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FcRγ Gene Editing Reprograms Conventional NK Cells to Display Key Features of Adaptive Human NK Cells

Summary Adaptive human natural killer (NK) cells display significantly enhanced responsiveness to a broad-range of antibody-bound targets through the engagement of CD16 compared to conventional NK cells, yet direct reactivity against tumor targets is generally reduced. Adaptive NK cells also display a distinct phenotype and differential expression of numerous genes, including reduced expression of signaling adapter FcRγ and transcription factor PLZF. However, it is unclear whether differential expression of specific genes is responsible for the characteristics of adaptive NK cells. Using CRISPR-Cas9, we show deletion of FcRγ in conventional NK cells led to enhanced CD16 responsiveness, abolished cell surface expression of natural cytotoxicity receptors, NKp46 and NKp30, and dramatically reduced responsiveness to K562 and Raji tumor cells. However, deletion of PLZF had no notable effects. These results suggest multiple roles for FcRγ and identify its deficiency as an important factor responsible for the functional and phenotypic characteristics exhibited by adaptive NK cells.


INTRODUCTION
Natural killer (NK) cells constitute the third largest population of lymphocytes and play important roles in host defense against malignancy and viral infection (Vivier et al., 2011). Upon recognition of tumors or virusinfected target cells via activation receptors, NK cells can rapidly release cytotoxic granules and produce inflammatory cytokines. Unlike B and T lymphocytes that express gene-rearranged antigen-specific receptors, NK cells only express germline-encoded receptors and are considered part of the innate immune system. However, many recent studies have revealed adaptive immune features of NK cells in humans as well as in animal models (Cooper et al., 2009;Guma et al., 2004;Hammer and Romagnani, 2017;O'Leary et al., 2006;Paust et al., 2010;Reeves et al., 2015;Rolle and Brodin, 2016;Sun et al., 2009;Zhang et al., 2013).
In humans, two distinct but largely overlapping NK cell subsets have been identified that exhibit adaptive immune features, such as clonal-like expansion and long-term persistence (Beziat et al., 2013;Cichocki et al., 2018;Foley et al., 2012;Hammer et al., 2018;Lee et al., 2015;Muccio et al., 2018;Schlums et al., 2015;Zhang et al., 2013). These subsets were initially identified by either a deficiency in expression of the signaling adapter protein FcRg or a high-level expression of the activation receptor NKG2C (Guma et al., 2004;Hwang et al., 2012). Epidemiological analyses indicate that the presence of these FcRg-deficient NK cells (termed g-NK cells), as well as NKG2C + NK cells that often co-express maturation marker CD57, is associated with previous infection by human cytomegalovirus (HCMV), a common herpesvirus that infects the majority of the human population (Guma et al., 2004;Zhang et al., 2013). Importantly, these adaptive NK cells display heightened potential for broad antiviral and antibacterial responses through enhanced function of CD16 (Costa-Garcia et al., 2015;Goodier et al., 2018;Hart et al., 2019;Lee et al., 2015;Schlums et al., 2015;Wu et al., 2013;Zhang et al., 2013;Zhou et al., 2015), a Fc receptor that can bind to the Fc portion of multiple subclasses of IgG (Bruhns et al., 2009). For instance, it has been shown that, compared to conventional NK cells, adaptive NK cells produce greater amounts of pro-inflammatory cytokines in response to cells infected with viruses (e.g., HCMV and HSV-1), as well as to the virus itself, in the presence of virus-specific antibodies. In contrast, adaptive NK cells generally show poor responsiveness to tumor targets, unless target-specific antibodies are provided (Beziat et al., 2012;Hwang et al., 2012;Liu