Association of NK cell subsets and cytotoxicity with FCGR3A gene polymorphism in functional NK cell deficiency

SUMMARY OBJECTIVE: The purpose of this study was to assess the association between clinical, laboratory, and functional analyses and polymorphism in the FCGR3A gene in individuals with functional NK cell deficiency. METHODS: A total of 15 functional NK cell deficiency patients and 10 age-matched healthy controls underwent NK cell subgroup, cytotoxicity, and FCGR3A whole-exome analysis with next-generation sequencing. RESULTS: Three different NK cell subsets (CD56brightCD16neg, CD56brightCD16int, and CD56dimCD16hi) were identified. No statistically significant difference was found in the ratio of CD56brightCD16neg cells between patients and controls. CD56brightCD16int and CD56dimCD16hi ratios were found to be significantly lower in patients. As a result of NK cell cytotoxicity analysis, a proportional decrease of K562 amount between patients and controls was found to be statistically significant (p<0.001). In the FCGR3A whole-exome analysis, all patients were found to be homozygous mutant for the c.526G > T (p.V176F) in exon 4, while three patients were homozygous wild type and 12 patients were heterozygous for the c.197T>A (p.L66H) in exon 3. CONCLUSION: In this study, a group of pediatric patients with suspected functional NK cell deficiency were evaluated and the findings indicated that NK subsets, cytotoxicity results, and FCGR3A gene polymorphism were found to be correlated with the clinical features. We conclude that this kind of study might contribute to follow-up the patients in time.

The FCGR3A (NM_000569) gene on chromosome 1 has five exons, is of 8345 bp size, and encodes CD16, a low-affinity receptor (50-70 kDa) that binds to the Fc region of IgG.FCGR3A is expressed in macrophages, γδ T cells, and mainly NK cells 6 .CD16 plays a role in antibody-dependent cellular cytotoxicity (ADCC) in NK cells 10 .The homozygous p.T230A substitution has no impact on CD16 expression but hinders detection with B73.1 monoclonal antibody (mAb), impairing NK cell cytotoxicity.This substitution is linked to functional NK cell deficiency (FNKD) 11,12 .FNKD is characterized by non-functional NK despite the normal range of mature NK cells in the PB and was first described in two patients with recurrent upper respiratory tract and herpes simplex virus (HSV) infections.Genetic and functional analysis of the patients showed that the ADCC function of NK cells was not impaired, their cytotoxicity was impaired, and p.L66H missense mutation was detected in the FcγRIIIA gene 2,13 .Although there is sensitivity to viral infections in FNKD, the most common finding is recurrent upper respiratory tract infection 12 .Although the population of CD56 dim and CD56 bright cells, which are NK cell subsets, is reported to be variable, there is still no clear data 14 .
A common polymorphism at position 176 (p.V176F) influences CD16's IgG Fc affinity 12 .From this point of view, it was aimed to investigate the relationship between NK cell subsets and NK cell cytotoxicity and FCGR3A polymorphism in FNKD.

Patients
The study included FNKD patients (n:15) admitted to our clinic between 2016 and 2018, who had viral infections (influenza, rhinovirus, respiratory syncytial virus A-B, herpes virus, and metapneumovirus), undetectable CD16 expression by NK cell and FCGR3A polymorphisms B73.1 mAb on NK cells (despite normal CD16 expression by 3G8 mAb), and age-matched healthy control (n:10).

NK cell cytotoxicity analysis
NK cell cytotoxicity was assessed using the NKTEST ® kit (Catalog number: 15991230, Glycotope Biotechnology, Heidelberg, Germany) with varying E:T ratios [NK as effector cells, (E); K562 as target cells, (T)] and analyzed by flow cytometry (BD Biosciences, Heidelberg, Germany) using the FACSDiva software version 6.1.3.

