FCGR3A and FCGR2A Genotypes Differentially Impact Allograft Rejection and Patients' Survival After Lung Transplant

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37–9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08–3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37-9.61), but it could not be associated with the level of CD16-mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08-3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of INTRODUCTION Lung transplantationi (LTx) remains a challenging therapeutic option for patients with end-stage pulmonary disease. Significant improvement in immunosuppressive strategies has led to decreased lung allograft loss in the early post-transplant period. When compared to other solid organ transplantation settings, LTx remains associated with the lowest survival rates with a median survival of 6 years after transplant (1). Chronic lung allograft dysfunction (CLAD) is the main cause of chronic lung allograft rejection and is characterized by an irreversible loss of lung function associated with a high prevalence of complications such as bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS) (2). Factors that relate to the HLA mismatch, to the graft procedure (ischemia, unilateral or bilateral surgery) and initial lung disease can lead to highly variable levels of recipient immune response to the lung allograft. Inflammatory biomarkers and antibodies, occurrence of acute cellular rejection (ACR), infections/colonization, auto-immunity, and air pollution have been analyzed for their potential predictive value in anticipating development of immunological responses associated with CLAD. Toll receptors, pro-inflammatory cytokines and non-classical HLA molecules, i.e., HLA-G and HLA-E polymorphisms or haplotypes, have been associated with CLAD (3)(4)(5)(6)(7)(8)(9), but the underlying mechanisms involved in this devastating outcome of LTx in a given recipient are still poorly understood. Antibodymediated rejection (ABMR) has been associated with a higher incidence of chronic lung allograft dysfunction (CLAD) and mortality after LTx but the specific mechanisms and histological or immunological biomarkers that allow to define the clinical ABMR entity still await further comprehension (10). Detection of preexisting or development of de novo donor-specific human leukocyte antigen (HLA) alloantibodies (DSAs) have been extensively investigated for their potential value as biomarkers of humoral responses that may predict adverse outcome of LTx.
Various studies suggest that the pre-transplant detection of circulating DSA is not associated with an increased risk of developing CLAD or related death if prospective cross-match testing was negative (11,12). The pretransplant detection of DSA and antibodies directed againd non-HLA antigens such as angiotensin type 1 receptor (AT1R) and endothelin type A receptor (ETAR) is reported to have a negative impact on lung transplant outcome (13).
Early detection of de novo DSA detected at 1 month after LTx has been significantly associated with a worse outcome (14). Multiplex solid phase single antigen bead assay (SAFB) detection of de novo DSAs, notably anti-HLA DQ DSA, have also been associated with acute cellular rejection (ACR) (15) CLAD (16)(17)(18). The presence of circulating DSA at the time of lung allograft biopsy has been identified as a risk factor for graft loss (19).
Although there is more and more direct and indirect evidence regarding the role of de novo DSA in CLAD occurrence, the mechanisms that sustain the variable toxicity of these anti-HLA antibodies to the lung allograft are still unclear. One of the wellknown cytotoxic mechanisms of DSA occurs through the IgGmediated activation of the complement cascade that results in C4d deposition within the graft. C4d staining in lung allograft biopsies is considered as a diagnosis criteria for ABMR (10,(20)(21)(22)(23)(24)(25)(26)(27)(28). However, although recent evidence suggests that detection of complement-binding DSA can be associated with lung allograft failure (29), the predictive value of complement-binding DSA on LTx clinical outcome remains to be firmly demonstrated.
IgG antibodies can also activate the immune system through ligation of functional Fc gamma receptors (FcyRs). Since these receptors are expressed by a variety of immune cells, including B cells, natural killer cells, platelets, dendritic cells and macrophages, FcR receptors constitute a major checkpoint that regulates the intensity of auto-and alloimmune responsiveness of the host in response to infectious and humoral threats (30,31). As infections and the immune alloreactivity of the recipient toward the transplant remain a leading cause of graft rejection and death during the first year after LTx, there is a need for biomarkers that may improve the monitoring of early humoral responses in LTRs (32)(33)(34) and open therapeutic perspectives to dampen antibody-driven inflammation in immunized patients (35,36).
Polymorphisms of FcγRIIA (FCGR2A [131R/H], rs1801274) FcγRIIIA (FCGR3A [158V/F], rs396991) genes, that respectively, encode for the CD32 and CD16 receptors for the Fc segment of IgG, have an impact on the level of expression and immune function of these activating FcγRs. Various reports have highlighted the clinical relevance of these SNPs in controlling the host immune response to monoclonal antibody therapy.
