Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development

This article is featured in In This Issue, p.373

The low-affinity FcγRIIIA (or CD16a) is an activating Fc receptor expressed by NK cells, macrophages, and monocytes. It is coded by the gene FCGR3A. Surface expression of CD16a is required for Ab-dependent cell-mediated cytotoxicity (ADCC). The efficacy of several clinically effective antitumor Abs involves the engagement of CD16a, which is required for ADCC (1–5). CD16a expression is acquired during NK cell maturation, which can be described in five sequential stages involving NK cell developmental intermediates (NKDIs) (6). Stages 1–3 are considered progenitor or immature stages and do not express CD16a. Stages 4 and 5 NK cells are considered mature. Expression of CD16a divides stage 4 (early mature) and stage 5 (late mature) NK cells (7–9). The NK cell population in secondary lymphoid tissues (SLTs) is primarily composed of CD16a⁻ NKDIs, including stage 4 NK cells, which are characterized by the CD56^bright/CD16a⁻ surface phenotype. SLTs also contain a minor population that is CD56⁺/CD16a⁺ and corresponds to a stage 5 NK cell phenotype. In the peripheral blood, this pattern is reversed: CD56^bright/CD16a⁻ stage 4 NK cells are the minor population, whereas CD56^dim/CD16a⁺ stage 5 NK cells are the predominant population. This suggests that CD56^bright/CD16a⁻ NKDIs develop in SLTs before full maturation and traffic into the periphery as stage 5 NK cells (7). Although the function of this receptor has been well characterized (3, 10–18), and its posttranslational regulation has been described (19), the mechanism(s) of FCGR3A regulation permitting the developmental acquisition of CD16a is not understood.

The lack of knowledge regarding FCGR3A regulation during human NK cell development is due, in part, to inherent difficulties in studying this gene. Cell lines expressing CD16a are notably lacking (20). The closest murine genes, Fcgr3 and Fcgr4, have limited homology (<50%) to human FCGR3A. Furthermore, in humans, a recent genomic segmental duplication event generated two genes, FCGR3A and FCGR3B, that retain a very high degree of sequence homology (>95%) across the gene locus (21). Abs that recognize CD16 generally recognize both gene products. Discrimination of these two homologs is highly relevant to immunological studies, because FCGR3B cannot mediate ADCC and instead functions as a sink for immune complexes (21, 22).

Despite their nearly identical genomic sequences, FCGR3 homologs are selectively expressed by specific cell types; FCGR3A is expressed by NK cells, monocytes, and macrophages, whereas FCGR3B is expressed by neutrophils (21). Previous work has shown that each FCGR3 homolog uses two distinct alternative promoters within its respective 5′-region to transcribe at least two unique transcripts (23, 24). In FCGR3A, both promoters confer selective activity in the NK cell line YT, whereas the FCGR3B promoter alternatively operates myeloid cells, indicating that lineage-specific factors are capable of selectively recognizing sequence differences between FCGR3 homologs (23, 24). However, the mechanism that endows this exquisite specificity and how it selectively develops in separate primary cell lineages are not understood.

To gain insight into mechanisms that might regulate FCGR3A, we focused our studies on NK cells. Epigenetic gene regulation is capable of tightly controlling gene expression in a developmental- and tissue-specific manner (25, 26) and may help to explain the tissue specificity, despite the very high degree of homology between FCGR3A/B. NKDIs do not quickly acquire CD16a surface expression, often requiring a week or more of expansion in vitro before significant CD16a expression is detectable by flow cytometry (27, 28). As the cells acquire CD16a expression, some level of posttranscriptional fine-tuning may also be required. To address this possibility, we further sought to identify microRNA (miRNA) regulators of FCGR3A.

In this article, we identify two types of regulation of CD16a in human NK cells: DNA methylation of the FCGR3A promoter and miR-218 targeting of FCGR3A mRNA. These mechanisms suggest that CD16a expression in repressed in stage 4 NK cells, primarily by DNA methylation silencing with concurrent high miR-218 expression. The time required to transition from stage 4 to stage 5 may be necessary to sufficiently modify the FCGR3A promoter methylation patterns and downregulate miR-218, culminating in robust CD16a expression in stage 5 NK cells.