et al., 2016). Moreover, adaptive NK cells show defective expression of natural cytotoxicity receptors (NCRs), NKp46 and NKp30, both of which are commonly expressed by conventional NK cells (Guma Figure 1. Deletion of FcRg in NK Cells Leads to Enhanced Cytokine Production in Response to CD16 Cross-linking (A) Contour plots show a mixture of NK (CD3 À CD56 + ), T (CD3 + CD56and CD3 + CD56 + ), and non-NK/non-T (CD3 À CD56 -) cells after expansion of PBMC samples enriched for NK cells. NK cells (gated, red) were further analyzed for FcRg expression following nucleofection with FCER1G-targeting Cas9/gRNA ribonucleoprotein (FCER1G-RNP) or nontargeting Cas9/gRNA ribonucleoprotein (ctrl RNP) after 10 days of in vitro culture. Data are representative of 5 independent experiments performed on NK cells from 10 donors. (B) Time course analysis of FcRg depletion in FCERIG-RNP-treated NK cells (red), with CD3 + CD56 -T cells (blue) overlaid for comparison. The staining pattern obtained using a matched isotype control antibody (iso ctrl) is shown. (C-E) Contour plots show flow cytometric analysis of (C) TNF-a, (D) IFN-g production, and (E) cell surface CD107a expression by FcRg + and FcRg À NK subsets from a representative donor without (À) or with (+) CD16 cross-linking (CXL).  et al., 2004;Hwang et al., 2012;Moretta et al., 2001). However, the molecular basis responsible for the altered functional and phenotypic characteristics of adaptive NK cells is unclear.
Recent gene expression profiling and epigenetic analyses have revealed differences in the expression of approximately 400 genes and widespread epigenetic changes between conventional and adaptive NK cells (Lee et al., 2015;Schlums et al., 2015). Flow cytometric analyses have confirmed altered expression of many genes at the protein level, including FcRg and PLZF. FcRg is an immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling adapter protein and is known to have physical association with CD16, along with CD3z, as homodimers or heterodimers. While CD3z contains 3 ITAMs, FcRg contains only 1 ITAM. FcRg and numerous other intracellular proteins are also deficient in adaptive g-NK cells, including transcription factors (e.g., PLZF and HELIOS) and signal transduction proteins (e.g., SYK and EAT-2). Through correlation studies, it has been suggested that deficiencies in SYK may contribute to some of the altered characteristics of adaptive NK cells (Lee et al., 2015). It was reported that epigenetic remodeling of the IFNG locus (Luetke-Eversloh et al., 2014), as well as elevated expression of the transcription regulator, ARID5B, correlates with enhanced IFN-g production by NKG2C + NK cells (Cichocki et al., 2018). Moreover, because of PLZF's potential involvement in chromatin structure modification and regulation of several genes differentially expressed in adaptive NK and conventional NK cells (Gleimer et al., 2012;Mathew et al., 2012), PLZF deficiency has been suggested to be an important factor contributing to the observed adaptive properties (Schlums et al., 2015). However, given the numerous epigenetic and gene expression differences in adaptive NK cells, it has been challenging to define the molecular mechanism(s) responsible for the enhanced CD16 responsiveness and reduced tumor responsiveness of adaptive NK cells.
In this study, using CRISPR/Cas9-mediated gene editing, we sought to determine the roles of FcRg and PLZF deficiencies in the functional response and phenotypic characteristics of primary human NK cells. Our data show that deletion of FcRg resulted in enhanced CD16 responsiveness but reduced NCR expression and tumor responsiveness. However, deletion of PLZF had no notable effects. These data provide evidence that deficiency of FcRg is an important factor responsible for the altered functional and phenotypic characteristics exhibited by adaptive NK cells.