The presence of a histidine (H) rather than an arginine (R) at position 131 results in higher affinity for IgG1 and IgG2. The FCGR2A [131H/H] genotype has been associated with higher efficacy of Rituximab-mediated B cell depletion strategies before transplant of ABO-incompatible organs. This FCGR2A [131H/H] SNP has also been identified as a susceptibility marker associated with the severity of community-acquired pneumonia (37).Genetic variation in the FCGR2A gene has also been shown to be associated with an increased prevalence of invasive pneumococcal diseases and respiratory infections after LTx (38,39), regardless of the site of pneumococcal infection (40). Pro-inflammatory effects associated with the FCGR2A [131R/R] genotype have also been reported as a consequence of activating signals that result from the dynamic interactions between CD32 and its cognate C-reactive protein (CRP) and immunoglobulin ligands.
The clinical relevance of CD16 inflammatory pathways in antibody-mediated rejection of kidney and heart allografts has also been illuminated by studies that deciphered the molecular landscape of immune and endothelial cell transcripts that associate with ABMR lesions evaluated in allograft biopsies (41)(42)(43). Ligation of the Fc domain of IgG has also been identified as a mechanism that allows CD16-dependant clearance of viral pathogens by immune NK cells (44). Recent studies suggest that the Fc fragment of DSA can indeed exert its adverse cytotoxic and inflammatory effects though a complement-independent mechanism that relies on level of expression of the NK-cell surface CD16 receptor and its affinity for the Fc fragment of alloantibodies. The intensity of host antibody dependent cell cytotoxicity (ADCC) is known to be conditioned by the capacity of NK effector cells to form conjugates with antibody-coated allogeneic cells, which is in part influenced by the FCGR3A [158V/F] genotype (45)(46)(47). We and others have recently shown that polymorphic variation in the FCGR3A genes is also likely to affect the pathogenic effects of IgG alloantibodies by controlling the level of the CD16 dependent recipient's immunological cytotoxic responses to allogeneic donor cells exposed to chronic alloantibody threat (35,(48)(49)(50)(51).
Genotypic variation in the FCGR3A receptor can thus impact the strength of FcR-dependent ADCC responses of NK cells and regulate their capacity to secrete inflammatory cytokines and release CD107a/Lamp1 + cytotoxic granules containing perforin and granzyme (50,52,53). The inter-individual variability in the functional CD16 receptor-dependent engagement by the Fc fragment of alloantibodies has been shown to condition the intensity of antibody-dependent cellular cytotoxicity (ADCC) of NK cells and the response to IgG immunotherapy.
The strength of FcR-mediated ADCC can also be affected by the isotype and glycosylation status of alloantibodies. The IgG1 and IgG3 isoforms have higher affinity for the CD16 FCGR3A [158V/V] variant when compared to FCGR3A [158F/F], thereby spurring increased effector cell activity in response to these IgG subclasses. We have shown that this high-affinity homozygous FCGR3A [158V/V] genotype is an independent predictor of cardiac allograft vasculopathy (48) and may be a clinically relevant underlying mechanism that sustains the level of DSAmediated allograft injury (48)(49)(50). The recent evidence that the NK cell infiltration of the graft can predict kidney graft failure also sustains the clinical relevance of these NK cell mediated mechanisms of allograft injury (54).
Considering the central contribution of polymorphisms affecting FcγRIIA and FcγRIIIA genes and receptor function in the individual shaping of antibody-mediated inflammatory and cytotoxic alloimmune responses of the recipient, deciphering the mechanisms and FcγR susceptibility profiles that may be associated with LTx outcome is necessary to optimize risk stratification (8).
This study thus aimed to investigate whether FcγR polymorphisms may be linked to the pathogenic mechanisms of DSA toxicity and be associated with adverse clinical complications that impair patient and allograft outcome after LTx.

Participants and Study Design
We conducted a retrospective single-center study enrolling 158 adult patients who underwent lung transplants (LTx) at the Marseille Lung Transplant Center between December 2006 and December 2013. All patients from the French cohort (COLT, Cohort in Lung Transplantation, l'Institut du Thorax, INSERM UMR1087/CNRS UMR 6291, CNIL 911142) were recruited in this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. A group of 184 healthy unrelated volunteer French bone marrow donors were also recruited to constitute a control cohort, allowing the analysis of the FcγR genotype. Blood donations were collected in the "Etablissement Francais du Sang, " in accordance with BSL-2 practices. A medical interview was carried out prior to blood donation to exclude donors with medical contraindications. This study was carried out in accordance with the French Public Health Code (art L1221-1), approved by an institutional ethics committee and conducted in compliance with the Good Clinical Practice Guidelines declaration of Helsinki and Istanbul.