All human cell work was performed with approval of The Ohio State University Institutional Review Board. Human NK cells were isolated from peripheral blood leukopaks of healthy individuals (American Red Cross) by negative selection with a MACSxpress NK Cell Isolation Kit, human (Miltenyi Biotec). Enriched cells were collected and labeled for FACS sorting. For DNA isolation of CD16⁻ and CD16⁺ NK cells, we gated on lymphocyte, followed by CD3⁻CD56⁺ gating, and then sorted for CD56^brightCD16⁻ or CD56^dimCD16⁺ populations, respectively. NK cells were sorted to >95% purity. Human neutrophils were enriched with CD66abce magnetic beads by positive selection (Miltenyi Biotec). Enriched cells were labeled for FACS with CD15 and CD16 Abs. For DNA isolation, we gated on the CD15⁺CD16⁺ population. Cells were sorted to >97% purity.

The following Abs were used to stain human peripheral blood cells: CD3 (SK7; BD Biosciences), CD14 (TÜK4; Miltenyi Biotec), CD15 (VIMC6; Miltenyi Biotec), CD16 (VEP13; Miltenyi Biotec), CD16 (3G8; BD Biosciences), and CD56 (N901; Beckman Coulter). Flow cytometry data were analyzed with FlowJo v7.6.1 (TreeStar).

YT (American Type Culture Collection [ATCC]), K562 (ATCC), and Jurkat (German Collection of Microorganisms and Cell Cultures) cells were cultivated in RPMI 1640/10% FBS (Life Technologies) and supplemented with Antibiotic-Antimycotic (Thermo Fisher Scientific). NKL cells were cultivated in RPMI 1640/10% FBS (Life Technologies) and supplemented with Antibiotic-Antimycotic (Thermo Fisher Scientific) and 150 IU/ml recombinant human IL-2 (rhIL-2) (Roche). HEK293T cells were obtained from the ATCC. HEK293T cells were cultured in DMEM/10% FBS (Life Technologies) and supplemented with antibiotics.

DNA was isolated using a Puregene Core Kit B (QIAGEN). One microliter of molecular-grade glycogen (Thermo Fisher Scientific) was added to each sample, and DNA was allowed to precipitate overnight at −20°C, followed by resuspension in water. DNA methylation analysis of the CD16 loci was carried out using the MassARRAY EpiTYPER assay (Agena Biosciences) (29). In short, genomic DNA was subjected to bisulfite treatment using an EZ DNA methylation kit (Zymo Research). Regions of interest were amplified with PCR primers specific for the FCGR3A or FCGR3B gene loci (primers are listed in Supplemental Table I) using the hg19 genome assembly from the UCSC Genome Browser (https://genome.ucsc.edu) (30). PCR products were transcribed in vitro and fragmented with RNase A (Agena Biosciences) to generate oligonucleotides that were subsequently analyzed via MALDI-TOF mass spectrometry. Ratios of unmethylated versus methylated oligonucleotide signals were used to calculate the percentage of DNA methylation for individual CpG dinucleotides. To examine the specificity of FCGR3A and FCGR3B assays, amplified sequences included non-CpG sequence variants that produced quantifiable differences in the mass spectra. Primers used for the final analysis were determined to be highly specific.

Luciferase reporter assays were carried out as described previously (31). Sequences of the FCGR3A and FCGR3B 5′ untranslated regions (UTRs) were amplified using PCR and primers specific for each gene (listed in Supplemental Table I) and inserted into the CpG-free reporter vector pCpGfree-promoter-Lucia (InvivoGen). Sequences were verified with Sanger Sequencing at the Plant-Microbe Genomics Facility (The Ohio State University). For methylation-specific assays, plasmids were methylated in vitro using CpG Methyltransferase (M.SssI) (Thermo Fisher Scientific). Luciferase assays were carried out in HEK293T cells using TransIT-LT1 Transfection Reagent (Mirus Bio), according to the manufacturer’s instructions. Jurkat, YT, and K562 cells were transfected with a Nucleofector device (Lonza) using an Amaxa Cell Line Nucleofector Kit V with programs X-001, O-017, and T-016, respectively. Luciferase activity was assessed 48 h after transfection on a DTX880 Multimode Detector (Beckman Coulter). Luciferase signals were normalized to cotransfected pGl4–CMV–firefly luciferase vector (Promega). Luciferase activity values were displayed relative to an unmodified pCpGfree-promoter-Lucia plasmid.

