is selected to be a CD56dimCD16+NK cell.

3. The method of claim 1, wherein the composition comprises amounts of oleic acid selected to be sufficient to enhance NK cell cytotoxicity.

4. The method of claim 1, wherein the composition comprises amounts of oleic acid selected to be sufficient to enhance antiviral NK cell effector function.

5. The method of claim 1, wherein the NK cell is selected to be one comprising a mutation in the myocyte enhancer factor 2C (MEF2C) gene.

6. The method of claim 5, wherein the NK cell is obtained from or present in a patient diagnosed with MEF2C Haploinsufficiency Syndrome (MCHS).

7. The method of claim 6, wherein the NK cell is present in a patient diagnosed with MEF2C Haploinsufficiency Syndrome and the composition is administered to the patient nonparenterally.

8. The method of claim 1, wherein the composition further comprises a cytokine.

9. The method of claim 1, wherein the cytokine is IL-15.

10. The method of claim 1, wherein the composition comprises a pharmaceutically acceptable carrier.

11. The method of claim 1, wherein the composition comprises at least 50, 100 or 200 uM oleate.

12. A method of making a composition, the method comprising combining oleate with an NK cell, wherein amounts of oleate present in the composition are selected to be sufficient to increase lipid content in the NK cell.

13. The method of claim 12, amounts of oleic acid are selected to be sufficient to enhance NK cell cytotoxicity.

14. The method of claim 12, wherein the method further comprises combining the NK cell and oleate with a cytokine.

15. A composition made by the method of claim 12.

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