The FCGR3A 158 V/V-genotype is associated with decreased survival of renal allografts with chronic active antibody-mediated rejection

Natural killer (NK) cells express the Fc-gamma receptor CD16 (FCGR3A) and could therefore mediate renal endothelial cell damage in cases of chronic-active antibody mediated rejection (c-aABMR). The V/V-genotype of the FCGR3A 158 F/V polymorphism is associated with increased CD16 expression and cytotoxicity by NK cells. This study evaluated whether this genotype is associated with the diagnosis of c-aABMR and renal allograft loss. The distribution of the FGCR3A 158 F/V-genotypes was not different for c-aABMR cases (N = 133) compared to control kidney transplant recipients (N = 116, P = 0.65). The V-allele was associated with increased median fluorescence intensity (MFI) of CD16 by NK cells (MFI 3.5 × 104 versus 1.3 × 104 for V/V and F/F-genotype, P < 0.001). Increased expression of CD16 correlated with CD16-dependent degranulation of NK cells (R = 0.4; P = 0.02). Moreover, the V/V-genotype was significantly associated with a higher glomerulitis score and an independent risk factor (HR 1.98; P = 0.04) for decreased allograft survival. Death-censored graft survival in c-aABMR cases at 3 years follow-up was 33% for the FCGR3A 158 V/V-genotype versus 62% for the F/F-genotype. In conclusion, the FCGR3A V/V-genotype increases CD16-mediated NK cell cytotoxicity and is associated with a higher glomerulitis score and decreased graft survival in cases with c-aABMR.

. Demographic and patient characteristics at baseline and time of diagnosis of c-aABMRh. Data represent median (IQR) and number (proportion of total), respectively. a Estimated glomerular filtration rate (eGFR): calculated using the CKD-EPI formula, multiplied by 1.159 when a patient has the African/Caribbean ethnicity.  2B). In addition, the CD16 MFI of NK cells was positively associated (Pearson's correlation coefficient; R = 0.40; P = 0.02) with the CD16-dependent degranulation potential of NK cells, expressed as CD107a URI (Fig. 2C). Summarizing, our results revealed the V-allele to be associated with an increased expression of CD16 on circulating NK cells and CD16-dependent NK cell function.

At time of transplantation
The V-allele of FCGR3A is associated with renal microvascular inflammation. The FCGR3A 158 F/V-genotypes were related to degree of microvascular inflammation [MVI] as measured by the Banff scores for glomerulitis (g) and peritubular capillaritis (ptc). Cases having the V-allele had significantly higher g-scores at time of renal allograft biopsy. The median g-score amounted to 2, 2 versus 1 for the V/V-, F/V-versus F/Fgenotype (P = 0.01), respectively. In early ABMR but not caABMR 14 , presence of DSAs were found to be associated with extent of microvascular inflammation 21 . We evaluated whether the presence of DSAs was associated with the degree of renal microvascular inflammation in cases with c-aABMRh in the this cohort. Although, the degree of MVI tended to be associated with presence of DSAs (P = 0.09), no significance was reached for the association with g (P = 0.27) or ptc (P = 0.68) scores. Only 28 out of 70 cases tested for DSAs were DSA-positive (Table 1), which limits the evaluation of association with FCGR3A 158 F/V-genotypes with level of inflammation for this subgroup. For DSA-negative c-aABMR cases (N = 42), the level of glomerulitis tended to be associated with the different genotypes (P = 0.05), i.e. similar to that observed for the total cohort.
In addition, ptc expressed as ptc score (P = 1.00) or as extent of ptc, i.e. being focal (< 50%) versus diffuse (≥ 50%) (P = 0.96) was not associated with FCGR3A 158 F/V-genotypes and this was also not observed for DSAnegative cases.
Positivity of C4d within PTC is associated with complement activation, which represent another mechanism by which DSAs can inflict damage to the renal allograft 1,7 . In our study, positivity for C4d was only detected in 21% of the cases diagnosed with c-aABMR (Table 1) and although associated with presence of DSAs (P = 0.001), no association with extent of microvascular inflammation was observed (P = 0.28). Moreover, FCGR3A 158 F/Vgenotypes were not associated to C4d-positivity (P = 0.57), which might relate to the fact that they represent different mechanisms of mediating damage to the graft.