Post-transplant Clinical Management
All recipients received a similar standardized immunosuppressive regimen in accordance with our institutional protocols. Induction therapy consisted of intravenous administration (IV) of rabbit anti-thymocyte globulins (rATG, Pasteur Merieux, Lyon, France) given for the first 3 post-operative days [except when daily lymphocyte count was below 200/mm 3 , and when there were cytomegalovirus (CMV) and/or EBV mismatches (i.e., seronegative recipient and seropositive donor)] and high dose methylprednisolone (6 mg/kg/d Day 1, 2 mg/kg/d Day 2 and Day 3, and 1 mg/kg/d thereafter). The standard triple maintenance immunosuppressive regimen consisted of tacrolimus (adjusted to maintain whole blood levels varying between 12 and 14 ng/ml), mycophenolate mofetil in 5 patients (adjusted to a white blood cell count above 4,000 mm 3 ), and steroids (prednisone) tapered to 0.25 mg/kg/d over the first 3 months and stopped around 12 months after surgery.
According to CMV recipient positive (R+) and donor positive/recipient negative (D+/R-) status, patients received CMV prophylaxis with IV ganciclovir switch to oral valganciclovir.
Postoperatively, recipients received prophylactic or preemptive anti-infection treatment (antibiotic, antiviral, and antifungal therapies) according to their preoperative and/or concomitant infectious status.
Recipients had regular visits to the transplant center for clinical radiological and functional evaluation. At our institution, surveillance transbronchial biopsies are routinely performed at the end of the first month, or earlier if clinically indicated. All transbronchial biopsies were graded for ACR (A grade) and lymphocytic bronchiolitis (B grade) (LB) by lung transplant pathologists. ACR and LB were defined, respectively, as perivascular or peribronchial mononuclear inflammation according to the International Society for Heart and Lung Transplantation (ISHLT) criteria. Histologic appraisal of ACR and LB was conducted in accordance with accepted ISHLT standards in terms of the minimum number of biopsy samples and exclusion of opportunistic infection (27,28). Pulmonary function tests (PFTs) were routinely conducted at our center on a monthly basis for the first 12 post-operative months, at M2 intervals in the second year and at M3 intervals thereafter. The baseline FEV 1 value was calculated as the average of the 2 best FEV 1 values at least 2 measure gap. Baseline values of total lung capacity (TLC) and FEV 1 /FVC were defined as the average of the 2 measurements obtained at the same time as the best 2 FEV 1 measurements. Chronic lung allograft dysfunction (CLAD) was defined according to the standardized international criteria (55). The phenotype BOS or RAS was specified according to ISHLT guidelines (56). The following histological patterns compatible with AMR were also analyzed: (i) neutrophil capillaritis and (ii) acute lung injury with or without diffuse alveolar damage and with or without organizing pneumonia.

HLA Antibody Screening and Identification Protocol
Recipient serum samples from 2006 to 2013 were collected routinely prior to transplant, at the minimum when the patient was placed on the waiting list and before the transplant procedure (D0: Day 0) and serially after LTx (at month 1 and 3). All sera samples obtained from the 158 recipients were further assessed using Luminex single-antigen flow beads (SAFB) to determine antibody specificity using Single Antigen-One Lambda reagents (LABScreen R Single Antigen class I or LABScreen R Single Antigen class II, One Lambda, Thermo Fisher Scientific, Canoga Park, California, USA) according to the recommendations of the manufacturers. The mean fluorescence intensity (MFI) used the baseline formula proposed by the Fusion TM v 3.2 software. All beads with a normalized MFI threshold >1,000 were considered positive. Since detected circulating alloantibodies were often directed against distinct HLA class I and/or class II antigens, the cumulative mean fluorescence intensity (cMFI) of DSA was calculated as the sum of the MFI for each of the individual DSAs detected. DSA were considered to be HLA antibodies directed against donor HLA antigen. Putative HLA-Cw and -DQA1 DSA were identified in accordance with the conventional linkage disequilibrium that is reported between HLA-B and HLA-C loci and between HLA-DQB1 and HLA-DQA1 loci, as respectively, described in the French population (57) and by supplementary extensive genotyping of HLA-genes in the recipient. Putative HLA-DR51, -DR52 and -DR53 DSA were determined in accordance with the linkage disequilibrium between DRB3, DRB4, DRB5 loci and DRB1 loci of recipient. The allelic specificities of HLA-Cw, DQA and -DR51, -DR52 and -DR53 DSA were assigned accordingly.

C1q Detection
All patients with HLA antibodies were tested for the presence of C1q. The binding level was determined by the C1qScreen TM assay per manufacturer instructions (One Lambda, Thermo Fisher Scientific, Canoga Park, California, USA). Fluorescence intensity was measured using Luminex-based LABScan TM 100 flow analyzer. C1q specific antibody specificity and binding levels were analyzed and determined through the Fusion TM v 3.2 software. C1q MFI > 1,000 was considered positive.

