Ablation of SYK kinase from primary human Natural Killer cells via CRISPR/Cas9 enhances cytotoxicity and cytokine production

Cytomegalovirus (CMV) infection alters natural killer (NK) cell phenotype and function toward a more memory-like immune state. These cells, termed adaptive NK cells, typically express CD57 and NKG2C but lack expression of the Fc receptor γ chain (Gene: FCER1G, FcRγ), PLZF, and SYK. Functionally, adaptive NK cells display enhanced antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. However, the mechanism behind this enhanced function is unknown. To understand what drives cytotoxicity and cytokine production in adaptive NK cells, we optimized a CRISPR/Cas9 system to ablate genes from primary human NK cells. ADCC by human NK cells is exclusively mediated by the CD16A (FcγRIIIA) signaling apparatus, which includes FcRγ, CD3ζ, SYK, SHP-1, ZAP-70, and the transcription factor PLZF. We ablated the genes encoding these molecules and tested subsequent ADCC and cytokine production. We found that ablating the FcRγ chain caused a modest increase in TNFα production. Ablation of PLZF did not enhance ADCC or cytokine production. Importantly, SYK kinase ablation significantly enhanced both cytotoxicity and cytokine production, while ZAP-70 kinase ablation diminished function. Ablation of the phosphatase SHP-1 resulted in mixed effects on function, with NK cells demonstrating enhanced cytotoxicity but reduced cytokine production. These results indicate that the enhanced cytotoxicity and cytokine production of CMV-induced adaptive NK cells is more likely due to the loss of SYK than the lack of FcRγ or PLZF. The lack of SYK expression may limit SHP-1-mediated inhibition of CD16A signaling, leading to enhanced cytotoxicity and cytokine production. In addition to providing mechanistic answers about CMV-induced adaptive NK cell functionality, our results indicate that NK chimeric antigen receptor (CAR) therapeutics that invoke ADCC signaling molecules (e.g., CD3ζ chain) may benefit from ablating SYK, while maintaining ZAP-70, to increase functionality.


