Abstract
Viral infection outcomes are sex-biased, with males generally more susceptible than females. Paradoxically, the numbers of anti-viral natural killer (NK) cells are increased in males compared to females. Using samples from mice and humans, we demonstrate that while numbers of male NK cells are increased compared to females, they display impaired production of the anti-viral cytokine IFN-γ. These sex differences were not due solely to divergent levels of gonadal hormones, since these differences persisted in gonadectomized mice. Instead, these differences can be attributed to lower male expression of X-linked Kdm6a (UTX), an epigenetic regulator which escapes X inactivation in female NK cells. NK cell-specific UTX deletion in females phenocopied multiple features of male NK cells, which include increased numbers and reduced IFN-γ production. Integrative ATAC-seq and RNA-seq analysis revealed a critical role for UTX in the regulation of chromatin accessibility and gene expression at loci important in NK cell homeostasis and effector function. Consequently, NK cell-intrinsic UTX levels are critical for optimal anti-viral immunity, since mice with NK cell-intrinsic UTX deficiency show increased lethality to mouse cytomegalovirus (MCMV) challenge. Taken together, these data implicate UTX as a critical molecular determinant of NK cell sex differences and suggest enhancing UTX function as a new strategy to boost endogenous NK cell anti-viral responses.
Introduction
Evolutionarily conserved sex differences exist in both innate and adaptive immune responses1, 2. While males are less susceptible to autoimmunity, they also mount a limited anti-viral immune response compared to females3. For instance, males have a higher human cytomegalovirus (HCMV) burden after infection, suggesting increased susceptibility to viral threats4. This has also been recently illustrated with the COVID-19 pandemic, in which the strong male bias for severe disease has been postulated to reflect sex differences in immune responses5. Multiple studies in humans and mice have recently reported differences in immune cell distribution and/or function in males vs. females6–10. However, the molecular basis for these differences, and the mechanisms by which these differences influence disease outcomes, remain incompletely understood.
Sex differences in mammals are defined not only by divergent gonadal hormones, but also by sex chromosome dosage1. Expression of a subset of X-linked genes, for example, is higher in females (XX) than males (XY). While females undergo random X chromosome inactivation (XCI) to maintain similar levels of X-linked protein expression between sexes, XCI is incomplete, with 3-7% of X chromosome genes escaping inactivation in mice and 20-30% escaping inactivation in humans11. As such, differential levels of X-linked gene expression in females vs. males have been linked to sex differences in a wide range of conditions including neural tube defects12 and autoimmune disease13.
As circulating type 1 innate lymphocytes, NK cells serve as an early line of defense against herpesvirus family members14. The importance of NK cells in anti-viral immunity is illustrated in patients with defective NK cell numbers or functionality, who are highly susceptible to infection by herpesviruses, such as HCMV and Epstein-Barr virus (EBV)15, 16. Similarly, NK cells are required for the control of mouse cytomegalovirus (MCMV) and other viral infections17–, 19, as mice with either genetic deficiency in NK cell function or loss of NK cell numbers have a significant increase in viral titers and mortality following MCMV infection20–25. Thus, NK cells are critical in anti-viral immunity across species.
Given this role for NK cells, it was therefore unexpected that NK cells are increased in virus-susceptible males6–10. Beyond NK cell numbers, other previously unappreciated sexually dimorphic NK cell feature(s) may instead account for sex differences with viral infections. Here, we show that NK cells in males are simultaneously expanded in number and deficient in effector function across mice and humans. These sex differences in NK cell composition and function are not completely due to hormonal differences, since these differences persisted in gonadectomized mice. Through expression screening, we identified the epigenetic regulator UTX (encoded by gene Kdm6a) as an XCI escapee that is expressed at significantly lower levels in male NK cells across mice and humans. Conditional ablation of UTX in female mouse NK cells, which mimics lower UTX expression in male NK cells, recapitulated NK cell phenotypes associated with male sex, such as increased NK cell numbers and lower production of IFN-γ. Furthermore, parallel Assay for transposase-accessible chromatin using sequencing (ATAC-seq) and bulk RNA sequencing (RNA-seq) of WT and UTXNKD NK cells revealed a critical role for UTX in regulating chromatin accessibility and transcription of gene clusters involved in NK cell fitness (Bcl2) and effector response (Ifng and Csf2). Notably, UTXNKD mice had increased mortality in response to MCMV infection, suggesting a critical role for UTX in the production of optimal anti-viral effector responses. Ultimately, our findings demonstrate that divergent UTX levels underlie sex differences in NK cell homeostasis and effector function. Enhancing UTX function may therefore represent a novel strategy for optimizing NK cell-mediated anti-viral immunity.