FCGR2A and FCGR3A Genotyping
The detection of single nucleotide polymorphisms (SNPs) allowed for the genotyping of FCGR3A ([158V/F], rs396991) and FCGR2A ([131R/H], rs1801274) as described (5,48) using the SNAPSHOT technique. Genomic DNA was extracted from a 200-µl whole blood sample using the QIAmp Blood DNA Mini kit (Qiagen, Courtaboeuf, France) according to manufacturer instructions. A previously described homemade primer extension method (58) was used to simultaneously analyze the SNPs of the FcγR genes. The forward and reverse primers sequences were, respectively: FCGR3A-F 5 ′ TCCTAA TAGGTTTGGCAGTG 3 ′ and FCGR3A-R 5 ′ AAATGTTCA GAGATGCTGCT 3 ′ , FCGR2A-F 5 ′ CCAGGAGGGAGAAAC CATC 3 ′ and FCGR2A-R 5 ′ CTCTTCTCCCCTCCCTACAT 3 ′ . The extension primers are, respectively: FCGR3A_176-F: 30T-CCTACTTCTGCAGGGGGCTT and FCGR2A_166F 57T-CCAAAAGCCACACTCAAAGA. Data were analyzed using GeneMapper 4.0 with specific detection parameters. Using an in-house computer program, output files (.txt) exported from GeneMapper 4.0 were automatically formatted into files readable by the "Phenotype" application of the Gene[Rate] computer tool package (http://geneva.unige.ch/generate). For each different allele obtained, PCR products were sequenced on both strands using the BigDye Terminator v1.1 Sequencing Kit (Applied Biosystems) and analyzed on an automated fluorescence-based ABI PRISM 3130 XL genetic analyzer according to manufacturer protocol. FcR polymorphisms at these 4 loci were analyzed for allele frequencies, or homozygous or heterozygous allelic combination of FcγR alleles defining the corresponding genotype.

HLA Genotyping
Recipients and deceased donors were genotyped for low resolution HLA-A, -B, -DRB1, and -DQB1 loci by LABType R SSO (One Lambda, Thermo Fisher Scientific, Canoga Park, California, USA) according to the manufacturer's specifications and the retrieved output was analyzed for allele identification using HLA Fusion TM v 1.2.1. Software (One Lambda, Thermo Fisher Scientific, Canoga Park, California, USA).

Phenotypic Analysis of Antibody-Dependent NK Cell Activation
Evaluation of the CD16-dependent alloreactive potential of DSA was assessed using the previously described NK cell humoral activation test (NK-CHAT) (50). The level of serum-induced CD16 engagement and the degranulating potential of NK cells was assessed by flow cytometry analysis of the level of CD16 cell surface expression within the CD3 − CD56 + NK cell compartment, gated within PBMC effector cells after exposure to target B cells that had been precoated in the presence of LTRs or control sera. Effector cells used in the standardized assay were prepared from a healthy donor displaying the FCGR3A [158V/V] encoding the high-affinity CD16 variant. The HLA typing of the two B cell lines used as targets were A2/A2, B44/B56, Cw1/Cw5, DR1/DR4, DR53, DQ5/DQ7 and A3/A3 B7/B35, Cw4/Cw5, DR10/DR15, DR51, DQ5/DQ6). Sera were selected for the presence of DSA recognizing a limited set of HLA antigens expressed by EBV target cell lines. Briefly, 500,000 target B cells (B-EBV cell lines expressing the cognate DSA target alloantigens detected in LTR) were incubated with control (CTL) unsensitized human male AB serum (Lonza) to block FcR, then rinsed and incubated for 15 min in the presence of 50% LTR serum [or CTL serum supplemented or not with 10 γg/ml of Rituximab IgG (positive control)] and then rinsed to remove unbound antibodies. Incubation of effector PBMC and precoated allogeneic B-EBV cell targets (1:1 ratio) was performed for 3 h at 37 • C in presence of GolgiStop (Becton Dickinson 554724) and CD107-PC5 (Becton Dickinson 555802). Cells were then washed and labeled with CD3-ECD (Beckman Coulter A07748), CD16-PE (Beckman Coulter A07766), CD56-PC7 (Beckman Coulter A21692) for 15 min at room temperature protected from light. After 1 wash step, cells were resuspended in 500 µl PBS 2% SVF. Data acquisition and analysis was performed on a Beckman Coulter Navios cytometer. The mean fluorescence intensity of CD16 and percent of NK cells expressing Lamp1/CD107a expression was analyzed within the CD3-CD56+ NK cell subset. The NK-CHAT CD16 down regulation index (CD16DRI) was evaluated as a ratio between CD16 MFI measured on NK cells incubated with target cells in presence of effector cell autologous CTL serum/CD16 MFI of NK cells incubated with target cells and recipient serum to be tested. CD16DRI = (MFI CD16 control serum)/(MFI CD16 test serum). Alternatively, when serum at time of LTx was available for the assay, the baseline CD16 expression was evaluated in reference to the DSA negative serum collected at time of LTx. Sera containing HLA-DQ7 DSA obtained from kidney transplant recipients (KTR) with antibody-mediated rejection (ABMR) at time of diagnosis, previously described to induce high levels of CD16-dependent NK cell alloreactivity, were introduced as positive controls of the experiments, indexing the CD16 down regulation induced by LTx sera.