Introduction
Natural killer (NK) cells are innate lymphocytes that can kill both cancerous and infected cells (Abel et al., 2018;Lanier, 2008). In peripheral blood, NK cells comprise 5 to 20% of lymphocytes, mainly surveying the bloodstream for potential target cells (Burrack et  Crosslinking of CD16A by antibodies bound to a target cell recruits Src-family kinases, such as LCK, to phosphorylate ITAMs on FcRg and/or CD3z. This phosphorylation recruits the tyrosine kinases SYK and/or ZAP-70 to ITAMs for subsequent signal transduction. SYK preferentially binds FcRg, whereas ZAP-70 preferentially binds CD3z (Shiue et al., 1995). SYK and ZAP-70 then phosphorylate their respective downstream targets (e.g., PI3K, VAV1, and phospholipase Cg isoforms) to further propagate the CD16A signaling pathway, ultimately cumulating in cytotoxicity and cytokine production (Freedman et al., 2015;Watzl and Long, 2010). While the molecules participating in CD16A-mediated signaling pathway are defined, the specific contributions of these signaling proteins to primary human NK cell function were unclear.  (Guma et al., 2006) and upon CMV reactivation in humans (Foley et al., 2012). After CMV reactivation, CD57+/NKG2C+/NKG2A-NK cells also exhibit enhanced cytokine production (Foley et al., 2012). These results led to the classification of CD57+/NKG2C+/NKG2A-NK cells as adaptive NK cells due to their adaptive qualities, such as longevity and antigen-specific expansion (albeit through a limited germline-encoded receptor). Adaptive NK cells are also hypothesized to play a protective role in cancer and transplant patients as a higher frequency of adaptive NK cells correlates with better clinical outcomes in individuals with reactivated CMV (Cichocki et al., 2019).
In addition to phenotypic differences, adaptive NK cells also show increases in ADCC and cytokine production compared to conventional NK cells (Hwang et al., 2012;Zhang et al., 2013). Zhang et al. showed that CMV infection was associated with loss of the FcRg chain.
FcRg-negative (FcRg-) NK cells showed increased degranulation (CD107a), IFNg, and TNFa production in response to opsonized infected cells over conventional NK cells . In addition to FcRg deficiency, adaptive NK cells have been shown to lack both SYK kinase and PLZF (Lee et al., 2015;Schlums et al., 2015). PLZF is a transcription factor that binds the promoter of genes encoding FcRg and SYK and likely plays a role in regulation of these genes (Correia et al., 2018;Schlums et al., 2015). In addition to CMV, other infectious diseases, such as HIV and malaria, can induce adaptive NK cell formation. HIV-and malariainduced adaptive NK cells also show enhanced ADCC and cytokine production. However, these cells have a different phenotype than their CMV-induced counterparts, such as maintained SYK expression, highlighting adaptive NK cell heterogeneity ( The absence of FcRg, PLZF, and/or SYK is hypothesized to be the mechanism responsible for enhancing the ADCC function of CMV-induced adaptive NK cells. Yet it was unclear whether these molecules are responsible for enhanced function or only correlate with it.
Here, we investigated the role of molecules associated with CMV-induced adaptive NK cellsincluding FcRg, PLZF, and SYK-in the ADCC signaling apparatus. We tested for redundancy in the ADCC signaling pathway by targeting molecules with similar functions as FcRg and Syk, such as CD3z and ZAP-70, respectively. We also sought to disentangle which molecules are associated with cytotoxic activity versus cytokine production after CD16A activation. Using a murine model could be one approach. However, murine NK cells do not perform ADCC well and the murine CD3z chain does not associate well with murine CD16 as an adapter protein.
This limits the utility of murine signaling protein knockouts (Aguilar et al., 2022). For humans, until recently, it has not been possible to ablate genes in primary human NK cells. Therefore, to accomplish these goals, we developed a CRISPR/Cas9 gene-editing protocol for thawed human primary NK cells, ablated genes that encoded molecules in the CD16A pathway, and tested the effect of gene ablation on ADCC and cytokine production.
We show that SYK kinase ablation enhances both ADCC and cytokine production, while ZAP-70 ablation shows the opposite phenotype. SYK ablation enhanced killing more so than degranulation and the increased killing capacity was maintained for four weeks post-expansion.
These results point toward a mechanism involving SYK for the enhanced function of CMVinduced adaptive NK cells. Overall, this study provides a mechanistic understanding of the enhanced ADCC function observed in CMV-induced adaptive NK cells and provides a new strategy for improving NK cell therapies by leveraging the ADCC pathway.

Lead Contact
Requests for information or resources should be directed to the lead contact, Geoffrey Hart (hart0792@umn.edu).

Materials Availability
This study did not generate unique reagents.