The FCGR3A 158 V/V-genotype is associated with decreased renal allograft survival. In order to evaluate whether this single nucleotide polymorphism within FCGR3A (158 F/V) is associated with renal allograft survival, Kaplan-Meier curves for the different FCGR3A 158 F/V-genotypes were generated for the c-aABMRh cohort. We evaluated survival of renal allografts at 3 years following diagnosis of c-aABMRh. Fiftytwo out of 133 (39%) c-aABMRh kidney transplant recipients lost their renal allograft due to graft failure and 6 out of 133 (5%) died with a functioning renal allograft ( Table 3). The presence or absence of DSAs was not associated with graft survival (P = 0.70) as was reported before by our group 14 and others [22][23][24][25] . Overall renal allograft survival was associated with the FCGR3A 158 F/V genotype (P < 0.01; Fig. 3) and similar results were obtained for DSA-negative cases (P = 0.04). The V/V-genotype showed a significant lower renal allograft survival compared to the F/V-as well as the F/F-genotype, using the pairwise between strata comparison (Mantel-Cox log rank statistical analysis) for the total cohort (P < 0.01 and P = 0.01, respectively) as well as DSA-negative cases (P = 0.03). Median (min-max) renal allograft survival times amounted to 2.1 (0.1-3), 2.7 (0.1-3) and 3.0 (0.4-3) years after diagnosis of c-aABMRh for the FCGR3A 158 V/V-, F/V-and F/F-genotype, respectively. Graft survival was not affected by the FCGR3A 158 F/V-genotype for the control cohort of kidney transplant recipients without rejection (P = 0.90; data not shown).
To determine whether the V/V-genotype was an independent factor contributing to the risk of renal allograft loss, relevant clinical characteristics from Table 1, like age of the kidney transplant recipient at transplantation, gender of kidney transplant recipient, age of the kidney donor, type of kidney donor (living or deceased and ABOcompatible or ABO-incompatible), re-transplantation and eGFR at time of the for-cause biopsy were included in a multivariate Cox regression model. Out of these variables only the FCGR3A 158 V/V-genotype (P = 0.04) together with eGFR at time of diagnosis (HR 0.94, 95% CI 0.91-0.96, P < 0.01) were independently associated with renal allograft survival). Chronic-aABMRh kidney transplant recipients having the FCGR3A 158 V/Vgenotype had a 1.98-fold (95% CI 1.01-3.82) increased risk (P = 0.04) for losing their allograft within 3 years.

Discussion
In this study, we investigated the association between a functional FCGR3A 158 F/V single nucleotide polymorphism and diagnosis of c-aABMR and renal allograft loss thereafter. The results show that the V-allele was linked to increased expression of CD16 on NK cells and CD16-mediated NK cell cytotoxic potential. In addition, this allele was associated with an increased glomerulitis score and the V/V-genotype was associated with a decreased renal allograft survival after the diagnosis of c-aABMR. However, the FCGR3A 158 V/V-genotype appeared not to be a risk factor per se for the development of c-aABMR after transplantation. This observation adds evidence for the pathogenicity of the V-allele as was shown in a previous publication that studied the impact of FCGR3A 158 F/V genotypes in kidney transplant recipients with c-aABMR 16 . In accordance with our study, the distribution of the F/V alleles was similar to the control population. Moreover, the V-allele was associated with a higher degree of peritubular capillarities (PTC) but did not affect the slope of eGFR loss and graft loss. As discussed by the authors, the latter finding is counterintuitive and contradictory to earlier studies, which showed a relation between the degree of MVI and graft loss [26][27][28] . In our cohort, we found a higher glomerulitis score to be associated with the V-allele of the FCGR3A 158 F/V single nucleotide polymorphism.
The V-allele was associated with increased levels of CD16 and CD16-mediated NK function as assessed by the CD16 DRI and CD107a URI. The V/V-genotype, but not the F/V-genotype was correlated with an increased risk for renal allograft loss. Unlike our study, where for-cause biopsies were taken, protocol biopsies were taken in the study by Arnold et al. 16 , allowing identification of cases with subclinical c-aABMR that may have a much better prognosis 29 . This may be an explanation for the discrepancy in clinical outcome associated with the FCGR3A 158 F/V genotypes between their cohort and ours. Furthermore, the fact that cases with c-aABMR having the V/V-or F/F-genotype tended to be more similar in baseline characteristics when compared to those having the F/V-genotype, could be another explanation. Nevertheless, we observed a significant increased risk for renal allograft for cases having the V/V-genotype compared to both those having the F/V-as well as F/F/-genotype.