Statistical Analyses
Variables used to perform univariate and multivariable analyses included preoperative donor variables (donor age, sex, CMV status, HLA typing), preoperative donor-recipient matching parameters (age, sex, CMV status, and HLA mismatch), operative variables such as ischemia time, type of procedure (single vs. bilateral LTx) and preoperative and post-operative recipient variables (initial lung disease, HLA typing, pre-and posttransplant immunization status, occurrence of AR BOS, RAS, CLAD, or infectious bacterial episodes occurring during the first year post-transplant), and notably during the early post-Tx period at months 1 (M1) and 3 (M3) after LTx ( Table 1).
Continuous variables were presented as median values and 25-75 interquartile ranges or mean ± sd according to their distribution evaluated using the Agostino & Pearson omnibus normality test performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla, California, USA). Categorical variables were presented as percentages. Fisher's exact test and the chi-square test were used to compare categorical data and t-tests or Mann Whitney tests were used for the comparison of continuous variables.
The primary endpoints of this study were overall survival (OS) and disease/CLAD-free survival (DFS). OS was defined as the interval between the date of transplant and the last follow-up visit or death. DFS was defined as the time interval from transplant to the first event: either the graft failure or diagnosis of acute rejection or CLAD in living recipients, or the death of the patient. The Kaplan-Meier method was used to estimate overall survival and rejection-free survival. The log-rank test was used to assess the univariate effects on OS and DFS. For all analyses, a 2-sided p < 0.05 was considered statistically significant. Variables with a p < 0.2 were also considered to construct multivariate regression models. Multivariate analyses were performed using Fine and Gray's proportional hazards regression model. All analyses were performed using IBM SPSS 15.0 software (SPSS Inc., Chicago, IL) and the cmprsk package (developed by Gray, June 2001) on R2.3.0 software (http://www.R-project.org).

Characteristics of the LTR Cohort
The demographic characteristics and clinical features of the 158 adult LTRs (median age: 42 years, 75 females and 83 males) are summarized in Table 1. Patients received a first LTx for cystic fibrosis (37%), emphysema (29%), pulmonary fibrosis (22%), or another diagnosis (12%). The median follow-up time was 38 months after LTx. Median survival time of the 94 patients who were alive at time of follow-up was 4.2 years. Sixty-four patients (40.5%) died during the study follow-up (median time before death 12.6 months, 25-75 interquartile range: 1.8-35.5 years) and 5 patients experienced CLAD-associated graft failure with indication of a second lung transplant or death. Among deceased patients, 32 patients died during the first year post-LTx, among which 12 patients died during the first month post-LTx and 9 between the first and third months post-LTx. Acute rejection diagnosis was confirmed in the lung transplant biopsy of 53 patients (36%), the first rejection episode occurring before the  (37) Fibrosis, n (%) 35 (22) Emphysema, n (%) 46 (29) Other, n (%) 18 (12) Transplantation type Bilateral LTx, n (%) 118 (74) Single LTx, n (%) 36 (24) Other, n (%) 4 (2) CMV Risk Immunization Status Distribution of FCGR3A and FCGR2A Polymorphisms Genotyping of the FCGR2A and FCGR3A polymorphisms was performed in 158 LTRs and analyzed in reference to a control cohort of 184 healthy donors (CTLs) (Figure 1) (Figure 2). The development of de novo DSA was observed at M3 in 6 LTRs with no detectable DSA at M1 (4 HLA class I, 1      who developed RAS-associated CLAD were homozygous for the FCGR3A-FF allele (p = 0.034). While the FCGR3A [158V/F] could not be significantly associated with the risk of CLAD, FCGR2A [131R/R] was identified as a susceptibility marker associated with lower CLAD-free survival of LTRs (n = 87, logrank test for equality of survivor functions p = 0.0123, Figure 3B). Multivariate Cox regression models adjusting covariables identified using univariate analysis further identified FCGR2A [131R/R] as an independent susceptibility marker associated with CLAD (OR 2.2; p = 0.022, 95CI 1.126-4.435) ( Table 4). Multivariate Cox analysis further showed that the FCGR2A [131R/R] genotype is significantly associated with the enhanced risk of developing a composite LTx adverse outcomes (CLAD, graft failure or death), independently of other factors such as detection of DSA at M3 or native emphysema lung disease ( Table 4).