Primary Cell Purification and Freezing
Primary red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were obtained and purified from deidentified adult blood donors (Memorial Blood Center). The work done in this study was approved by the Institutional Review Board (IRB) of the University of Minnesota. The cytomegalovirus (CMV) status of each donor was reported by Memorial Blood Center. RBCs were purified from whole blood by filtration (Memorial Blood Center, 4C4300) and resuspended at 50% hematocrit in RPMI-1640 + 25 mM HEPES, L-Glutamine, and 50 mg/L Hypo-Xanthine (Kd Medical, 50-101-8907). PBMCs were isolated using Ficoll (MP Biomedicals, ICN50494) centrifugation and the remaining RBCs were lysed using ACK (Lonza, BP10-548E). Once isolated, PBMCs were counted and resuspended at 2x10 8 cells/mL in 10mL of PBS + 2% FBS (PEAK Serum, PS-FB1) + 1 mM EDTA (SC Buffer). NK cells were then isolated from these PBMCs using a magnetic negative selection kit (STEMCELL Technologies, 17955). Briefly, PBMCs were stained with 500µl of the manufacturer's antibody cocktail, which is less concentrated than the manufacturer's protocol, and 30 µl of additional biotinylated anti-CD3 (STEMCELL Technologies, 18051) was added to the sample to improve purity. Samples were incubated at RT for 10 minutes. After incubation, 1 mL of magnetic beads was added to the sample, which was then vortexed 2-3 times and incubated for 10 minutes at RT. Post-incubation, 35 mL of SC Buffer was added to the cells, which were then placed on a magnet and incubated at RT for 10 minutes. After incubation, the liquid-which contained purified NK cells-was removed from the tube without touching the magnet. NK cells were then added to a new 50 mL conical and placed back on the magnet for 10 minutes at RT. After 10 minutes, purified NK cells were removed and counted. For cryopreservation, NK cells were resuspended in RMPI-1640 (Fisher, SH3002701) + 50% FBS (PEAK Serum, PS-FB1) at 10 7 cells/mL. 500 µL of the suspension was transferred to a cryovial. When all tubes were ready to freeze, 500 µl of FBS (PEAK Serum, PS-FB1) +15% DMSO was added to cells in cryovials. The cells were immediately transferred to a pre-cooled Mr. Frosty (Thermo-Fisher, 5100-0001) and then placed in a -80° C freezer. After 24-48 hours, the cells were transferred to a vapor-phase liquid nitrogen freezer for later use.

NK Cell Expansion
Cryopreserved vials of primary NK cells were thawed by placing them in a water bath at 37 °C until the vial was 95% thawed. The cells were then transferred to a 15 mL conical. Prewarmed RP10 was added dropwise at a rate of 1 mL/minute while the conical was swirled. After adding 3 mL of RP10, 6 more mL of pre-warmed RP10 was added and the cells were centrifuged at 650 x g. The cells were washed once with RP10, centrifuged, and resuspended in 5 mL of X-VIVO TM 15 (Lonza, BE02-060F) + 10% FBS (PEAK Serum, PS-FB1) (X-Vivo 10) + 2 ng/mL IL-15 (NCI). Cells were then transferred to a 6-well plate and incubated overnight. After incubation, 3x10 6 NK cells were mixed with 6x10 6 irradiated K562-aAPC cells in 30 mL X-Vivo 10 + 50 IU/mL IL-2 (NCI). K562-aAPCs were irradiated at 100 grays (RS-2000 X-Ray irradiator). The cells were added to a G-Rex © 6-well plate (Wilson Wolf, 80240M) and incubated at 37 °C for six days.

CRISPR/Cas9 Gene-editing
Single-guide RNAs (sgRNA) were designed using Synthego's CRISPR Design tool NK cells were centrifuged and resuspended in P3 Primary Cell Nucleofector © Solution (Lonza, V4XP-3032) at 3.33x10 8 cells/mL. 5x10 6 cells were mixed with RNPs and transferred without forming bubbles to a 16-well Nucleocuvette © Strip (Lonza, V4XP-3032). Unless otherwise stated, the cells were nucleofected with an Amaxa 4D-Nucleofector TM (Lonza) using code CA137. Post-nucleofection, the cells were rested for 15 minutes at RT. 80 μL of prewarmed X-Vivo 10 was added to each nucleofection well. The cells were rested for 30 more minutes at RT, then the total volume was transferred to a G-Rex © 24-well plate (Wilson Wolf, 80192M) with 1.9 mL of pre-warmed X-Vivo 10. Each well was then washed with 100 µl of media, which was transferred to the cell suspension. The cells were rested once more for 30 minutes at RT before 4 mL of X-Vivo 10 + 4.5 ng/mL of IL-15 (NCI) was added to each well (3 ng/mL IL-15 final). The cells were then incubated at 37 °C for six days.

