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The X-linked epigenetic regulator UTX controls NK cell-intrinsic sex differences

Nature Immunology volume 24, pages 780–791 (2023)Cite this article

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Abstract

Viral infection outcomes are sex biased, with males generally more susceptible than females. Paradoxically, the numbers of antiviral natural killer (NK) cells are increased in males. We demonstrate that while numbers of NK cells are increased in male mice, they display decreased effector function compared to females in mice and humans. These differences were not solely dependent on gonadal hormones, because they persisted in gonadectomized mice. Kdm6a (which encodes the protein UTX), an epigenetic regulator that escapes X inactivation, was lower in male NK cells, while NK cell-intrinsic UTX deficiency in female mice increased NK cell numbers and reduced effector responses. Furthermore, mice with NK cell-intrinsic UTX deficiency showed increased lethality to mouse cytomegalovirus. Integrative multi-omics analysis revealed a critical role for UTX in regulating chromatin accessibility and gene expression critical for NK cell homeostasis and effector function. Collectively, these data implicate UTX as a critical molecular determinant of sex differences in NK cells.

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Fig. 1: Sex differences in IFN-γ production and NK cell numbers are independent of gonadal hormones.
Fig. 2: X-linked UTX displays sexually dimorphic gene expression independent of sex hormones.
Fig. 3: UTX suppresses NK cell fitness.
Fig. 4: UTX enhances NK cell effector function and is required for survival against viral infection.
Fig. 5: UTX controls NK cell homeostasis and IFN-γ production independent of demethylase activity.
Fig. 6: Global changes in NK cell chromatin accessibility and transcription mediated by UTX.

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Data availability

Sequencing datasets are accessible from the Gene Expression Omnibus under accession number GSE185065. Source data are provided with this paper. Further information and requests for resources and reagents should be directed and will be fulfilled by the corresponding authors.

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Acknowledgements

We thank members of the T.E.O. and M.A.S. laboratories for helpful discussion. We thank the UCLA Technology Center for Genomics and Bioinformatics for RNA-seq library preparation and the Cedars Sinai Applied Genomics, Computation, and Translational Core Facility for ATAC-seq library preparation. T.E.O. is supported by the National Institutes of Health (NIH; AI145997) and UC CRCC (CRN-20-637105). We thank the blood donors and UCLA/CFAR Virology Core Laboratory for providing human PBMCs for study, funded by UCLA CFAR grant 5P30 AI028697. We thank M. Lechner for use of a BioRender license to produce the schematic in Fig. 6. M.A.S. is supported by the NIH (NS107851, AI143894, DK119445), Department of Defense (USAMRAA PR200530) and National Organization of Rare Diseases. M.I.C. is supported by Ruth L. Kirschstein National Research Service Awards (GM007185 and AI007323), and a Whitcome Fellowship from the Molecular Biology Institute at UCLA. L.R. is supported by the Warsaw fellowship from the MIMG department at UCLA. J.H.L. is supported by the NIH NIAMS (T32AR071307) and the UCLA Medical Scientist Training Program (NIH NIGMS T32GM008042). A.P.A. is supported by the NIH (HD100298).

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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

    Mandy I. Cheng, Joey H. Li, Luke Riggan, Bryan Chen, Rana Yakhshi Tafti, Scott Chin, Timothy E. O’Sullivan & Maureen A. Su

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

    Mandy I. Cheng, Joey H. Li, Luke Riggan, Rana Yakhshi Tafti, Timothy E. O’Sullivan & Maureen A. Su

  3. Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA

    Luke Riggan, Feiyang Ma & Matteo Pellegrini

  4. Institute for Genomics and Proteomics, University of California Los Angeles, Los Angeles, CA, USA

    Feiyang Ma & Matteo Pellegrini

  5. Department of Integrative Biology & Physiology, Laboratory of Neuroendocrinology of the Brain Research Institute, University of California Los Angeles, Los Angeles, CA, USA

    Haley Hrncir & Arthur P. Arnold

  6. Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Maureen A. Su

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Contributions

M.I.C., L.R., J.H.L., R.Y.T., H.H., A.P.A., T.E.O. and M.A.S. designed the study; M.I.C., J.H.L., R.Y.T., L.R. and S.C. performed the experiments; M.I.C., F.M., B.C. and M.P. performed bioinformatics analysis; M.I.C., M.A.S., J.H.L. and T.E.O. wrote the paper.