Overall, the findings of this study indicate that the V/V variant allows for increased activation of the NK cell after FCGR3A ligation which may cause more inflammation and progressively more tissue damage to the www.nature.com/scientificreports/ renal allograft with c-aABMR. The possible pathogenetic role of CD16 is underlined by the finding of increased transcript levels of CD16 in biopsies diagnosed with ABMR [8][9][10] . A review recently, published by Miyairi 30 , summarizes literature on the role of NK cells as critical effectors during ABMR. Detection of NK cell infiltration in biopsies is limited by the number of specific markers and staining antibodies available. CD16 was used to stain for NK cells in lung grafts 31 , CD56+ NK cells were detected in peritubular microvasculature during ongoing biopsy-indicated ABMR 8 and others have used CD57 and NKp46 to identify NK cells in biopsies of ongoing AMR and describe their association with microvascular inflammation [32][33][34] . Interestingly, activation of NK cells in absence of DSAs also strongly associated with severity of glomerulitis and peritubular capillarities and less with C4d deposition 35 . Recently, infiltrates in biopsies of kidney transplant recipients diagnosed with c-aABMR were immunophenotypically characterized 36,37 . NK cells were found at relatively low frequencies but increased proportions of NK cells within the intravascular glomerular as well as peritubular compartment were observed in cases with ABMR when compared to TCMR.
Therefore, present data suggest that NK cells and in particular the FCGR3A (CD16) + are most likely involved in the pathogenesis of c-aABMR.
Of note is that myeloid cells like macrophages also express CD16 and they are among the cells found in the renal microvasculature and interstitium in cases of c-aABMR 37 . Increased macrophage-associated transcripts were described for individuals with the V-allele compared to the F/F-genotype in the ABMR cases by Arnold et al. 16 . This implies that in addition to NK cells, expression of the V/V variant of FCGR3A by myeloid cells may also contribute to increased inflammation and damage as CD16 appears to be indispensable for ADCC by monocytes 38 .
Several other studies have investigated the FCGR3A polymorphism in solid organ transplantation other than kidney transplantation. In heart transplantation, Paul et al. demonstrated that this polymorphism contributed to an early (non-invasive) evaluation of risk stratification for cardiac allograft vasculopathy (CAV), an important cause of late mortality after heart transplantation 12 . These clinical data are supported by the observation that increased expression of CD16 and CD16-mediated NK function associated with the V/V-variant were observed heart transplant recipients 12 as well as renal transplant recipients 16 . In accordance with these findings we previously showed increased CD16 expression by circulating NK cells in cases with c-aABMR 11 . This FCGR3A polymorphism was also found to stratify patients at risk for acute rejection in the first 3 months after transplantation, in a cohort of lung transplant recipients 39 . They also observed the V/V-genotype to behave different from F/V-genotype, i.e. to significantly reduce the acute-rejection free survival when compared to F/V-and F/F-genotype. Taken together, the current data suggest that FCGR3A polymorphism is associated with rejection-related complications across various types of solid organ transplantation.
One of the limitations of our retrospective study involves the lack of DSAs from the complete c-aABMR cohort. Although DSA-positive and -negative c-aABMR are similar with respect to renal allograft survival, there might be differences in terms of underlying mechanisms of damage to the renal allograft. However, in our cohort, we did observe similar findings for the DSA-negative cohort compared to the total cohort with respect to this FCGR3A 158 F/V SNP and level of microvascular inflammation as well as renal allograft survival. Furthermore, antibodies directed to non-HLA might also contribute to damage to the renal allograft but this information is lacking. Another limitation of the present study is the lack of a validation cohort, allowing confirmation of our findings in a similar large group of c-aABMR cases.
Concluding, the V-allele of the FCGR3A 158 F/V single nucleotide polymorphism leads to increased CD16 expression and upregulated cytotoxicity of NK cells. Clinically this is associated with increased microvascular inflammation in the glomerulus and significantly decreased renal allograft survival of grafts diagnosed with c-aABMR for cases having the V/V-genotype. Assessment of this FCGR3A 158 F/V SNP may be of importance with respect to interpretation of results from studies. Furthermore, it may add to the risk stratification for graft loss in kidney transplant recipients by identifying patients that might benefit from a more intense immunosuppressive regime.