Results
NK cells display sexually dimorphic phenotypes independent of gonadal sex hormones
Due to the critical role of NK cells in anti-viral immunity and increased male susceptibility to cytomegalovirus (CMV) and other herpesvirus family infections4, it was surprising that multiple studies have reported that human males display increased NK cell numbers6–10. A recent investigation examining spleens of C57BL/6 mice also reported increased numbers of NK cells in males vs. females26. Consistent with this, our data show that splenic NK cells are increased in frequency (Fig. 1a,b) and absolute numbers (Fig. 1c) in male C57BL/6 mice compared to females. These findings suggest that other sexually dimorphic features beyond NK cell numbers may account for increased male susceptibility to viral infections. In response to viral infection, NK cells are critical for early production of proinflammatory cytokines, particularly IFN-γ27. To test if sex differences exist in NK cell-intrinsic function, we compared effector cytokine production in NK cells from female vs. male mice ex vivo. Stimulation with IL-12 and IL-18, cytokines known to induce robust IFN-γ production by NK cells28, resulted in lower IFN-γ production by male NK cells (Fig. 1d-f). Similarly, stimulation of activated human NK cells (TCRβ- CD3- CD56+) isolated from peripheral blood mononuclear cells (PBMCs) with IL-12 and K562 leukemia cells resulted in lower %IFN-γ+ (Fig. 1g) and IFN-γ MFI (Fig. 1h) in male compared to female NK cells. Thus, while NK cell numbers are increased, male NK cell effector function is consistently reduced in both mice and humans.
Female or male sex is based on a composite of gonadal hormones (e.g., estrogens or androgens) and sex chromosomes (e.g., 46XX or 46XY)1. Previous studies demonstrated direct effects of gonadal hormones in regulation of IFN-γ production by NK cells29, but it remains possible that NK cell sex differences can also be attributed to sex chromosome complement. To test this possibility, we gonadectomized mice to abolish the effect of sex hormones. Gonadectomy failed to eliminate sex differences in NK cell frequency (Fig. 1i), absolute numbers (Fig. 1j) and IFN-γ protein production in response to cytokine stimulation (Fig. 1k), indicating that gonadal hormones are not solely responsible for sex differences in NK cells. Thus, we hypothesized that chromosomal complement, in particular X chromosome dosage, may also play an important role.
X-linked UTX escapes X-inactivation and has higher expression in female NK cells
While 46XX females undergo X chromosome inactivation (XCI) to control dosages of X-linked genes, a subset of genes escapes XCI (termed XCI escapees), often resulting in higher expression in females compared to males. Thus, XCI escapees are prime candidates for mediating phenotypic sex differences in NK cells. Five genes (XIST, DDX3X, KDM6A, EIF2S3, KDM5C) have previously been identified as XCI escapees in both humans and mice30. XIST was excluded from further analysis because it is not expressed in male cells due to its known role in X chromosome inactivation in female cells1. All 4 remaining genes were significantly downregulated in male vs. female NK cells, from humans (Fig. 2a) and mice (Fig. 2b). The greatest differential expression in both human and mouse NK cells was seen with Kdm6a (also known as UTX) (Fig. 2a,b). Male NK cells also expressed lower UTX protein levels compared to female NK cells in mice (Fig. 2c,d). These data indicate that expression levels of Kdm6a (UTX) is sex-biased in NK cells.
In NK cells derived from gonadectomized mice, differences persisted in Kdm6a transcript levels (Fig. 2e) and UTX protein levels (Fig. 2f). Additionally, using the four core genotype (FCG) mouse model, which uncouples sex chromosome complement (XX or XY) and gonadal sex organ (ovaries or testes)31, Kdm6a transcript levels were also lower in mice with one X chromosome (XY) independent of gonadal composition (Extended Data Fig. 2a). Together these findings suggest that increased UTX expression in female mice is not due to hormonal effects and instead point to a primary role for X chromosome dosage.