Corresponding authors

Correspondence to Timothy E. O’Sullivan or Maureen A. Su.

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Competing interests

T.E.O. is a scientific advisor for Modulus Therapeutics and Xyphos companies that have financial interest in human NK cell-based therapeutics. The other authors declare no competing interests.

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Nature Immunology thanks Mihalis Verykokakis, Edith Heard, and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the rest of the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Sex differences in IFN-γ production in response to IL-12/18.

a) Representative dot plots showing gating strategy to identify CD3− TCRβ- NK1.1+ mouse NK cells. b) Representative contour plots, (c) percentage IFN-γ+, and (d) normalized IFN-γ MFI of female and male WT NK cells with cultured no treatment (NT) or IL-12 (20 ng/mL) and IL-18 (10 ng/mL) for 4 hours (n = 14 per group). e) Representative contour plots of CD3−CD56+ female (n = 6) and male (n = 7) human NK cells cultured and stimulated with 10 ng/mL of IL-12 for 16 hours in the presence of K562 cells. f) Representative dot plots of splenic NK cells (CD3−TCRβ- NK1.1+) in gonadectomized female and male mice. g) Representative contour plots of total splenic NK cells isolated from gonadectomized female and male mice and cultured with no treatment (NT) or IL-15 (50 ng/mL) and IL-12 (20 ng/mL) for 4 hours. h) Representative contour plots and i) percentage CD27−CD11b− (DN), CD27+CD11b− (CD27 SP), CD27+CD11b+ (DP), and CD27−CD11b+ (CD11b SP) of total splenic NK cells from female and male mice (n = 7 per group). j) Representative contour plots and k) percentage CD27−CD11b− (DN), CD27+CD11b− (CD27 SP), CD27+CD11b+ (DP), and CD27−CD11b+ (CD11b SP) of total splenic NK cells from gonadectomized mice (n = 6 per group). Data are representative of 2-3 independent experiments. Samples were compared using unpaired two-tailed Student’s t test and data points are presented as individual mice with the mean ± SEM (N.S., Not Significant; **, p < 0.01; ****, p < 0.0001). Specific p-values are as follows: c:[NT = 0.987; IL-12+IL-18 = 0.00125]; d:[NT = 0.4144; IL-12+IL-18 < 0.0001]; i:[DN > 0.999; CD27 SP = 0.9514; DP = 0.8995; CD11b SP = 0.6517]; k:[DN = 0.0951; CD27 SP = 0.225; DP = 0.161; CD11b SP = 0.789].

Source data

Extended Data Fig. 2 UTX expression in Four Core Genotypes mice and maturation in UTX mouse models.

a) Relative expression of Kdm6a (encodes the protein UTX) by RT-qPCR in Four Core Genotypes mice, in which male or female gonads present are independent of XX or XY chromosome composition, normalized to female WT expression (XX with ovaries) (XX ovaries: n = 4; XY ovaries: n = 5; XX testes: n = 3; XY testes: n = 5). b) Relative UTX MFI in splenic NK cells isolated from female WT (n = 19), male WT (n = 19), female UTXHet (n = 7), and female UTXNKD (n = 4) mice. c) CD11b and CD27 expression within NK cells isolated from female WT (n = 13), male WT (n = 7), and female UTXHet (n = 8) mice. d) Representative western blot showing protein expression of UTX in NK cells isolated from female WT and UTXNKD mice compared to β-actin loading control. Data are representative of 2-3 independent experiments. Samples were compared using a) unpaired two-tailed Student’s t test, b,c) one-way ANOVA with Tukey’s correction for multiple comparisons. Data points are presented as individual mice with the mean ± SEM (N.S., Not Significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Specific p-values are as follows: a:[Ovaries – XX vs. XY < 0.0001; Testes – XX vs. XY = 0.0016]; b:[F WT vs. M WT = 0.003; F UTXNKD vs. F UTXHet and F WT vs. F UTXNKD < 0.0001; M WT vs. F UTXHet = 0.7435; F UTXHet vs. F WT = 0.0004]; c > 0.999.