R: ACT T TGGGAAGCCAAGGCTG; primers for exons 4-5; F: CCATGCTCAGTAAATTACTTGGTG, R : AT T TA G G A ATA AT TG T T T T T T T T TC C C ) .
Polymorphisms detected in two different regions of the FCGR3A gene were validated by Sanger sequencing (for c.526G > T (p.V176F); F: ACTTTTGGGGACCTCCTGGT, R: TCACAGCTGGAAGAACACTGC; for c.197T>A (p.L66H); F: TGGGACCACACATCATCTCA, R: CAAAGGCTGTGGTGTTCCTG).
After primer design, optimization was performed, and a PCR pool was created.Purification of the PCR pool was done using the NucleoFast ® 96 PCR kit (Cat.no.740786, Macherey-Nagel GmbH, Germany).The resulting DNA was quantified (Nanodrop ND-1000, Thermo Inc.) and standardized to 0.5 ng/μL.DNA library preparation utilized the NexteraXT DNA Library Prep Kit (Cat.no.FC-131-1024, Macherey-Nagel Gmbh, Germany).Illumina Miseq NGS (Illumina, San Diego, CA, USA) was used for sequencing, and data analysis was performed with the MiSeq Reporter Software (Illumina Inc.) and IGV 2.5.0 software (Broad Institute) using the hg19 human reference genome (Figure 1) 15,16 .

Statistical analysis
Normality assumption was assessed using the Shapiro-Wilk test.Two-way repeated-measures ANOVA and Mann-Whitney U test compared concentration changes in patient and control groups in NK cell cytotoxicity.Mauchly's test of sphericity evaluated the sphericity assumption.The within-subjects effects table assessed concentration-dependent changes, main effects, and interactions with groups.Simple effect tests compared concentrations within each group.Bonferroni correction controlled multiple comparisons.Results included F-test values and partial eta square (η 2 ) for effect size.Pearson correlation examined the relationship between NK cell subgroups and cytotoxicity.p<0.05 indicated statistical significance.Analyses were performed by the JASP Team (Version 0.11.1;2019) software.

RESULTS
A total of 15 patients (11 males and 4 females) and 10 agematched controls (5 males and 5 females) were included.There was no significant difference in age and gender (p>0.05).The admission age was a median of 9.5 months (2-144±42), and the study age was a median of 2 years (2-15±5).Seven patients presented with frequent illness, five patients with recurrent bronchiolitis, two patients with recurrent resistant fever, and one patient with persistent wounds on the face and recurrent fever.When the clinical histories of the patients are evaluated, it is noteworthy that the most common findings are fever, pneumonitis, and bronchiolitis.Eleven patients had a history of hospitalization due to infection.Six patients had consanguineous marriages, and eight patients had a family history of death from an unknown cause in infancy.In addition, as recurrent infections observed in patients may be related to dysfunction of anatomical and physiological barriers and allergic diseases, these findings were also evaluated and these conditions were excluded.Other immunological tests (e.g., immunoglobulin levels, lymphocyte subsets, vaccine responses, isohemagglutinin titers) were normal.

NK and NK cell subgroup analysis
Patients had significantly lower total NK cell rates (6.7%) compared with controls (18.8%,p=0.002).CD16 expressions were also significantly lower in patients (p<0.001).

NK cell cytotoxicity
In the control group, the decrease in target cell ratio was 3.07fold between E:T4 (13%) and E:T3 (40%) and 4.07-fold between E:T4 and E:T2 (53%).In the patient group, the average decrease was 1.25-fold between these rates.These results suggest reduced NK cell cytotoxicity in the patient (Figure 2B).
Correlation analysis was performed for E:T2 and E:T3 concentrations in NK cell cytotoxicity.A stronger correlation was observed in E:T2 for overall cytotoxicity.Positive correlations were found between CD56b right CD16 int and CD56 dim CD16 hi subsets and NK cell cytotoxicity (p=0.044 and p=0.018).A non-significant negative correlation was found between CD56 bright CD16 neg subset and cytotoxicity (p=0.433)(Table 1).