UTX deletion recapitulates male NK cell phenotypes of frequency and IFN-γ production
To determine if UTX mediates sex differences in NK cells, we generated mice with a conditional deletion of UTX in NK cells (Kdm6afl/fl x Ncr1Cre+, referred to as UTXNKD hereafter) with WT (Kdm6afl/fl x Ncr1Cre-) littermates used as controls. To control for gonadal hormone and sex chromosome effects, comparisons were made only in female mice (female WT vs. female UTXNKD littermates). We confirmed decreased UTX protein expression in NK cells from UTXNKD mice using flow cytometry (Extended Data Fig. 2b,c). Similar to male mice, female UTXNKD mice displayed increased splenic NK cell frequency (Fig. 3a,b) and absolute numbers (Fig. 3c) in the spleen, blood, lungs, liver and bone marrow, demonstrating that this increase was not tissue specific. Furthermore, IFN-γ protein production in response to IL-12 and IL-18 stimulation was decreased in NK cells from UTXNKD vs. WT mice (Fig. 3d-f). These results implicate UTX in limiting NK cell numbers and promoting IFN-γ production, suggesting divergent UTX levels may play a causal role in NK cell sex differences.
Global changes in NK cell chromatin accessibility and transcription mediated by UTX
Recent studies have identified NK cell regulatory circuitry (regulomes) that prime innate lymphoid cells for swift effector responses even prior to NK cell activation32, 33. As an epigenetic modifier, UTX can alter transcription by organizing chromatin at regulatory elements of target gene loci34. To investigate the UTX-mediated modifications on chromatin accessibility and gene expression in NK cells, we performed Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in tandem with bulk RNA sequencing (RNA-seq) on sort-purified WT (CD45.1+) and UTXNKD (CD45.2+) NK cells from WT:UTXNKD mixed bone marrow chimeras (mBMCs) (Fig. 4a). Using mBMCs allowed for an internally controlled experiment to minimize environmental confounding factors. Principle Component Analysis (PCA) of both ATAC-seq and RNA-seq data revealed that samples clustered together by genotype (Fig. 4b), indicating that loss of UTX results in profound changes in both the chromatin landscape and transcriptome of NK cells. ATAC-seq revealed 3569 peaks decreased and 2113 peaks increased in accessibility in UTXNKD compared to WT NK cells (Log2 Fold Change > ±0.5, adjusted p-value < 0.05, FDR < 0.05) (Supplementary Table 1). Moreover, RNA-seq identified 577 decreased and 377 increased genes in UTXNKD vs. WT (Log2 Fold Change > ± 0.5, adjusted p-value < 0.05, FDR < 0.05) (Supplementary Table 2). Thus, these data suggest UTX plays an active role in controlling the NK regulome at baseline.
Integrative analysis of ATAC-seq and RNA-seq identified 400 genes that are both differentially accessible and expressed with a significant positive correlation (Spearman correlation: R = 0.62, p < 2.2×10-16) between the mean log2 fold change of ATAC-seq peaks and log2 fold change of RNA-seq expression (Extended Data Fig. 3a). Fuzzy c-means clustering35 of both the ATAC-seq and RNA-seq datasets identified six major clusters which were significantly decreased (Clusters 1, 2, 3, and 6) or increased (Clusters 4 and 5) in accessibility (Fig. 4c) and expression (Fig. 4d) in UTXNKD NK cells. For functional enrichment analysis, g:Profiler36 was used to analyze clusters of differentially expressed genes identified by RNA-seq (Fig. 4e). Major pathways such as immune system process, cytokine production, IFN-γ production, lymphocyte activation, and immune effector process were associated with decreased expression in UTXNKD (Clusters 1, 2, 3, and 6) (Fig. 4e). At the same time, pathways such as developmental process, biosynthetic process, and metabolic process were significantly associated with increased expression in UTXNKD (Clusters 4 and 5) (Fig. 4e). Collectively, these findings implicate UTX in the coordinate regulation of genes associated with NK cell homeostasis and effector function.
Furthermore, UTX is known to interact with transcription factors (TFs) to coordinate target gene transcription34. To identify putative UTX TF partners, we performed HOMER (Hypergeometric Optimization of Motif Enrichment)37 TF motif analysis on each cluster of significant differentially accessible peaks (Fig. 4f). TFs associated with modulating effector function during viral infection such as Runt (Runx1 and Runx2)38 and T-box (Eomes, T-bet, Tbr1 and Tbx6)39 family TFs were more significant and had a higher percentage of target motifs associated with clusters displaying decreased accessibility in UTXNKD (Clusters 1, 2, 3, and 6) (Fig. 4f). Conversely, TFs associated with proliferation, differentiation, and metabolism in the zinc finger family TFs (KLF1, KLF5, KLF6, KLF14, Sp2 and Sp5)40 were more significantly associated with clusters displaying increased accessibility (Clusters 4 and 5) (Fig. 4f). These data suggest that UTX poises the chromatin accessibility of several genes at steady state known to influence NK cell fitness and effector responses, while also controlling genome-wide accessibility of transcription factor binding sites implicated in these processes.