CRISPR/Cas9-mediated Deletion of FcRg in Human NK Cells
Although NK cells express a limited repertoire of germline-encoded activation receptors, they are able to recognize and respond to a broad-range of targets in the presence of target-specific antibodies through the expression of CD16 (Bruhns et al., 2009;Costa-Garcia et al., 2015;Hwang et al., 2012;Lanier, 2008;Zhang et al., 2013). To determine the role of FcRg in the function of CD16, we initially sought to silence FcRg expression in human NK cells using a siRNA-mediated knockdown approach. However, this strategy resulted in only transient and partial (approximately 50%) reduction of FcRg protein level with no notable change in functional responsiveness of NK cells (data not shown). As an alternative approach to achieve more stable and more complete depletion of FcRg, we explored CRISPR/Cas9-mediated gene editing.
To obtain sufficient quantities of NK cells for gene editing and subsequent functional analysis, NK cells were enriched from peripheral blood mononuclear cell (PBMC) samples specifically chosen for their lack of detectable FcRg-deficient (hereafter termed g-NK) cells, and these enriched samples were expanded in vitro for 10-14 days. Expanded cells were subjected to nucleofection of FCER1G (encoding FcRg)-targeting Cas9/gRNA ribonucleoprotein (FCER1G-RNP). After subsequent culturing, flow cytometric analysis revealed the appearance of a distinct subset of CD3 À CD56 + NK cells deficient for FcRg expression (Figure 1A). This effect was specific to FCER1G-RNP, as there was no change in FcRg expression in cells treated with non-targeting control RNP (ctrl RNP). Moreover, the expression of CD3z, another CD16-associated signaling adapter protein that is homologous to FcRg in function and sequence and encoded by a Figure 1. Continued Numbers represent the relative percentages of FcRg + or FcRg À subset that produced indicated cytokine or displayed CD107a. Line graphs show the relative percentages of FcRg + or FcRg À subset that produced indicated cytokine or displayed CD107a from several donors in response to CD16 CXL (n = 10). Circles connected by a line designate data obtained from the same donor sample.

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iScience 23, 101709, November 20, 2020 3 iScience Article gene located close to FCER1G on chromosome 1 (Eiseman and Bolen, 1992), was unaffected by FCER1G-RNP treatment ( Figure 1B). Kinetic analysis showed a partial reduction of FcRg expression detectable 3 days post-nucleofection, but approximately 1 week was needed to reveal a distinct population having complete depletion ( Figure 1B). Since it is estimated that CRISPR/Cas9-mediated gene editing occurs within 12 hr post-nucleofection (Brinkman et al., 2018;Kim et al., 2014), our data suggest that pre-existing FCER1G transcripts and FcRg proteins have relatively long lifespans. Therefore, subsequent analyses of FCER1G gene-edited cells were performed at least 10 days post-nucleofection. Under this condition, we were able to obtain on average approximately 60% efficacy in the production of FcRg À NK cells.