Source data

Extended Data Fig. 3 UTX regulates NK cell fitness.

a) Representative flow cytometry plots of splenic NK cells from female WT, male WT, and female UTXHet, and female UTXNKD mice. b) Representative contour plots and c) percent of WT(CD45.1+) or UTXNKD(CD45.2+) cells of total splenic T (left) and NK (right) cells in 1:1 WT:UTXNKD mBMC mice (n = 3). d) Percentage of Ki67+ cells in blood of 4:1 WT:UTXNKD mBMCs (n = 28), injection ratio used to normalize WT and UTXNKD NK cell numbers. e) Representative histogram (left) and quantification (right) of CFSE expansion, division and proliferation indices calculated using FlowJo’s Proliferation tool of CFSE-labeled splenic NK cells isolated from WT and UTXNKD mice stimulated ex vivo with IL-15 (50 ng/mL) for 4 days (n = 4). f) Schematic showing adoptive transfer of CTV-labeled congenically distinct WT (CD451x2) and UTXNKD (CD45.2+) NK cells transferred into WT (CD45.1+) recipients at a 1:1 ratio with analysis of CTV dilution and WT:UTXNKD ratio on D7 by flow cytometry. g) Representative histograms showing CTV dilution of congenically distinct WT (CD451x2) and UTXNKD (CD45.2+) NK cells transferred into WT (CD45.1+) recipients before transfer (left) and on day 7 post-transfer (right). h) Representative histograms and i) percentage of cleaved caspase 3+ splenic NK cells from female WT and male WT treated ex vivo with IL-15 (5 ng/mL) and DMSO (Female WT: n = 7; Male WT: n = 8) or 2.5 uM Nutlin-3a (Female WT: n = 3; Male WT: n = 4) for 24 hours. j) Representative histograms and k) percentage of cleaved caspase 3+ splenic NK cells from gonadectomized female and male mice (n = 6) treated ex vivo with IL-15 (5 ng/mL) and DMSO or 2.5uM Nutlin-3a for 24 h. l) Representative histograms of Bcl-2 (left) and Bim (right) of flow cytometry in splenic NK cells from female WT and UTXNKD mice (n = 5). “Ctrl”:unstained flow cytometry controls. Data are representative of 2-3 independent experiments. Samples were compared using unpaired two-tailed Student’s t test with Welch’s correction and data points are presented as individual mice with the mean ± SEM (N.S., Not Significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001). Specific p-values are as follows: c:[T cells=0.705; NK Cells=0.0202]; d < 0.0001; e:[Expansion=0.0192; Division=0.0253; Proliferation=0.0032]; i:[DMSO = 0.0096; Nutlin-3a = 0.0473]; k:[DMSO = 0.001; Nutlin-3a = 0.0013].

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Extended Data Fig. 4 UTX enhances effector function independent of gonadal hormone and maturation.