LTx DSAs Have No Major Impact on CD16-Dependent NK Cell Cytotoxic Activation
Considering our previous finding that identifies the potential value of FCGR3A [158V/F] in predicting cardiac allograft vasculopathy (48), we further investigated whether the presence of association of acute rejection events with high affinity FCGR3A [158V/V] genotype could be associated with the DSA-mediated pathogenic effects that promote acute rejection mechanisms of lung allograft. Using the NK-cellular humoral activation test (NK-CHAT), previously designed to index the level of DSAmediated engagement of CD16 and unravel potential ADCCdriven pathogenic effects of circulating DSA (41), we therefore aimed to investigate whether circulating DSA detected at M1 or M3 in LTRs have the potential to stimulate NK cell alloreactivity. Evaluation of the DSA-induced down regulation of NK cell CD16 expression (CD16 Down Regulation Index or CD16DRI) was performed in a standardized NK-CHAT assay analyzing alloreactivity of NK cells toward serum-coated CD20 + B lymphocytes expressing the cognate HLA target alloantigen, using 23 serum samples obtained from 20 distinct LTRs (Table 5). Thirteen sera were collected at M1 and ten at M3. Four sera had detectable DSA that persisted at M3. Seven DSA at M1 and Four DSA at M3 were C1q positive.
Surprisingly, the CD16DRI values induced by the DSA + LTx sera were very low when compared to those obtained in response to Rituximab. These CD16 DRI values could not be associated with the intensity of DSA MFI and C1q binding activity of DSA. Furthermore, the CD16DRI of LTRs (LTR 17,18,and 20) with HLA DQ7 DSA were lower than those obtained of ABMR kidney transplant recipients (KTR) with HLA DQ7 DSA and with similar MFI (Figure 4).
In contrast to the results previously observed in the kidney and heart transplant setting, the association of the FCGR3A [158V/V] genotype with acute rejection appears to be independent of the presence of circulating DSA and the DSA MFI levels. In contrast to the KTR serum, NK-CHAT evaluation of the serum of immunized LTRs did not reveal significant alloreactive DSA toxicity and did not allow for determination of their pathogenic potential in eliciting CD16 and NK-cell cytotoxic activation.

FCGR2A Polymorphism Impacts Patients and Allograft Survival
Occurrence of acute and chronic events or infectious episodes was not shown to have a major impact on overall survival in the analyzed cohort.
Early development of DSA that persist at M3 (n = 21) were associated with lower graft or patient survival rates (p = 0.002) and were associated with the presence of the low-affinity F allelic Our results show evidence that the FCGR3A [158V/V] genotype can be identified as a genetic marker that predisposes LTRs to acute rejection, independently of their DSA immunization status. Fifteen out of thirty patients with the FCGR3A [158V/V] genotype indeed developed acute rejection during the first trimester. Since this profile has been associated with higher NK cell responsiveness, we further investigated how it may relate to the NK cell mediated cytotoxic effects of DSA toward the lung allograft. Various studies have shown that the detection of circulating DSA with an ability to ligate the C1q component and activate the complement cascade can be associated with a greater risk of acute rejection and allograft loss after heart or kidney transplant. These complement-dependent mechanisms of DSA-mediated cytotoxic effects have also been recently documented in the lung transplant setting (19,20). As previously reported (29), all C1q DSA detected at M3 were directed toward donor HLA-DQ alloantigens and C1q positive were shown to be significantly associated with acute rejection in the study cohort. A limitation of our study is the lack of elements allowing the distinction of cellular acute rejection (ACR) from ABMR. Endothelial deposition of C4d and microvascular inflammation were shown to be reliable markers of ABMR in renal and cardiac allografts, but the clinical relevance of C4d staining within lung allograft biopsies still remains controversial for lungs. These diagnostic criteria for ABMR were not always available on a routine basis at the time of evaluation until the Banff nomenclature released more accurate histologic features that characterize lung allograft grafts biopsies of DSA-positive LTRs. However, the association between the FCGR3A [158V/V] genotype and presence of circulating DSA at M1 or at M3 did not appear to confer a higher risk of acute rejection, whatever their MFI and their C1q binding activity status, thus suggesting that the graft damage associated with the FCGR3A [158V/V] genotype is independent from DSA