Deletion of FcRg Increases Cytokine Production in Response to CD16 Stimulation
To evaluate whether FcRg deletion affects the functional responsiveness to CD16 stimulation, we examined cytokine production by the gene-edited FcRg-null NK cells and NK cells expressing normal levels of FcRg (hereafter termed FcRg À subset and FcRg + subset, respectively) following incubation with immobilized anti-CD16 mAb. Flow cytometric analysis showed that the FcRg À subset produced significantly greater amounts of TNF-a compared to the corresponding FcRg + subset in the same culture (n = 10, p < 0.01; Figure 1C). In addition to TNF-a, CD16 stimulation resulted in significantly more IFN-g production by FcRg À subset (n = 10, p < 0.01; Figure 1D). Of these donors, 7 were HCMV seronegative donor samples which also exhibited statistically significant increases in TNF-a and IFN-g production (p < 0.05). However, there was no consistent difference in the degranulation response between FcRg À and FcRg + subsets, as measured by cell surface expression of CD107a ( Figure 1E). These data demonstrate that FcRg deletion in NK cells results in increased cytokine production but does not alter degranulation activity in response to CD16 stimulation, suggesting that FcRg may function to modulate CD16-mediated cytokine production.
Given that CD16 is known to associate with either FcRg-FcRg or CD3z-CD3z homodimer or FcRg-CD3z heterodimer in human NK cells (Lanier, 2008), exclusive association with CD3z-CD3z homodimer may yield biochemical signaling that is more effective for cytokine production. Thus, when both adapters are co-expressed, FcRg may negatively regulate the CD16-CD3z signaling pathway by competing for association with CD16 and/or interaction with downstream signaling molecules. The lack of any detectable difference in CD107a expression between FcRg À and FcRg + subsets could be explained by a lower activation threshold for degranulation (Bryceson et al., 2009).
We have previously demonstrated that g-NK cells in PBMC produced greater quantities of cytokines compared to the corresponding conventional NK cells upon CD16 cross-linking, whereas degranulation responses between these groups of cells were similar (Hwang et al., 2012). Given that the current study tested in vitro expanded NK cells to examine the effects of FcRg deletion, we also tested CD16 responsiveness of g-NK and conventional NK cells after expansion in the same culture conditions as the gene-edited cells. Consistent with observations of fresh NK cell populations, in vitro expanded g-NK cells produced significantly greater amounts of both TNF-a and IFNg compared to the corresponding conventional NK cells that had been expanded in the same culture conditions (n = 7, p < 0.05; Figures 1F and 1G), whereas no significant difference in degranulation was observed ( Figure 1H). When these data were compared with gene-edited FcRgand FcRg + subsets, a similar trend in cytokine production between g-NK and conventional NK cells was observed, although g-NK cells in certain donors appeared to produce dramatically higher levels of cytokine ( Figures 1C, 1D, 1F, and 1G). In light of the genetic heterogeneity among donors, as well as differential expression of numerous genes between g-NK and conventional NK cells, determining the specific contribution of FcRg loss to the enhanced CD16-mediated cytokine production of g-NK cells is challenging with currently available technology. However, given the notable positive impact of CRISPR/ Cas9-mediated FcRg deletion in the absence of other gene expression differences ( Figures 1C and 1D), our data suggest that FcRg-deficiency is an important factor responsible for the enhanced CD16-mediated cytokine production by g-NK cells.

Deletion of FcRg Decreases Functional Responsiveness to Tumor Target Cells
In contrast to enhanced CD16 responsiveness, g-NK cells respond relatively poorly to the prototypical NK cell-sensitive leukemic cell line K562, as evidenced by a lower degranulation response and lower cytokine production compared to conventional NK cells (Hwang et al., 2012). Similarly, NKG2C + NK cells were also shown to respond poorly to this tumor target relative to conventional NKG2C À NK cells (Beziat et al., 2012;Liu et al., 2016). We evaluated the potential impact of FcRg deletion on tumor responsiveness by examining FcRg À and FcRg + subsets for degranulation and cytokine production following incubation with K562 tumor cells. Compared to the FcRg + subset, the FcRg À subset showed dramatic reduction in both degranulation ll OPEN ACCESS iScience 23, 101709, November 20, 2020 iScience Article activity and cytokine production (Figures 2A-2C). Similar results were obtained following incubation with Raji tumor cells; FcRg À subset showed a reduced degranulation response compared to the FcRg + subset ( Figure 2D), as well as a reduced cytokine response ( Figures 2E and 2F). Taken together, these results indicate that FcRg plays an important role in functional response of conventional NK cells to both K562 and Raji tumor cells. Thus, FcRg deficiency is likely a major factor responsible for the reduced responsiveness of g-NK cells to tumor cells.