a) Representative contour plots of total NK cells from female WT, male WT, female UTXHet, and female UTXNKD mice cultured with IL-15 (50 ng/mL) and IL-12 (20 ng/mL) for 4 h. b) Absolute number of IFN-γ+ NK cells from female WT, male WT, and female UTXHet mice stimulated with no treatment (NT) or IL-15 (50 ng/mL) and IL-12 (20 ng/mL) for 4 h (n = 8). c) Specific lysis of MHC Class I deficient MC38 (Target) cells by female WT (n = 4), male WT (n = 5), and gonadectomized female (n = 3) and male (n = 6) NK cells for 16 h at 4:1 effector:target ratio, normalized to lysis by female WT. d) Representative histograms and e) MFI of CD107a (n = 5 per group), granzyme b (GzmB) (Female:n = 5; Male:n = 4), and perforin (n = 5) of female WT and male WT NK cells incubated with IL-15 (50 ng/mL) only or additionally stimulated with plate-bound anti-NK1.1 antibody (PK136). “Ctrl” refers to matched unstained control for flow cytometry. Representative f) contour plots of IFN-γ and g) histogram of GzmB expressing total splenic NK cells on D1.5 post MCMV infection of 4:1 WT:UTXNKD mixed bone marrow chimeras (mBMCs), ratio used to normalize cell numbers between genotypes. h) IFN-γ protein production in UTXNKD compared to WT NK cells within maturation subsets: CD27−CD11b− (DN), CD27+CD11b− (CD27 SP), CD27+CD11b+ (DP), and CD27−CD11b+ (CD11b SP) isolated from 4:1 WT:UTXNKD mBMC 1.5 days post-MCMV infection (n = 6). i) Representative contour plots of total NK cells derived from 1:1 WT:iUTX-/- mBMC mice on day 1.5 post MCMV infection, normalized to WT (n = 6). Data are representative of 2-3 independent experiments. Samples were compared using paired two-tailed Student’s t test and data points are presented as individual mice with the mean ± SEM (N.S., Not Significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Specific p-values are as follows: b:[F WT vs. M WT = 0.0162; M WT vs. UTXHet = 0.968; F WT vs. F UTXHet = 0.0288]; c:[F WT vs. F Gonadectomy=0.245; F WT vs. M WT = 0.0086; M WT vs. M Gonadectomy=0.998; F Gonadectomy vs. M Gonadectomy=0.0247]; e:[CD107a – NT = 0.54 and NK1.1 = 0.772; GzmB – NT = 0.0004 and NK1.1 = 0.00129; Perforin – NT = 0.0101 and NK1.1 < .0001]; h:[DN = 0.03281; CD27 SP = 0.0186; DP = 0.0231; CD11b SP = 0.0114].

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Extended Data Fig. 5 UTY is expressed but not sufficient to compensate for loss of UTX in NK cell homeostasis and effector function.

a) Expression in transcripts per million of KDM6A (encodes the protein UTX) and KDM6C (encodes the protein UTY) using DICE database RNA-seq data on sorted NK cells from human females (n = 36) vs. males (n = 54). b) Relative expression of Kdm6a (encodes the protein UTX) and Kdm6c (encodes the protein UTY) by RT-qPCR in splenic NK cells isolated from female WT, male WT, and gonadectomized female and male mice (n = 6 per group). c) Representative flow cytometry dot plots and quantification of d) frequency and absolute numbers of NK cells in spleen of male WT and UTXNKD mice (n = 8 per group). e) Representative flow cytometry contour plots and quantification of f) percentage IFN-γ + and normalized IFN-γ MFI of total NK cells from male WT vs. male UTXNKD mice with no treatment (NT) or in response to IL-15 (50 ng/mL) and IL-12 (20 ng/mL) stimulation for 4 hours ex vivo, MFI normalized to male WT (n = 8 per group). g) Representative flow cytometry contour plots and quantification of h) percentage IFN-γ+ and normalized IFN-γ MFI of total NK cells from male WT vs. male UTXNKD mice with no treatment (NT) or in response to IL-12 (20 ng/mL) and IL-18 (10 ng/mL) stimulation for 4 hours ex vivo, MFI normalized to male WT (n = 8 per group). Data are representative of 2-3 independent experiments. Samples were compared using paired two-tailed Student’s t test and data points are presented as individual mice with the mean ± SEM (N.S., Not Significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Specific p-values are as follows: a < 0.0001; b:[UTX - Female vs. Male=0.0008; rest<0.0001]; d:[%NK = 0.0003; No. NK = 0.002]; f:[%IFN-γ+- NT = 0.112; 15 + 12 = 0.00133; IFN-γMFI – NT = 0.145; 15 + 12 < 0.0001]; h[%IFN-γ+- NT = 0.112; 12 + 18 = 0.00135; IFN-γMFI – NT = 0.155; 12 + 18 < 0.0001].