Total RNA from the indicated cell types was extracted using the Total RNA Purification Plus Kit (Norgen Biotek). For miRNA analysis, we used an nCounter miRNA Expression Assay (NanoString Technologies) interrogating a panel of 800 miRNAs. The microarray data presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo) under accession number GSE106469. miRNA expression was also determined using TaqMan MicroRNA Assays (Thermo Fisher Scientific). cDNA from mRNA was generated using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) or SuperScript III reverse transcriptase (cell lines). Real-time PCR was done using Power SYBR Green Master Mix reagent (Thermo Fisher Scientific) and a ViiA 7 Real-Time PCR System (Applied Biosystems). Relative gene expression was calculated as 2^−Δct, with Δct = (ct target − ct housekeeping gene), where ct stands for threshold cycle. Data were individually normalized to housekeeping gene expression values of β-actin, GAPDH, TATA box binding protein, and hypoxanthine phosphoribosyltransferase 1; the average of the four normalized expression values was used per sample. Quantitative assessment of the ratio of FCGR3A/FCGR3B expression was performed using the MassARRAY iPLEX application (Agena Biosciences) designed for the accurate measurement of allele-specific expression (32). Briefly, five primer sets were used to amplify cDNA from purified CD16⁺ NK cells and neutrophils that targeted FCGR3A and FCGR3B equally, but flanked at least one discriminatory nucleotide (primers are listed in Supplemental Table I). Oligonucleotides were annealed to the PCR products immediately 5′ to the discriminatory nucleotide, followed by single-base extension. Mass spectrometry was used to determine the ratio of FCGR3A/FCGR3B sequences; the reported values represent the average of the five assays.

Infections were modified from a previously published protocol (33). Briefly, low-passage (5–20) HEK293T cells were transfected with pCDH CD511B-1 (System Biosciences) and two packaging plasmids containing VSV-G and gag/pol/tat/rev. pCDH expression vectors contained no insert or mir-218-2 plus 200 bp upstream and downstream. Virus media were concentrated by centrifugation (20,000 × g). Human NK cells isolated by FACS sorting were cultured overnight in RPMI 1640/5% human AB serum/Antibiotic-Antimycotic supplemented with 450 IU/ml rhIL-2. NK cells were infected with virus in 96-well round-bottom plates at a multiplicity of infection of 2–10. Plates were centrifuged for 2 h at 1800 rpm at 32°C and placed in a 37–C incubator overnight. After resting the cells in virus-free media, NK cells were subjected to a second round of infection with virus overnight. The next day, virus media were removed again and replaced with RPMI 1640/5% human AB serum/Antibiotic-Antimycotic supplemented with 450 IU/ml rhIL-2, and cells were cultured for 48 h. NK cells were stained, sorted for live GFP⁺ cells, and used for RNA expression assays.

NK cells acquire CD16a as part of their maturation process; less mature NK cells tend to be found in SLTs such as the lymph nodes and more mature NK cells tend to be found in the peripheral blood. To study the acquisition of CD16a by NK cells, we sorted NK subpopulations that precede and succeed this process and obtained CD16a⁻ and CD16a⁺ subpopulations at >95% purity (Fig. 1). We next assessed DNA methylation of the proximal promoter region of FCGR3A in these NK cell subsets. Because FCGR3B displays a different tissue-specific expression pattern, yet a high degree of promoter sequence homology compared with FCGR3A, we included both genes in our analysis to identify important regulatory features. FCGR3A and FCGR3B genes are separated by ∼81 kb on chromosome 1q23.3 and show nearly identical exon/intron architectures and promoter sequences (Fig. 2A). Both genes are annotated in the RefSeq database as being potentially expressed from at least two alternative transcriptional start sites: transcript variants 1 and 3 for FCGR3A and variants 1 and 2 for FCGR3B (with variants 3 and 2 being homologous between FCGR3A and FCGR3B, respectively). There are two promoters annotated for FCGR3A: proximal promoter (Pprox)-A (−198 to −10 bp) and medial promoter-1 (Pmed1)-A (−942 to −850 bp) (23, 24). Corresponding regions in FCGR3B were termed Pprox-B and Pmed1-B. To separately interrogate the profile of DNA methylation across both genes, we used the quantitative MassARRAY system and PCR primers that exploit sequence variations between FCGR3A and FCGR3B genes. Analysis of primary sorted CD16a⁺ and CD16a⁻ NK cell subsets revealed that CD16a⁺ NK cells show lower methylation levels within the FCGR3A 5′-region compared with CD16a⁻ NK cells (Fig. 2B). CpGs exhibiting the greatest difference between subsets were focused in Pmed1-A and immediate downstream sequences. The Pprox-A region was partially unmethylated in CD16a⁻ NK cells, whereas the region in the vicinity of Pmed1-A only showed low methylation in CD16a⁺ NK cells. FCGR3B was highly methylated throughout the entire 5′ region in CD16a⁻ and CD16a⁺ NK cells, consistent with selective expression of FCGR3A in the NK lineage (21). We next investigated the NK cell lines YT and NKL, which are negative and positive for FCGR3A mRNA, respectively. YT cells generally showed high methylation of FCGR3A (and FCGR3B), whereas NKL cells showed lower levels of methylation in FCGR3A, including CpGs immediately proximal and within Pmed1-A (Fig. 2C). These data show that pronounced lower levels of DNA methylation are found at the FCGR3A promoter versus the FCGR3B promoter in CD16a⁺ NK cells, and differential surface expression of CD16a is associated with reduced methylation of CpGs surrounding the Pmed1-A promoter.