UTX coordinately regulates chromatin accessibility and expression of apoptosis pathway genes
The observed expansion of NK cell numbers in UTXNKD mice (Fig. 3a-c) could either be due to higher NK cell proliferation or increased resistance to apoptosis. However, UTXNKD NK cells paradoxically displayed a lower proportion of cells expressing the cell division marker Ki67 (Extended Data Fig. 4a). UTXNKD NK cells also showed less CFSE dilution in response to IL-15, a cytokine known to induce NK cell proliferation (Extended Data Fig. 4b,c). Thus, these results suggest that higher NK cell numbers observed in UTXNKD mice were not due to increased proliferation. In contrast, increased survival is likely the cause of expanded NK cell numbers in UTXNKD mice.
Among the differentially accessible and expressed genes in NK cells lacking UTX were those important in controlling apoptosis. An anti-apoptotic gene, Bcl2, showed increased accessibility and expression in UTXNKD vs. WT NK cells (Fig.5a, Extended Data Fig. 3b).
Previous studies in mice have demonstrated that Bcl-2 can inhibit the pro-apoptotic function of Bim to promote NK cell survival41, thus, we interrogated the expression of these proteins in UTXNKD NK cells. While naïve UTXNKD NK cells showed increased intracellular protein expression of Bcl-2 compared to WT NK cells (Fig. 5b,d), UTX-deficient NK cells also displayed a modest increase in intracellular Bim levels (Fig. 5c,e). Importantly, the Bcl-2:Bim ratio was significantly higher in UTXNKD NK cells (Fig. 5f), suggesting UTX-deficiency likely results in a higher proportion of pro-survival proteins present in NK cells. Notably, male NK cells also displayed a significant increase in Bcl-2:Bim ratio, which may underlie the expanded NK cell numbers observed in male mice (Fig. 5g). These results implicate UTX in regulation of NK cell fitness to restrict numbers at homeostasis.
UTX is critical for NK cell IFN-γ production and effector function
Since NK cells are early responders to MCMV, rapid effector molecule production is critical for NK cell mediated anti-viral control. ATAC-seq and RNA-seq of NK cells from WT:UTXNKD mBMCs identified various chromatin accessibility and transcript changes at steady state (Fig. 4b,c). Specifically, genes involved in the NK effector response (Ifng and Csf2)27, 42 showed decreased accessibility and expression in UTXNKD compared to WT NK cells (Fig. 6a, Extended Data Fig. 3b-d). Moreover, qRT-PCR verified decreases in Ifng transcript levels in UTXNKD vs. WT NK cells at rest (Fig. 6b). Similarly, male NK cells, which express lower levels of UTX (Fig.2c,d), also had a similar decrease in Ifng transcript levels at rest (Fig. 6c). Thus, through shaping of the chromatin landscape, UTX controls levels of effector gene transcripts available prior to immune challenge.
To determine whether UTX deficiency also led to decreased chromatin accessibility and transcription of effector genes in NK cells during infection, we performed ATAC-seq on NK cells isolated from MCMV-infected WT:UTXNKD mBMCs on D1.5 post-infection (PI). Similar to naïve UTX-deficient NK cells, D1.5 PI UTXNKD NK cells also showed decreased chromatin accessibility at the Ifng and Csf2 loci (Fig. 6d,e, Extended Data Fig. 3e,f). qRT-PCR confirmed decreased Ifng and Csf2 transcripts in NK cells from UTXNKD mice at D1.5 PI (Fig. 6f). Similarly, UTXNKD NK cells showed decreased IFN-γ protein expression in UTXNKD NK cells on D1.5 PI indicating that UTX expression in NK cells is required for optimal IFN-γ production following viral infection (Fig. 6g,h). To confirm whether dosage of UTX expression in mature NK cells associates with NK cell production of IFN-γ during viral infection, we generated transgenic mice to achieve tamoxifen-inducible UTX deletion (Rosa26ERT2CRE/+ x Kdm6afl/fl: iUTX-/-). Tamoxifen administration in WT:iUTX-/- mBMCs resulted in differential degrees of UTX protein loss, and displayed a positive correlation between intracellular UTX levels and IFN-γ production on D1.5 PI (Extended Data Fig. 5). Since IFN-γ production by NK cells is critical for protection against MCMV27, we challenged WT and UTXNKD mice with a sublethal dose of MCMV and monitored survival. While WT mice controlled MCMV infection (n = 8/8 survived), UTXNKD mice rapidly succumbed to infection (n = 3/8 survived) (Fig. 6i). These results demonstrate a requirement for NK cell-intrinsic UTX in the control of effector molecule production and protection against MCMV infection.