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Extended Data Fig. 6 Integrative ATAC, RNA, anti-UTX CUT&Tag sequencing analysis reveal concomitant changes in chromatin accessibility and transcription mediated by UTX.

a) Schematic of mBMC generated by 1:4 WT (CD45.1+) and UTXNKD (CD45.2+) bone marrow into a lymphodepleted host (CD451x2). After 6wks of reconstitution, splenic NK cells were sorted for ATAC-seq and RNA-seq library preparation. b) Principal component analysis (PCA) of (left) ATAC-seq and (right) RNA-seq changes in WT and UTXNKD NK cells. c) Volcano plot of differentially expressed genes by RNA-seq plotted by Log2FC of UTXNKD vs. WT (x-axis) and Log2 Mean Expression (y-axis). Dotted lines represent Log2FC cut offs (0.5 and 1). Red dots are genes in NK cell effector and developmental pathways. d) Scatter plot highlighting differentially accessible and expressed genes (FDR and p-value<0.05) colored by fuzzy c-means cluster (see Fig. 6). Mean log2FC of ATAC accessibility peaks (y-axis) and log2FC (x-axis) of RNA-seq transcript levels. Best fit regression line (red) with standard error (light red ribbon) (SEM). Positive correlation calculated by two-tailed Spearman correlation of dataset (R = 0.62, p < 2.2×1016). e) Heatmap displaying expression of cell death genes between WT and UTXNKD NK cells FDR < 0.05, adjusted p-value<0.05, and log2FC > 0.5. f) PCA analysis of anti-UTX CUT&Tag in sort-purified WT and UTXNKD NK cells (n = 3). g) Pathway analysis on UTX-bound genes that are decreased (red) or increased (blue) by expression by RNA-seq using Enrichr (p-value by Fisher’s exact test). h) Log2FC in UTXNKDvs.WT of ATAC accessibility (y-axis) plotted by either decreased (>-0.5Log2FC) (blue) or increased (>+0.5Log2FC) (purple) expression by RNA-seq (x-axis) of the UTX bound genes with significant accessibility and expression differences. i) Correlation plot of Log2FC of UTXNKDvs.WT RNA-seq values compared to corresponding ATAC-seq values for each UTX-bound gene. Linear regression was performed (black line) with the standard error 95% confidence intervals plotted (dotted red lines). Two-tailed Pearson’s correlation was performed (r = 0.5165; p-value<0.0001). j) HOMER motif analysis of ATAC-seq peaks grouped by transcription factor family (top) and transcription factor (bottom). Point size indicates percentage of target sequences featuring motif and red gradient indicates -log10(p-value) of enrichment. k) HOMER Motif analysis performed on UTX-bound peaks. % Target Sequences refers to percent of target motifs identified by the HOMER algorithm out of the background motifs. j,k) Cumulative binomial distribution statistical analysis was performed.

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Supplementary Data Tables

Supplementary Data Table 2: Table of the differentially accessible peaks between WT and UTX-NKD NK cells. Descriptive statistics are provided. DAG analysis was performed using a two-sided Wilcoxon rank sum test. Supplementary Data Table 3: Table of the differentially expressed genes between WT and UTX-NKD NK cells. Descriptive statistics are provided. DEG analysis was performed using a two-sided Wilcoxon rank sum test. Supplementary Data Table 4: Table of anti-UTX CUT&Tag peaks in WT and UTX-NKD NK cells.

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Unprocessed blots for the protein ladder, UTX, and actin from Extended Data Fig. 2d.

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Cheng, M.I., Li, J.H., Riggan, L. et al. The X-linked epigenetic regulator UTX controls NK cell-intrinsic sex differences. Nat Immunol 24, 780–791 (2023). https://doi.org/10.1038/s41590-023-01463-8

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