Degranulation and Cytokine Production Assay with Red Blood Cells as NK Targets
RBCs were opsonized with polyclonal anti-RBC rabbit antibody (Rockland, 1094139) and combined with NK cells at a ratio of 1 NK:5 RBC in RP10 + 1 µg/mL brefeldin A (BFA) + 2 µM monensin + 1:200 anti-CD107a, which measures degranulation (Biolegend, 328625). As a negative control, NK cells were also incubated with non-opsonized RBCs. The cells were then incubated at 37 °C for 5 hours. After five hours, the cells were centrifuged, washed, and stained for flow cytometry.

ICE Analysis
Synthego's Inference of CRISPR Edits (ICE) (https://ice.synthego.com/) was used to analyze gene ablation. For ICE analysis, DNA from gene-ablated and control samples was isolated using QIAGEN's DNAeasy kit (Qiagen, 69506) according to the manufacturer's instructions. PCR primers were designed to amplify a region of 500 base pairs (bp) around the cut site. The forward primer was designed 100-200 bp upstream from the cut site to capture it with high-quality sequencing. Additionally, we designed sequencing primers that were nested 5-50 base pairs inside the amplicon to improve sequencing efficiency. After amplifying the sgRNA cut site, approximately 100 ng of the PCR product was submitted to the UMN Genomics center along with 6.4 pmol of a sequencing primer for Sanger sequencing. The primers used are listed in Supplementary Table 1.

DELFIA Killing Assay with SKOV-3 cells as NK Targets
DELFIA killing assays were performed according to manufacturer instructions (PerkinElmer, AD0116). Briefly, SKOV-3 (HER-2 +) cells were resuspended at 10 6 cells/mL in SKOV-3 Media. To label the cells, 1.5 μL/mL of Bis(acetoxymethyl)-2-2:6,2 terpyridine 6,6 dicarboxylate (BATDA) was added to the suspension. The cell suspension was then incubated for 30 minutes at 37 °C. After labeling the cells, they were washed and transferred to a roundbottom 96-well plate at a concentration of 8x10 3 cells/well. Anti-HER2 (Trastuzumab) was then added to the well at a concentration of 3 or 10 µg/mL along with either primary NK or NK-92 (effector) cells at various E:T ratios. The plate was spun at 400 x g for one minute and incubated at 37 °C for 2 hours with 5% CO2. After 2 hours, the plate was centrifuged at 500 x g for five minutes. 20 μL of supernatant was then transferred to a 96-well DELFIA Yellow Plate and combined with 200 μl of europium. Signal was measured by time-resolved fluorescence using a BioTek Synergy 2. To measure maximum release, BADTA-labeled target cells were incubated with 10μL of lysis buffer per the manufacturer's instruction. To measure spontaneous lysis, BADTA-labeled SKOV-3 cells were cultured in parallel without effector cells or antibodies.
When possible, samples were run in technical triplicates.

Incucyte Killing Assay with SKOV-3 cells as NK Targets
On the day prior to the killing assay, SKOV-3 NLR cells (HER-2 +) were resuspended at 4x10 4 cells/mL in RP10. 100 µl of the cell suspension was added to each well of an Incucytecompatible flat-bottom 96-well plate. The SKOV-3 NLR cells were then incubated overnight at 37 °C to allow them to adhere to the bottom of the well.
The following day, NK cells were resuspended at 1.2x10 6 cells/mL in RP10. 60 µl of NK cells were added to a new 96-well plate. Anti-HER2 was then diluted to 20 µg/mL in RP10 and 60 µL of the solution was added to the NK cells. To prevent cells from localizing to the edge of the well, both the 96-well plate containing the NK cells/anti-HER2 and the plate containing the SKOV-3 NLR cells were incubated at 37 °C for 15 minutes. 100 µl of NK cells/anti-HER2 was then added to the plate containing SKOV-3 NLR cells for an effector: target (E: T) ratio around 15:1 and a final anti-HER2 concentration of 5 µg/mL. The cell mixture was then loaded into an Incucyte SX5 (Sartorius). Immediately after loading the plate, a t = 0-hour image was taken to ensure cells were not localized to the edge of the well. After confirming cells were dispersed throughout the well, four consecutive images were taken per well once per hour for 24 hours. As controls, SKOV-3 NLRs and NK cells without anti-HER2 were imaged as well as SKOV-3 NLRs without NK cells. Samples were run in technical triplicates.