Discussion
Sex is a critical biological variable in determining outcomes to viral infections3. This was recently illustrated with COVID-19, in which male sex was identified as a major risk factor for severe disease5. Moreover, recent studies have linked NK cell dysfunction within severe COVID-19 disease43. Given the importance of NK cells in anti-viral immunity, understanding the root causes of sex differences in NK cell biology will have far-reaching implications in optimizing anti-viral immunity. In this study, we demonstrated that lower expression of UTX in XX UTXNKD NK cells mimics levels in XY NK cells, which may contribute to increased NK cell numbers and decreased IFN-γ production in males (Extended Data Fig. 6a). UTX is expressed at lower levels in male NK cells across mice and human and this observation is independent of gonadal hormones in mice. NK cell UTX is required for controlling NK cell fitness, modulating accessibility of transcription factor binding motifs, increasing chromatin accessibility at effector gene loci, and poising NK cells for rapid response to virus infection (Extended Data Fig. 6b). Together, these findings support a model in which divergent UTX expression contributes to sex differences in NK cell numbers and effector function.
Our findings indicate that UTX restricts NK cell numbers at steady state, since NK cells are increased at baseline in UTXNKD mice. This is in contrast to UTX deficiency in other immune cell types, which have been reported to result in moderate (CD8+ and CD4+ T cells) or severe (iNKT) decreases in peripheral cell numbers44–46. Interestingly, T cell-specific UTX-deficiency is associated with CD8+ T cell accumulation during viral infection. Thus, it is possible that UTX-mediated gene programs that inhibit CD8+ T cell numbers during inflammation are shared by NK cells at rest46. Indeed, increased Bcl-2 levels were observed in both UTX-deficient NK cells and UTX-deficient CD8+ T cells, suggesting that UTX down-regulates this anti-apoptotic factor in both innate and adaptive cytotoxic lymphocytes.
NK cell-mediated effector functions during viral infection include cytokine production (IFN-γ) and cytotoxic molecule expression47. Our results from simultaneous ATAC-seq and RNA-seq suggest that UTX poises the chromatin landscape of NK cells at rest to quickly respond to viral challenge by increasing accessibility and transcription of effector loci such as Ifng and Csf2 prior to viral infection27, 42. Decreased IFN-γ protein levels were seen at D1.5 post MCMV-infection in UTX-deficient NK cells, suggesting decreased effector functionality during inflammation. These results support a previous study that suggests the existence of an NK cell regulome27, 42, in which NK cell chromatin accessibility is actively maintained at steady-state and demonstrates a critical role for UTX in its maintenance before and during inflammation.
As a histone demethylase, UTX has intrinsic catalytic ability to demethylate H3K27me3 (a repressive histone mark) to poise chromatin for active gene expression48. In addition to its catalytic activity, UTX functions in multiprotein complexes with other epigenetic regulators (e.g. SWI/SNF, MLL4/5 and p300) to mediate chromatin remodeling in a demethylase-independent manner48, 49. In CD8+ T cells, UTX binds to enhancer and TSS of effector genes to promote effector gene programs in a demethylase-independent manner46. In contrast, demethylase activity in iNKT cells is required for the development and function and H3K27 methylation correlated with gene programs important for CD4+ T follicular helper cell development44, 50. Thus, the molecular mechanisms by which UTX functions appears to be immune cell specific. A previous study treated human NK cells with a small molecule inhibitor of H3K27me3 demethylases (GSK-J4) and found reduced cytokine expression (IFN-γ, TNF-α, GM-CSF, and IL-10) in response to in vitro stimulation51. However, GSK-J4 is not a specific tool for studying UTX-mediated mechanisms because it also inhibits Jmjd3, another H3K27me3 demethylase as well as having non-specific effects on other histone demethylases52. Thus, further studies using more precise genetic modification strategies are needed to understand the mechanisms by which UTX functions in NK cells.