To further investigate the role of DNA methylation in the selective expression of FCGR3A, we sorted primary neutrophil samples, which selectively express FCGR3B (21). We found that CpGs in the FCGR3A promoter that selectively demonstrated low methylation in CD16a⁺ NK cells were highly methylated in neutrophils, whereas two regions of low methylation in FCGR3B were observed (Fig. 2D). These regions corresponded to Pprox-B and CpGs adjacent to, but not within, Pmed1-B. Similar to CD16a⁻ NK cells, neutrophils also displayed low methylation of Pprox-A within FCGR3A, despite both cell types lacking expression of FCGR3A. These data show that, in neutrophils, the selective expression of FCGR3B is associated with a decrease in FCGR3B methylation, primarily at Pprox-B, corresponding to the known promoter usage (34), and, like CD16a⁻ NK cells, low methylation of Pprox-A is not associated with expression.

To demonstrate the importance of methylation differences between transcriptional start sites, we next evaluated the expression of the transcript variants within FCGR3A and FCGR3B genes in primary cells. Because the 5′ exon sequences of FCGR3A and FCGR3B are virtually identical, the sequence identity between genes does not allow the discrimination of variant expression from each gene promoter individually. Thus, we first ensured that the relative expression of FCGR3A versus FCGR3B in our samples matched reported cell type–specific expression patterns. For this, we measured the expression of FCGR3A relative to FCGR3B using a highly quantitative and accurate MassARRAY method for determining transcript expression ratios (32). We confirmed that CD16a⁺ NK cells are virtually only expressing FCGR3A, and neutrophils are expressing only FCGR3B (Fig. 2E). Next, using quantitative RT-PCR primers that are specific for variant 1 (FCGR3A and FCGR3B) or variants 3 (FCGR3A) and 2 (FCGR3B), we found that CD16a⁺ NK cells are strongly expressing variant 3 (and variant 1, to some extent); inversely, neutrophils are strongly expressing variant 1 and a small amount of variant 2 (Fig. 2F). Taken together, these data show that primary CD16a⁺ NK cells primarily use the Pmed1-A promoter, whereas neutrophils mainly use Pprox-B, similar to previous observations in cell lines (23).

To functionally evaluate the role of DNA methylation in the control of the selective activity of FCGR3A and FCGR3B variant promoters, the proximal 5′ region from each gene was split into four sections and cloned individually into a promoter-less luciferase-expressing plasmid devoid of CpG dinucleotides (Fig. 3A). Luciferase plasmids were transfected into YT cells (NK lineage), K562 cells (myeloid lineage), and into Jurkat and HEK293T cells as negative controls for non–CD16a-expressing lineages. As found previously (23, 24, 35), the Pmed1-A region, but not Pmed1-B, was selectively activated in YT cells (Fig. 3B). K562 cells selectively activated Pmed1-B, but not Pmed1-A. Other cloned regions did not possess inherent promoter activity in either lineage, including Pprox promoters that are known to require additional flanking enhancer sequences (23, 24). Importantly, in vitro methylation of Pmed1 sequences abolished their activity in both lineages (Fig. 3B), corroborating the findings in primary cells that DNA methylation of Pmed1-A is likely involved in the regulation of CD16a expression in the NK lineage.