Material and methods
Study population. For this study we included 141 kidney transplant recipients, diagnosed with clinical c-aABMR (within our hospital) in the period from 1998 to 2019 of which snap-frozen PBMCs prior to transplantation were stored in the kidney transplant biobank. Patients in whom c-aABMR was diagnosed following a transplantectomy, were excluded from analysis (N = 8). As a control population we selected 116 kidney transplant recipients from the biobank, transplanted within the same period, without a diagnosis of c-aABMR. All kidney transplantations were performed across a negative CDC crossmatch. Demographic and clinical parameters were collected for the c-aABMR cohort at time of transplantation and diagnostic biopsy. All renal biopsies were done on indication (eGFR loss or proteinuria) and re-evaluated by an experienced renal pathologist based on the 2015 Banff classification 40 . Donor-specific anti-HLA antibodies (DSAs) were measured using the Lifecodes Lifescreen Deluxe (LMX) kit according to the manufacturer's manual (Immunocor Transplant Diagnostics Inc., Stamford, CT, USA). Samples positive for either HLA class I (HLA-A, -B or -C) or HLA class II (HLA-DQ or -DR), were further characterized with the Luminex Single Antigen assay, using LABscreen HLA class I and II antigen beads (One Lambda, Canoga Park, GA, USA). Before 2009, DSAs were not routinely assessed but 40% of the tested cases (N = 70) were DSA-positive. When histologic criteria were met, a diagnosis of c-aABMRh, termed suspicious for c-aABMR in the Banff 2015 criteria, was made in accordance with recent publications 13,14,[40][41][42] .
Renal allograft function was determined by calculating the estimated glomerular filtration rate (eGFR) using the CKD-EPI formula. www.nature.com/scientificreports/ Follow up of c-aABMRh kidney transplant recipients was until 1st of January 2020 and graft loss/failure was defined as the need for dialysis or a re-transplantation. The date of diagnosis of c-aABMRh and date of graft failure were used to calculate the graft survival upon diagnosis.
Kidney transplant recipients gave written informed consent and the study was approved by the Medical Ethical Committee of the Erasmus MC (MEC-2012-022 for pre-transplant material and MEC-2015-222 for the collection of data from cases of c-aABMRh and controls). The study was conducted in accordance with the Declaration of Helsinki and the Declaration of Istanbul.
Peripheral blood mononuclear cell isolation. Prior to and at time of the for-cause biopsy, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by using Ficoll-Paque (GE HealthCare, Uppsala, Sweden). Two million PBMCs of the sample, obtained prior to transplantation, were snapfrozen in liquid nitrogen for isolation of genomic DNA (see below). The remaining PBMCs were washed, frozen at 10 × 10 6 /vial in RPMI-1640 with Glutamax (ThermoFisher Scientific, Landsmeer, The Netherlands) supplemented with 100 IU/mL penicillin/streptomycin and 10% heat-inactivated pooled serum and 10% dimethyl sulphoxide (Sigma Aldrich, Darmstadt, Germany) in liquid nitrogen until further use.
FCGR3A 158 F/V (rs396991) genotyping. Genomic DNA was extracted from 2 × 10 6 snapfrozen PBMC using the QIAamp DNA Mini isolation kit (Qiagen, Venlo, The Netherlands) according to manufacturer's instruction. FCGR3A rs396991 genotype was determined by the StepOnePlus Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) using Taqman SNP Genotyping assay (assay ID C_25815666_10; ThermoFisher Scientific Inc, Bleiswijk, The Netherlands) and Taqman Universal PCR Master Mix according to manufacturer's instruction.

Statistical analyses.
Normally distributed data are expressed as mean ± SD, non-normally distributed data as median and IQR. Continuous variables of c-aABMRh and control kidney transplant recipients were compared using unpaired T test or Mann-Whitney U test. Discrete data were analyzed as frequencies with Chi-square test or Fisher's exact test. Demographic as well as patient characteristics are depicted as median and IQR, number and proportion of total, respectively. Comparisons of characteristics between the different FCGR3A 158 F/V-genotypes was done using Chi-square test for discrete data and Kruskal-Wallis for continuous data. Death-censored graft survival was assessed taking into account the FCGR3A 158 F/V-genotype for the c-aABMRh cohort by Kaplan-Meier analysis with log-rank statistics for difference between strata (pooled and pairwise). Multivariate Cox regression analysis, using the Enter method as well as the Forward and Backward stepwise Likelihood Ratio method, was used to evaluate the significance and contribution of the FCGR3A 158 F/V-genotype and other relevant clinical characteristics with respect to renal allograft survival. The level of glomerulitis (g) and peritubular capillaritis (ptc) as well as the combination thereof representing the total level of microvascular inflammation (MVI) were correlated to the FCGR3A 158 F/V-genotype. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software La Jolla, CA,USA) and IBM SPSS statistics for Windows, version 25 (SPSS Inc. IL, USA). The significance level (P value) was two-tailed and an α of 0.05 was used for all analyses.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.