UTX has been reported to interact with lineage specific TFs in T cells to target particular effector loci34. Our studies using HOMER motif analysis revealed potential interactions between UTX and TFs associated with modulating NK cell effector function during viral infection (Runx1, Runx2, Eomes, T-bet, Tbr1, and Tbx6). Moreover, these analyses also point to UTX interactions with TFs associated with NK cell proliferation, differentiation, and metabolism (KLF1, KLF5, KLF6, KLF14, Sp2 and Sp5). In line with a physiologic role for TFs in UTX-mediated control of chromatin accessibility, TF binding motifs were strongly enriched in ATAC-seq gene clusters. Further studies will be needed to experimentally verify these interactions in mouse and human NK cells.
NK cells are critical for control of HCMV infection in humans because NK cell-deficient individuals develop disseminated herpesvirus infections16, 53. Sex differences in immune response to multiple viruses have been reported, including immune responses to HCMV53. Our data support a model in which sex differences in anti-viral immunity can partially be explained by differences in UTX expression in NK cells, given the increased susceptibility of UTXNKD mice to MCMV challenge. UTX deficiency has also been associated with Kabuki Syndrome and Turner Syndrome44, 54, two human conditions associated with immune dysregulation and increased infections. Our findings suggest the possibility that UTX deficiency in human NK cells may contribute to decreased viral immunosurveillance observed in these patients, although future work will be needed to support this hypothesis.
Weighing factors that define patient subsets with different immune responses will allow us to move past a “one-size-fits-all” therapeutic approach to a precision medicine paradigm. Understanding sex differences in NK cell function and their molecular underpinnings is an important step toward incorporating sex as a biological factor in treatment decisions. In males with severe viral illness, for instance, enhancing NK cell UTX activity may provide therapeutic benefit. We expect that these insights will be important not only in the setting of viral infections, but also in other infections and cancer, where NK cells also play an important role. These findings may also have important implications for adoptive cellular therapies, in which NK cells are the subject of intense interest55, 56.
Author 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., R.T.Y., F.M., and M.P. performed bioinformatics analysis; M.A.S, M.I.C. and T.E.O. wrote the manuscript.
Competing Interests Statement
T.E.O. is a scientific advisor for Xyphos Inc., a company that has financial interest in human NK cell-based therapeutics.
Contact for Reagent and Resource Sharing
Further information and requests for resources and reagents should be directed and will be fulfilled by the Corresponding Authors, Timothy O’Sullivan (tosullivan{at}mednet.ucla.edu) and Maureen Su (masu{at}mednet.ucla.edu)
Method Details
Mice
Mice were bred at UCLA in accordance with the guidelines of the institutional Animal Care and Use Committee (IACUC). The following mouse strains were used in this study: C57BL/6 (CD45.2) (Jackson Labs, #000664), B6.SJL (CD45.1) (Jackson Labs, #002114), Rosa26ERT2Cre, Ncr1Cre, Kdm6afl/fl and FCG mice. For experiments with gonadectomy, procedure was performed by Jackson Laboratories Surgical Services. For experiments in UTXNKD mice, only female mice were used to control for Y chromosome and sex hormone independent effects. Thus, experiments were conducted using 6-8 week old age-matched females in accordance with approved institutional protocols. For comparisons between male and female WT, we used 6-8 weeks age-matched littermates. Mixed bone marrow chimeras (mBMCs) were generated by depleting host CD45.1 x CD45.2 mice by intraperitoneal (i.p.) injection of busulfan (1mg/mL) at 20mg/kg for 3 consecutive days, which were then reconstituted 24 hours later with various mixtures of bone marrow cells from WT (CD45.1) and knockout (CD45.2) donor mice in the presence of an anti-NK1.1 antibody (at 1 mg/ml; clone: PK136) to deplete any remaining mature NK cells.
MCMV infection
MCMV (Smith) was serially passaged through BALB/c hosts three times, and then salivary gland viral stocks were prepared with a dounce homogenizer for dissociating the salivary glands of infected mice 3 weeks after infection. Experimental mice in studies were infected with MCMV by i.p. injection of 7.5 x 103 plaque-forming units (PFU) in 0.5 mL of PBS. Mice were monitored and weighed daily and sacrificed when body weight dropped over 20% from initial weight.