Ablation Level Cutoffs
Data points from samples with less than 25% protein ablation were excluded from all analyses to ensure the gene-ablated population was substantial enough to analyze. For expansion experiments, the cutoff was only applied at day 6 (e.g., if a sample was above 25% protein ablation at day 6 but below 25% at day 13, the data from day 13 was included). Samples between 25-49% ablation were kept for ADCC assay analysis as the gene-ablated cells can be gated on via flow cytometry. Unlike flow cytometry-based ADCC assays, killing assays utilized a target cell count readout, precluding specific analysis of gene-ablated cells. Thus, killing assay data from samples with less than 49% protein ablation were excluded.

LEGENDScreen TM Cutoffs
Proteins that were expressed in fewer than 10% of cells from each subject at each timepoint were classified as not expressed in NK cells. These proteins were excluded from the final analysis.

Fold-change Data
Data were transformed using fold-change and log2 fold-change to ease the visualization of trends. Fold-change was calculated by dividing the gene-ablated value by the no RNP value for the measurement in question.

Gene Ablation Analysis
Flow cytometry was used to assess the extent of CRISPR/Cas9 gene-ablation at the protein level. To calculate the protein ablation percentage for single-gene ablations, the following formula was applied: To calculate the protein ablation percentage for double-gene ablations, the following formula was used:

Incucyte Killing Assay Analysis
Incucyte Base Analysis Software (Satorius, v2019B) was used to analyze killing assays.
Images with SKOV-3 NLRs + NK cells, SKOV-3 NLRs alone, and NK cells alone were chosen from the 0-hour, 12-hour, and 24-hour images. Image parameters were then adjusted to ensure the software detected SKOV-3 NLRs but not NK cells. Graphs were created by normalizing each well's SKOV-3 NLR count at each timepoint to its time 0-hour count.