DISCUSSION
FNKD patients were assessed for NK cell subsets, cytotoxicity, and FCGR3A polymorphism.The study aimed to investigate low cytotoxicity in patients compared with controls, focusing on FCGR3A polymorphism.Correlations were found among NK subgroups, cytotoxicity, gene polymorphism, and clinical features.
The literature suggests variations in NK cell subsets with gene polymorphism in NKD 10,17 .CD16 expression differences in cases with these polymorphisms are not well studied.Our study detected CD16 epitope loss and consistent NK cell subset results.NK cell rates were similar between patients and controls (p=0.002)and within the normal range for age, aligning with the literature 18,19 .
NK cell subsets analysis revealed no significant difference in CD56 bright CD16 neg between groups, but significant differences were observed in CD56 bright CD16 int and CD56 dim CD16 hi subsets.Limited literature is available on the normal ranges of NK cell subsets.Angelo et al., reported CD56 bright CD16 neg as 6.9-8.56% in 40 healthy individuals 5 .In our study, the control group had a lower rate of CD56 bright CD16 neg (2.04%) compared with the literature.Notably, our study involved a pediatric group, which may contribute to the variation in CD56 bright CD16 neg levels compared with Angelo et al.'s study conducted with adult donors 5 .
CD56 bright cells are precursors of CD56 dim NK cells 6,20,21 and exhibit high proliferative capacity [21][22][23] .The lack of significant difference in CD56 bright CD16 neg between patients and controls suggests normal development of NK cells up to the CD56 bright stage.CD56 bright NK cells have potent cytokine secretion, while CD56 dim subsets are responsible for natural cytotoxicity 18,19 .CD56 dim CD16 dim cells were more degranulated than CD56 dim CD16 bright cells in PB.In patients, low CD56 dim CD16 hi cells were consistent with the previous literature 22,24 .
The literature suggests that the CD56 dim subset is responsible for NK cell cytotoxicity 18,19 .However, this is the first study to examine this in the pediatric group, and there are no data on the effectiveness of different gating methods.Correlation analysis showed a negative correlation between CD56 bright CD16 neg NK subset and cytotoxicity and a positive correlation between CD56 bright CD16 int and CD56 dim CD16 hi .Results align with patient cell counts.CD56 bright CD16 int counts (patients: 3.7% and controls: 22.5%) and CD56 dim CD16 hi counts (patients: 1.3% and controls: 3.2%) differed significantly (p<0.001),indicating their role in NK cell cytotoxicity.
CD16's role in NK cell cytotoxicity was demonstrated.CD56 dim CD16 neg expression negatively affects cytotoxicity, while CD16-expressing cells have a positive impact.CD56 bright CD16 neg subgroup negatively affects cytotoxicity.Correlation analysis implies that NK cell cytotoxicity can be assessed without specific analysis.
FCGR3A gene sequencing revealed exon 3 variations.Heterozygous genotype was found in 12 patients, while three patients had homozygous normal genotype.Clone B73.1 did not detect CD16 expression in any of these patients, implying that additional unidentified polymorphism may cause epitope loss 11,25 .Epitope loss with B73.1 mAb is not solely caused by the p.L66H polymorphism, and other polymorphism/mutations may also contribute.Findings align with Lenart et al.'s study, indicating the presence of additional gene mutations causing CD16 epitope loss 25 .Polymorphic changes were observed in exon 4. Transversion in the FCGR3A gene led to the increased binding affinity of NK cells to IgG1 or IgG3 antibodies, affecting NK cell-mediated ADCC.Extreme polymorphism in this region has been observed in different populations but lacks data on the Turkish population.Patients in the study exhibited homozygous wild-type or heterozygous genotypes for exon 3 and mutant homozygous genotype for exon 4.

CONCLUSION
This study assessed patients with suspected FNKD using comprehensive functional and genetic analyses.NK cell cytotoxicity analysis, despite its complexity, plays a crucial role in FNKD diagnosis.Correlating NK cell subsets with cytotoxicity results can aid in predicting NK cell cytotoxicity.FCGR3A gene sequencing involved a limited number of patients and controls, but detecting mutations is essential for disease diagnosis and patient monitoring.

Figure 1 .
Figure 1.Presentation of the detailed data interpretation and analysis.

Figure 2 .
Figure 2. (A) Distribution of NK subgroups in patients and controls.(B) Variation of different ratios in target cell amount in NK cell cytotoxicity analysis (*statistically significant; E:T: effector: target cell ratio).