Isolation and enrichment of mouse NK cells
Mouse spleens, livers, lungs, and blood were harvested and prepared into single cell suspensions as described previously57. Splenic single cell suspensions were lysed in red blood cell lysis buffer and resuspended in EasySep™ buffer (Stemcell). To avoid depleting Ly6C+ NK cells we developed a custom antibody cocktail as follows: splenocytes were labeled with 10 μg per spleen of biotin conjugated antibodies against CD3 (17A2), CD19 (6D5), CD8 (53-6.7), CD88 (20/70), Ly6G (1A8), SiglecF (S17007L), TCRβ (H57-597), CD20 (SA275A11), CD172a (P84) and magnetically depleted from total splenocyte suspensions with the use of anti-biotin coupled magnetic beads (Biolegend)58.
Ex vivo stimulation of lymphocytes
∼5 x 105 NK cells were stimulated for 5 hours in complete RPMI media (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, 0.01% 55 mM 2-mercaptoethanol), Brefeldin A (1:1000; BioLegend) and Monensin (2uM; BioLegend) with or without recombinant mouse IL-12 (20 ng/ml; Peprotech) and recombinant mouse IL-18 (10ng/ml; Peprotech). Cells were cultured in complete RPMI media alone as a negative control (No Treatment or NT).
Human NK cell culture and Stimulation
Human peripheral blood mononuclear cells (PBMCs) from anonymous healthy donors were obtained from leukoreduction filters after platelet apheresis from the UCLA Virology Core. NK cells were isolated using the EasySep Human NK Cell Isolation Kit (Stem Cell Technologies) following manufacturer instructions. Following isolation, cells were maintained in 24-well G-Rex plates (Wilson Wolf) in NK MACS media (Miltenyi Biotech) supplemented with human IL-2 (100 IU/mL, Peprotech) and human IL-15 (20 ng/mL, Peprotech) at a plating density of 5×106 cells per well. For cytokine stimulation, 14 d IL-2/IL-15 activated human NK cells were plated with K562 leukemia cells at an effector:target (E:T) ratio of 2.5:1 in addition to human IL-2 (100 IU/mL, Peprotech), human IL-15 (20 ng/mL, Peprotech), human IL-12 (10 ng/mL, Peprotech), and/or human IL-18 (100 ng/mL, Peprotech) in complete RPMI media (Thermo Fisher). NK cells were stimulated with cytokines for 16 h before analysis by flow cytometry.
Proliferation assays
CellTrace™ CFSE (Thermo) stock solution was prepared per the manufacturers’ instructions and diluted at 1:10,000 in 37C PBS. Isolated NK cells were incubated in 0.5mL of diluted CFSE solution for 5 minutes at 37C. The solution was quenched with 10X the volume of complete RPMI media. Cells were then washed and plated with 50 ng/ml of recombinant mouse IL-15 (Peprotech) and cultured for 4 days to assess proliferation. Flow cytometry was used to quantify CFSE dilution 4 days post-stimulation. Samples were compared to levels of CFSE labeled on Day 0. FlowJo’s proliferation tool was used to model and measure number of divisions in addition to expansion, division, and proliferation indices.
Flow Cytometry and Cell Sorting
Cells were analyzed for cell-surface markers using fluorophore-conjugated antibodies (BioLegend, eBioscience). Cell surface staining was performed in FACS Buffer (2% FBS and 2 mM EDTA in PBS) and intracellular staining was performed by fixing and permeabilizing using the eBioscience Foxp3/Transcription Factor kit for intranuclear proteins or BD Cytofix/Cytoperm kits for cytokines. Flow cytometry was performed using the Attune NxT Acoustic Focusing cytometer and data were analyzed with FlowJo software (TreeStar). NK cells were identified as CD3- TCRβ- NK1.1+ cells: see gating strategy in Extended Data Fig. 1. Cell surface and intracellular staining was performed using the following fluorophore-conjugated antibodies: CD45.1 (A20), CD45.2 (104), NK1.1 (PK136), TCRβ (H57-597), CD3 (17A2), IFN-γ (XMG1.2), Ly6C (HK1.4), BCL2 (BCL/10C4), UTX (N2C1 - GeneTex), Goat anti-rabbit H&L (Abcam - ab6717), BIM (c34c5), CD90 (30-H12), Ki-67 (16A8). Isolated splenic NK cells were sorted using Aria-H Cytometer to > 95% purity.