Figure 2. Deletion of FcRg Yields Reduced NK Cell Responsiveness to K562 and Raji Tumor Cells
(A) Contour plots show flow cytometric analysis of CD107a expression by FcRg + and FcRg À NK subsets from a representative donor following incubation in the absence (À) or presence (+) of K562 tumor cells. Numbers represent the relative percentages of FcRg + or FcRg À subset that displayed CD107a. Line graph shows the relative percentages of FcRg + or FcRg À subsets that displayed CD107a from several donors in response to K562 stimulation (n = 8). Circles connected by a line designate data obtained from the same donor sample.
(B and C) Contour and dot plots show the percentages of FcRg + and FcRg À subsets that produced (B) TNF-a or (C) IFN-g in response to K562 stimulation. (D-F) Line graphs show the relative percentages of FcRg + and FcRg À subsets that displayed (D) CD107a or (E) produced TNF-a or (F) IFN-g in response to Raji stimulation. (G) Contour plots and line graphs show the percentages of FcRg + and FcRg À subsets that produced IFN-g in response to stimulation with IL-12 plus IL-18. Wilcoxon signed-rank test; *p < 0.05, **p < 0.01, n.s., not significant.

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iScience 23, 101709, November 20, 2020 5 iScience Article In addition to altered responsiveness to CD16 and tumor stimulation, g-NK cells display altered responsiveness to stimulation with IL-12 and IL-18, i.e., although conventional NK cells can produce copious amounts of IFN-g following incubation with these cytokines, IFN-g production by g-NK cells is dramatically reduced (Schlums et al., 2015). However, both FcRg + and FcRg À subsets showed comparable IFN-g production with respect to percentage and median fluorescence intensity in response to stimulation with IL-12 and IL-18 by generating ample amounts of IFN-g (n = 6; Figure 2G), indicating that FcRg deletion does not affect IL-12 and IL-18 driven cytokine production. Thus, the impaired IL-12/18-mediated responsiveness of g-NK cells is likely controlled by a mechanism that is independent of FcRg deficiency.

Role of FcRg in the Expression of CD16, NKp46, and NKp30
It has been shown that co-expression of CD16 with either FcRg or CD3z in heterologous expression systems can support cell surface expression of CD16 (Blazquez-Moreno et al., 2017;Lanier et al., 1991). To evaluate the specific contribution of FcRg to cell surface expression of CD16 on primary NK cells, we compared CD16 expression on FcRg À and FcRg + subsets. Flow cytometric analysis showed that CD16 expression is lower on the FcRg À subset compared to the FcRg + subset for all donors tested ( Figure 3A). Based on the median fluorescence intensities, CD16 expression was reduced on average by 53% in the FcRg À subset ( Figure S1A), indicating that normal CD3z expression in the FcRg À subset ( Figure 1B) cannot compensate for the absence of FcRg and does not support normal levels of CD16 expression. This, coupled with the previous observation of lower CD16 expression on g-NK cells relative to conventional NK cells (Hwang et al., 2012), supports the conclusion that FcRg deficiency is also responsible for reduced expression of CD16 on g-NK cells. Of note, NK cells in FcRg knockout mice do not express any detectable cell surface CD16 (Takai et al., 1994), suggesting a differential role of FcRg in humans and mice. Importantly, the enhanced CD16-mediated cytokine production of the FcRg À subset ( Figures 1C and 1D) was not due to higher CD16 expression. In the absence of FcRg bearing 1 ITAM, the potential exclusive association between CD16 and homodimer of CD3z bearing 3 ITAMs may induce higher cytokine production. This possibility warrants further investigation.
As FcRg is also known to be associated with NKp46 and NKp30 (Sivori et al., 2019), we examined cell surface expression of these activation receptors on FcRg + and FcRg À subsets. The FcRg À subset showed a dramatic reduction in NKp46 expression (87% reduction on average), compared to FcRg + subset ( Figures  3B and S1B). Moreover, the FcRg À subset also showed a dramatic reduction in NKp30 expression (92% reduction) (Figures 3C and S1C). These results indicate that FcRg plays a crucial role in supporting cell surface expression of both NKp46 and NKp30. Given that both NKp46 and NKp30 were shown to associate with CD3z as well (Pende et al., 1999;Vitale et al., 1998), it is likely that CD3z alone in the FcRg À subset cannot compensate for the absence of FcRg and can only support a minimal level of expression of these receptors. Nonetheless, our results provide strong evidence that FcRg deficiency is responsible for reduced expression of these receptors by g-NK cells, as well as by NKG2C + NK cells (Guma et al., 2004;Hwang et al., 2012;Schlums et al., 2015). Since NKp46 and NKp30 are considered major tumor recognition receptors, the abolished expression of these receptors likely resulted in the reduced responsiveness of FcRg À subset to K562 and Raji tumor cells. Consistent with this possibility, both K562 and Raji cells were shown to express B7-H6, a ligand for NKp30 (Brandt et al., 2009). Taken together, our results suggest that FcRg deficiency in g-NK cells is a major factor contributing to their reduced responsiveness toward tumor target cells through limiting the cell surface expression of NKp30 and NKp46.
In contrast to CD16, NKp46, and NKp30, we observed that the expression of other markers, including NKG2A, CD2, CD11a, NKG2C, CD57, ILT2, and Bcl-2, all of which are differentially expressed between g-NK and conventional NK cells (Hwang et al., 2012;Lee et al., 2015;Zhang et al., 2013), were unaffected by FcRg deletion (Figure 3D), suggesting that altered expression of these molecules in g-NK cells is controlled by other mechanisms.