immunization status. This finding was unexpected, as we have previously shown that the intensity of antibody-dependent CD16 activation of NK cells, indexed by the non-invasive NK-CHAT, is significantly enhanced in recipients that bear the FCGR3A [158V/V] genotype and constitute a relevant cytotoxic mechanism sustaining toxic effects of DSA and allograft vasculopathy (48,50). In contrast, NK-CHAT evaluation of DSA from LTRs actually shows evidence that the engagement of CD16 by LTx DSA is low when analyzed in reference to anti-HLA class II DSA that are found in the serum of kidney transplant recipients at time of ABMR diagnosis and that exhibit comparable HLA-DQ7 specificities and MFI values. This low potential of LTx DSA to induce CD16-mediated NK cell activation appears to be independent of their C1q binding activity and could not be associated with adverse outcome of LTx in the present study. As CD16 exhibits higher affinity for IgG1 and IgG3 alloantibodies, this failure of DSA to engage CD16 mediated cytotoxic functions may be due to the IgG2 and/or IgG4 isotypes of DSA, which have been shown to exhibit lower affinity for CD16. This finding may also indicate that the glycosylation pattern of IgG1 DSA interferes with the FcγRsmediated recognition of the Fc fragment of IgG by immune cells or complement factors. These hypotheses are supported by the observation that only 30% of circulating DSA detected at M1 and at M3 were found to bind C1q in this study cohort and by previous findings that report high IgG4 levels in patients with cystic fibrosis lung disease (59). These data suggest that, in contrast to previous findings, the FCGR3A [158V/V] susceptibility genotype does not appear as a major mechanism of CTL were exposed to B-EBV cell lines previously coated with DSA-negative serum. This allowed for evaluation of the baseline expression level of CD16 expression (CD16MFI) within the CD3-CD56+CD16+ NK cell compartment when PBMC were exposed to B cell targets. As a positive control indexing the level of IgG-induced CD16 down regulation (CD16DRI), the same B-EBV cell lines were coated in the presence of 10γ/ml Rituximab (anti CD20 IgG) prior to exposure to the effector PBMC. This allowed for calculation of the CD16 down regulation index (CD16DRI: baseline CD16MFI NK cells exposed to B cells coated with CTL DSA-negative serum/CD16 MFI of NK cells exposed to the same B targets pre-coated in presence of Rituximab). In this context, CD16DRI in response to Rituximab = 19, i.e., 90/4.8. (B) The CD16 DRI of NK cells was evaluated in response to the serum collected at M1 in one LTR (LTR 1, Table 5) with detectable levels of DSA recognizing the HLA-A3 and -B7 expressed on the B cell targets. Non-immunized serum (DSA-negative) collected from LTR 1 at time of LTx (D0) was used as the reference baseline CD16 MFI value to calculate the CD16DRI. Despite a cumulative MFI of DSA >17,000, DSA detected at M1 in LTR1 failed to induce CD16-dependent NK cell alloreactivity (CD16DRI = 1). (C) The CD16 DRI of NK cells was evaluated in response to the serum collected at time of ABMR diagnosis in one kidney transplant recipients (KTR 1, Table 5) with detectable levels of DSA recognizing the HLA-DQ7 (MFI: 13,000) expressed on the B cell targets. The CD16DRI of KTR1 (CD16DRI: 45) was greater than that observed in response to the Rituximab (CD16DRI:19, A). (D) The CD16 DRI of NK cells was evaluated in response to the serum collected at time of ABMR diagnosis in a second KTR (KTR 2, Table 5) with detectable levels of DSA recognizing the HLA-DQ7 (MFI: 9,000) expressed on the B cell targets. The CD16DRI of KTR1 (CD16DRI: 30) was greater than that observed in response to the Rituximab (CD16DRI:19, A). (E) The CD16 DRI of NK cells was evaluated in response to the serum collected at M3 (time of acute rejection) in a second LTR (LTR 17, Table 5) with detectable levels of DSA recognizing the HLA-DQ7 (MFI: 13,400) expressed on the B cell targets. Non-immunized serum (DSA-negative) collected from LTR 17 at D0 was used as the reference baseline CD16 MFI value to calculate the CD16DRI (CD16DRI = 1). The MFI DSA of LTR 17 (MFI: 13,400) was similar to the MFI DSA of ABMR KTR 1 serum as illustrated in (C). (F) The CD16 DRI of NK cells was evaluated in response to the serum collected at M3 (time of acute rejection) in a third LTR (LTR 20, Table 5) with detectable levels of DSA recognizing the HLA-DQ7 (MFI DSA: 9,000) expressed on the B cell targets. The DSA-negative serum of LTR 20 collected at D0 was used as a baseline CD16 MFI value to calculate the CD16DRI (CD16DRI = 1.9). The MFI DSA of LTR 20 (MFI: 9,000) was similar to the MFI DSA of ABMR KTR 2 serum evaluated in (D).