RNA-seq and ATAC-seq library construction and analysis
RNA was isolated from the cells using RNeasy Mini kit (Qiagen) and used to generate RNA-seq libraries followed by sequencing using Illumina HighSeq 4000 platform (single end, 50bp). The reads were mapped with HISAT2 (version 2.2.1) to the mouse genome (mm10). The counts for each gene were obtained by HtSeq59, in print, online at doi:10.1093/bioinformatics/btu638). Differential expression analyses were carried out using DESeq260 (version 1.24.0) with default parameters. Genes with adjusted p value <0.05 were considered significantly differentially expressed. Sequencing depth normalized counts were used to plot the expression values for individual genes.
ATAC-seq libraries were produced by the Applied Genomics, Computation, and Translational Core Facility at Cedars Sinai in the following manner: 50,000 cells per sample were lysed to collect nuclei and treated with Tn5 transposase (Illumina) for 30 minutes at 37°C with gentle agitation. The DNA was isolated with DNA Clean & Concentrator Kit (Zymo) and PCR amplified and barcoded with NEBNext High-Fidelity PCR Mix (New England Biolabs) and unique dual indexes (Illumina). The ATAC-Seq library amplification was confirmed by real-time PCR, and additional barcoding PCR cycles were added as necessary while avoiding overamplification. Amplified ATAC-Seq libraries were purified with DNA Clean & Concentrator Kit (Zymo). The purified libraries were quantified with Kapa Library Quant Kit (KAPA Biosystems) and quality assessed on 4200 TapeStation System (Agilent). The libraries were pooled based on molar concentrations and sequenced on an Illumina HighSeq 4000 platform (paired end, 100bp).
ATAC-seq fastq files were trimmed to remove low-quality reads and adapters using Cutadapt61 (version 2.3). The reads were aligned to the reference mouse genome (mm10) with bowtie262 (version 2.2.9). Peak calling was performed with MACS263 (version 2.1.1). The peaks from all samples were merged into a single bed file, peaks from different samples that were closer than 10bp were merged into a single peak. HTseq59 (version 0.9.1) was used to count the number of reads that overlap each peak per sample. The peak counts were analyzed with DESeq260 (version 1.24.0) to identify differentially accessible genomic regions. Peaks with adjusted p-value < 0.05 were considered significantly differentially accessible. The peak counts were visualized with Integrated Genome Browser, (version 9.1.8).
Fuzzy c-means clustering was used for both ATAC-seq and RNA-seq using significant (p-value and FDR <0.05, Log2FC +/- 0.5) normalized counts generated from DESeq2. MFuzz package (version 3.14) within R was used to perform this analysis into 6 clusters with a membership score of >0.5. The differentially accessible ATAC peaks were analyzed using the findMotifsGenome.pl function from HOMER37 (version 4.9.1) of each cluster to identify enriched cis-regulatory motifs of transcription factors. Pathway analysis of clustered RNA-seq data was performed using g:Profiler using the g:GOSt function. Top relevant pathways were selected from KEGG Biological Pathways and Gene Ontology Pathways (Biological Processes and Molecular Function).
Statistical Analyses
For graphs, data are shown as mean ± SEM, and unless otherwise indicated, statistical differences were evaluated using a Student’s t-test. For Kaplan-Meir survival curve, samples were compared using the Log-rank (Mantel-Cox) test with correction for testing multiple hypotheses. A p-value < 0.05 was considered significant. Graphs were produced and statistical analyses were performed using GraphPad Prism and ggplot2 library in R. Spearman Correlation on best fit regression line was performed using ggpubr library in R.
Data Availability
Sequencing datasets are accessible from GEO with accession number GSE185065. Data can be accessed by reviewers using the access token ilqlqswavvqhtir.
Acknowledgements
We thank members of the O’Sullivan and Su labs for helpful discussion. We thank the UCLA Technology Center for Genomics and Bioinformatics for RNA sequencing library preparation and the Cedars Sinai Applied Genomics, Computation, and Translational Core Facility for ATAC sequencing library preparation. T.E.O. is supported by the NIH (AI145997) and UC CRCC (CRN-20-637105). 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 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). A.P.A. is supported by NIH HD100298.
Footnotes
Figure 1f and 1k Revised.