Deletion of PLZF Does Not Alter Responsiveness to CD16 or IL-12/18 Stimulation
In addition to FcRg deficiency, altered expression of other genes may also contribute to the altered functional responsiveness exhibited by adaptive NK cells. Among such alterations, PLZF deficiency has been suggested to be an important factor contributing to the observed adaptive properties (Schlums et al., 2015). iScience Article in expression of PLZF appeared approximately 1 week post-nucleofection ( Figure 4A). The cytokine production assay demonstrated that following incubation with immobilized anti-CD16 mAb, PLZF À NK cells produced similar levels of IFN-g and TNF-a as PLZF + NK cells (n = 7; Figures 4B and 4C). Degranulation responses were also comparable ( Figure 4D). In addition, stimulation by IL-12 and IL-18 resulted in equivalently high levels of IFN-g by both PLZF À and PLZF + NK cells (data not shown). We also observed no difference in the expression of components for IL-12 or IL-18 receptors ( Figure S2). Finally, we observed that FcRg, CD16, NKp46, and NKp30 were all normally expressed by PLZF À NK cells (data not shown). Taken together, unlike the deletion of FcRg, the available data do not demonstrate any notable functional or phenotypic changes resulting from the deletion of PLZF.

Concluding Remarks
Collectively, our results demonstrate that CRISPR/Cas9-mediated deletion of FcRg in human NK cells led to enhanced CD16 responsiveness but reduced tumor responsiveness in association with reduced expression iScience Article of NCRs. However, deletion of PLZF did not elicit notable changes. Importantly, our results also provide strong evidence that FcRg deficiency is an important factor responsible for both enhanced CD16-mediated cytokine production and reduced tumor responsiveness exhibited by adaptive human NK cells. However, it should be noted that our data do not exclude the possibility that factors other than FcRg deficiency may further contribute to the altered functional responsiveness of adaptive NK cells. Future studies utilizing the CRISPR-Cas9 gene editing approach will be helpful in identifying such factors, thereby deepening our understanding of the mechanism(s) contributing to adaptive NK cell functionality and illuminating potential development for clinical application.

Limitations of the Study
In the gene knock-out experiments, NK cells were cultured in vitro before gene editing to expand the number of available cells and then again after editing to completely deplete any pre-existing target proteins before analysis. It is likely that during in vitro culture, expression of many genes, including those involved in functional responses of NK cells, may be altered. Therefore, it is possible that in addition to the disrupted expression of the target genes being edited in this study, other genes with altered expression may have contributed to the phenotypic and functional characteristics exhibited by the gene-edited NK cells.

Resource Availability Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sungjin Kim (sjikim@ucdavis.edu).