To identify the basis for lineage-specific expression of Pmed1, we aligned Pmed1-A and Pmed1-B sequences and found that variants are strongly enriched at CpG dinucleotides. Differences generally disrupt CpGs in FCGR3B versus FCGR3A, including CG > TG transitions at CpG1 and CpG2 and an 8-bp indel that disrupts CpG6 (Fig. 3C). To test whether these CpGs are involved in selective regulation, we introduced C > T mutations for CpG1 and CpG2 in Pmed1-A FCGR3A, which enhances the likeness toward FCGR3B, and then retested these regions in our luciferase-based expression assay (Fig. 3D). Mutation of these CpGs abolished selective activity in YT cells. Remarkably, these mutations permitted Pmed1-A to be expressed in K562 cells, because increased likeness toward FCGR3B in the pattern of CpG sequences was associated with increased activity in K562 cells. This suggests that, in addition to the regulatory function provided by CpG methylation, the CpGs themselves are required to direct selective expression of FCGR3A versus FCGR3B.

To identify miRNA regulators of FCGR3A, we followed the strategy described by Witkos et al. (36). We compiled a list of 53 potential miRNA regulators by using TargetScan, DIANA, PITA, and mirSVR prediction tools. Separately, we obtained a miRNA expression array of 800 miRNAs in mature human peripheral blood NK cell populations defined as CD56^brightCD94^high and CD56^dimCD94^low, which overlap significantly with CD56^bright/CD16a⁻ and CD56^dim/CD16a⁺ (Supplemental Fig. 1) (37). We screened for miRNAs that were highly expressed in CD16a⁻ NK cells and poorly expressed in CD16a⁺ NK cells. We next cross-referenced this list with the list of miRNAs compiled from the prediction tools and identified three miRNAs: miR-92a, miR-133a, and miR-218 (Fig. 4A). Each of these miRNAs has putative recognition sites in the 3′UTR of FCGR3A: miR-92a at UTR positions 255–262, miR-133a at UTR positions 458–471, and miR-218 at UTR positions 843–850 (Fig. 4B). We found that this pattern could be validated by real-time PCR in freshly sorted CD16a⁻ and CD16a⁺ primary NK cells for miR-133a and miR-218, but not for miR-92a (Fig. 4C).

To determine whether these putative miRNA regulators could interact with the 3′UTR of FCGR3A mRNA, we performed luciferase assays. We cloned the entire 3′UTR of FCGR3A into the luciferase vector pmirGLO downstream of the firefly luc gene, which is driven by a constitutively active promoter. Following cotransfection with an expression vector for one of each of the candidate miRNAs, we found that miR-218 showed a trend toward negative regulation of luciferase activity (Fig. 4D).

To validate the activity of miR-218 against FCGR3A, we overexpressed miR-218 in primary human NK cells by lentiviral infection. Transduced cells were sorted based on GFP expression and analyzed by flow cytometry for CD16 expression (Supplemental Fig. 2A). The average transduction efficiency (percentage of GFP⁺) was 58.0 ± 23.9% for empty vector and 13.8 ± 9.2% for miR-218 (Supplemental Fig. 2B). We found that miR-218 overexpression resulted in decreased CD16a surface expression in NK cells infected with miR-218 virus compared with control virus (Fig. 5A, 5B). We confirmed that miR-218 virus-infected cells did overexpress miR-218 (Fig. 5C) and also found that this overexpression inversely correlated with FCGR3A mRNA expression (Fig. 5D).

In this article, we describe two important modes of epigenetic control of CD16a expression in primary NK cells that involve regulation at the transcriptional and posttranscriptional levels. Previous articles have described that Pmed1-A promotes specific expression of FCGR3A in NK cells (23, 24, 35); however, sequence features alone cannot fully account for the lineage- and stage-specific expression in primary cells. In our study, Pmed1-A and the immediate surrounding sequences were found to be the focal point of reduced methylation in CD16a⁺ versus CD16a⁻ NK cells, as well as in the CD16a⁺ NK cell line NKL. Reduced Pmed1-A methylation was associated with expression of the FCGR3A variant 3 transcript, which initiates from Pmed1-A. Interestingly, Pprox-A displayed low methylation, regardless of CD16a expression, indicating that the initiation of FCGR3A variant 1 requires additional factor(s) that are not active in CD16a⁻ NK cells. Indeed, previous work has shown that Pprox-A requires an enhancer element for activity (23, 24, 35). Based on the coordinated expression of FCGR3A variants 1 and 3, we propose that the unmethylated Pmed1-A may provide this function and enhance the expression of variant 1. In further support of the role of DNA methylation in the repression of FCGR3 genes, we made the novel observation that focally reduced methylation of Pprox-B was specifically associated with FCGR3B variant 1 expression in neutrophils. The lack of activity of Pprox-B in K562 luciferase experiments suggests that Pmed1-B may operate as an enhancer for Pprox-B; however, one cannot exclude that K562 cells are lacking the necessary transcriptional machinery to activate this promoter. Together, these findings indicate that a lineage-specific reduction in DNA methylation of key regulatory elements within the FCGR3A and FCGR3B promoter regions permits cell type–specific CD16a versus CD16b expression.