DELIFA Spontaneous Lysis Calculation
For each sample, specific lysis was used to report ADCC activity. The calculation for specific lysis is shown below: Graphing and Statistical Analysis for each code. However, viable cell recovery varied. Of the codes tested, CA137 showed the highest average recovery at 3.3x10 6 cells ( Figure S1D). We concluded that using CA137 results in the best balance of effective gene ablation and cell recovery, leading us to use CA137 for the rest of the experiments. By evaluating a compilation of experiments that used CA137, we found that cell counts six days post-CRISPR often exceeded our starting cell count, indicating that the cells recovered well from nucleofection ( Figure S1E). We also tested CRISPR/Cas9 deletion efficiency by molecular and protein-level methods to evaluate concordance. Indel analysis of sequencing data (Inference of CRISPR Edits (ICE)) and flow cytometry analysis of our target gene protein expression typically showed similar levels of gene ablation ( Figure S1F).
Representative gating schemes and protein ablation frequency for all data are shown in Figure   S2.
We then assessed the phenotype and function of primary human NK cells post-CRISPR.
To expand primary human NK cells, we incubated them for seven days by co-incubating them with to evaluate the effect of our expansion protocol on cell phenotype, we barcoded primary human NK cells from 7 subjects and examined the expression of 350 surface proteins pre-and postexpansion using Biolegend's LEGENDScreen TM . As expected, we observed that many proteins were significantly up-and down-regulated during expansion ( Figure S1G). However, we did not see loss of CD16A expression, which indicated that our expanded cells would retain ADCC function. We confirmed this by measuring ADCC-induced degranulation and cytokine production with an anti-RBC ADCC assay, in which red blood cells (RBCs) coated with antihuman RBC polyclonal antibodies were used as target cells. On day 6 post-CRISPR, NK cells yielded high levels of degranulation (CD107a+) and IFNg production ( Figure S1H-I). Overall, these results show that our expansion and CRISPR/Cas9 protocol generates gene-ablated primary NK cells that can be utilized in functional assays ( Figure 1A).
Ablating FcRg increases TNFa, but PLZF ablation does not affect cytotoxicity or cytokine production in cellular ADCC assays.
We used our CRISPR/Cas9 gene-editing protocol to test whether the loss of proteins associated with adaptive NK cells affected ADCC and cytokine production. A representative gating scheme used for analyzing ADCC assays with gene-ablated NK cells is shown in Figure S3.   Figure 1C-D, 1G). TNFa was expressed in a significantly higher frequency of FcRg-negative cells ( Figure 1C) but this was not true for CD3z-negative cells ( Figure 1D). Overall, though ablating the FcRg chain increases TNFa production during ADCC, neither ablation of the FcRg nor the CD3z chain consistently alters ADCC function.
Adaptive NK cells also lack PLZF (ZBTB16) expression (Schlums et al., 2015). PLZF controls expression of genes associated with adaptive NK cell activity, such as FcRg and SYK (SYK), leading to some speculation that PLZF may be the regulator of adaptive NK cell development (Schlums et al., 2015). Without PLZF, the FcRg and other genes may not be transcribed, leading to stronger ADCC and cytokine production. We hypothesized that ablation of PLZF may trigger the adaptive NK cell transcriptional program, resulting in enhanced ADCC and cytokine production in PLZF-ablated primary NK cells. To test this hypothesis, we ablated PLZF individually or PLZF together with FcRg ( FcRg/PLZF). We found that there was no significant difference in cytotoxicity or cytokine production with PLZF or FcRg/PLZF-ablated cells six days post-CRISPR/Cas9 in the anti-RBC ADCC assay ( Figure 1E-G). However, we were concerned that testing PLZF-ablated cells six days post-CRISPR/Cas9 may be too soon to observe an effect. Since PLZF is a transcription factor, we speculated that changes in transcription could take longer than six days post-CRISPR/Cas9 to manifest. We therefore tested  Figure S5).

SHP-1 ablation increases degranulation but decreases cytokine production
We next explored other molecules in the CD16A signaling pathway, including SHP-  that SYK and/or ZAP-70 mediates ADCC signal strength. However, the mechanism behind this finding was initially unclear. The enhanced function of SYK-ablated cells was independent of changes in CD16A expression ( Figure S5). This led us to examine SHP-1 regulation of ADCC signal strength as the mechanism. SHP-1 is a phosphatase that can modulate signaling in multiple pathways. Relevant to this study, SHP-1 can dephosphorylate ZAP-70 in T cells (Brockdorff et al., 1999;Plas et al., 1996). In addition, SHP-1 has been shown to mediate ITAM observed. In contrast, with ZAP-70 ablated, SYK may bind FcRg at a higher rate than with ZAP-70 present. This could facilitate more ITAMi-through SHP-1-than in canonical NK cells, resulting in the decrease in cytotoxic function we observed. This model is depicted in Figure 6.
Overall, our results indicate that ITAMi inhibits the ADCC pathway in NK cells, is facilitated by SYK and SHP-1, and could be a mechanism behind the enhanced ADCC observed in adaptive NK cells.
Interestingly, IFNg production from SHP-1 ablated NK cells was consistently lower than control NK cells-the opposite result of SYK ablation. One explanation could be different

Declaration of interests
The authors declare no competing interests related to the content of this work.   Figure 2D and FcRg-ablated samples in Figure 1G was stratified by CMV status (E-F). Wilcoxon matched-pairs signed rank test (A-C). Significance shown in D carried over from tests run for A-C. Unpaired t-test (E-F). *: p < 0.05; lack of stars indicates nonsignificance.     line indicates the minimum protein ablation % at day 6 for a sample to be included (25%). Red lines represent the median. One-way ANOVA with Tukey's multiple comparisons test. *: p < 0.05; lack of stars indicates non-significance.