The activity state of a particular promoter or genomic element is dependent upon two primary factors: the epigenetic structure, which controls accessibility, and the presence of specific interacting trans-activating factors. We observed that the activity of Pmed1-A and Pmed1-B was restricted to YT and K562 cells, respectively, supporting the specific recognition of these sequences in NK and myeloid lineages, as found previously (23, 24, 35). These experiments reveal that YT cells, being of the NK lineage, possess the transcriptional machinery to selectively activate exogenous Pmed1-A; however, YT cells do not express endogenous FCGR3A, in concordance with the observation of high endogenous Pmed1-A methylation. Indeed, in vitro methylation of Pmed1-A silenced its activity. Because YT cells represent an earlier stage of NK development (38), our findings suggest that the transcriptional machinery required may already be active in earlier stages of NK cell development. We propose that Pmed1-A methylation prevents interaction with the transcriptional machinery and that Pmed1-A demethylation may function as the switch for CD16a expression during the transition from stage 4 to stage 5 NK development.

All sequence variations between Pmed1-A and Pmed1-B involve CpG dinucleotides. Interestingly, a greater number of CpGs are retained in the Pmed1-A region compared with Pmed1-B, concomitant with the greater importance of Pmed1-A for FCGR3A than for FCGR3B in NK cells. Conversely, the Pprox-B promoter that is primarily used by neutrophils retains two CpGs not present in Pprox-A. Remarkably, mutation of one or two variant CpGs in Pmed1 was sufficient to disrupt the lineage specificity of wild-type Pmed1-A and Pmed1-B sequences. These findings highlight the dual importance of CpGs in the Pmed1 region, functioning as an off switch when methylated and concurrently endowing selective activity when unmethylated.

In our survey of miRNA expression in peripheral blood–derived NK cells, we found that the majority of differentially expressed miRNAs were more highly expressed in the mature CD16a⁺ NK cell population than in the less mature CD16a⁻ population. This greatly narrowed our search for putative direct negative regulators of FCGR3A mRNA. For three miRNAs, we observed consistent, but modest, differences in miRNA expression between CD16a⁻ and CD16a⁺ NK cells. Direct validation of miR-218 expression in CD16a⁺ and CD16a⁻ primary NK cells demonstrated significant differences in relative expression levels. This difference in scale may be due, in part, to differences in experimental technique, as well as to partly overlapping populations. CD56^bright/CD94^hi NK cells correspond in many ways to CD16a⁻ stage 4 NK cells, but they have some level of CD16a (9, 37). This would explain the modest differences in miRNA expression between NK populations in the expression array and the stark differences in the direct validation by quantitative PCR using CD16a⁻ and CD16a⁺ NK cells. In addition to the difference in miR-218 expression between CD16a⁺ and CD16a⁻ NK cells, we further observed direct antagonism of miR-218 against CD16a with overexpression studies in primary NK cells. These results suggest that a layer of miRNA regulation functions in conjunction with FCGR3A methylation–based silencing, potentially for the purpose of controlling spurious transcription that may occur despite promoter repression.

In summary, we propose a system of regulation of FCGR3 homologs that confers profound cell-type specificity, despite minimal differences in regulatory sequences, and facilitates strong and fine regulation of FCGR3A expression in NK cells. We have found that well-defined genetic elements with distinct regulatory functions cooperate with epigenetic modifications to ultimately control the cell type–specific expression. This knowledge may provide avenues for improving NK-dependent antitumor strategies, such as ADCC-based therapeutics and the use of engineered NK cells.

The authors have no financial conflicts of interest.