DSA-mediated lung allograft injury. CD16-dependent activation of NK cells in FCGR3A [158V/V] individuals that develop acute rejection may nevertheless be mediated by allo-or autoantibodies that target non-HLA antigens and may not be revealed in the standardized NK-CHAT assay revealing CD16 cellular activation toward B lymphocyte cell targets. Several reports have indeed described ABMR lesions of the graft that occur in the absence of detectable levels of circulating DSA in the serum of kidney or heart transplant recipients. Antibodies directed against endothelial antigens or stress-induced antigens, such as vimentin, collagen V, Kα1 tubulin, AT1R, and MICA have also been reported in transplant recipients but the role of these non-HLA antibodies in the destruction and accelerated dysfunction of lung allograft remains poorly addressed (60). These reports have nevertheless raised interest in chronic injury resulting from humoral responses targeting non-HLA antigens, as these may be underestimated by the standard monitoring of patients' immunization status which is mainly restricted to the detection of anti-HLA alloantibodies (52). While the rate of acute rejection during the first year was previously identified as a risk factor for CLAD occurrence, occurrence of acute rejection in the first trimester could not be associated with an enhanced risk of developing chronic rejection nor with patient or graft survival in the present cohort. The F allelic variant of CD16 with low affinity for the Fc fragment of IgG was associated with the early development during the first month post-LTx of DSA that persist at M3. Persistence of DSA at M3 was thus less frequent in FCGR3A [158V/V] LTRs and was shown to be associated with lower survival times in LTR.
Our observations also identify a link between the presence of circulating DSA at M3 and the FCGR2A [131R/R] genotype, thus suggesting that FCGR2 polymorphisms may actually be associated with the persistence of harmful DSA at M3 rather than in the development of anti-HLA antibodies per se. The FCGR2A [131R/R] genotype was reported to be associated with shorter allograft survival in immunized kidney transplant recipients (KTR) (61,62). We find that, independently of the risk associated with the FCGR2A [131R/R] genotype, detection and persistence of DSA at M3 constitute independent predictors of the adverse clinical composite outcome comprising CLAD or the patient's death. The persistence of DSA has been reported as a risk factor linked to BOS and to LTR death (16). A recent report showed that a majority of patients who were positive for de novo DSA during the first year after LTx developed BOS and were at higher risk of graft failure or death (63). In line with these reports, our study suggests that, independently of the presence of the susceptible FCGR2A [131R/R] genotype, the presence at M1 of DSA with high affinity for donor antigens (MFI DSA M1 > 18,750) can be identified as a risk factor associated with lower survival of LTRs.
The role of the complement-dependent pathogenicity of DSA is less well-documented in the lung transplant setting (19,20). In a murine model of BOS, complement activation by antibodies to HLA class I was not required for the development of obliterative airway disease (OAD) that is similar to BOS in human LTx. Interestingly, in this study, at 90 days after LTx, only one out of 5 BOS patients had C1q DSA detected by SAFB whereas 3 out of 11 stable patients had C1q DSA. However, in our study, C1q DSA detected at M1 and M3 were mostly directed against HLA class II antigenic targets (90%) and do not appear as major contributors associated with the development of CLAD.
Although this is a limitation of the study, we cannot exclude the idea that auto-antibodies that target non-HLA antigens which were associated with the development of DSA, such as autoantibodies against K-α1 tubulin (K-α1T) and collagen V (ColV) (64), can participate in the chronic FcR-dependent reaction of recipient cells toward the lung allograft. This is supported by studies that report that development of col (V)specific TH-17 cells may contribute to the pathogenesis of BOS (25,59). Indeed, our standardized NK-CHAT evaluation of the DSA induced NK-cell activation was conducted toward B cell lines that express cognate HLA alloantigens. As B cells may not be relevant to evaluate the deleterious impact of non-HLA alloantibodies toward the lung allograft, this is a limit that prompts further studies that uses serum-coated lung epithelial or endothelial cells targets to evaluate NK cell ADCC.
FCGR2A [131R/H] is considered to be a heritable risk factor for a variety of infectious and inflammatory autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, malaria, multiple sclerosis, and anti-neutrophil cytoplasmic auto-antibody positive systemic vasculitis (65). The CD32 membrane receptor is expressed by a variety of immune cells that orchestrate the humoral immune response to pathogens, including B-lymphocytes, natural killer cells, macrophages, mast cells, and neutrophils. Its capacity to recognize IgGs bound to pathogens or infected cells has a protective effect against infections. While the FCGR2A allelic isoforms exhibit similar affinity for IgG1, the FCGR2A [131H/H] is the only FcγR variant that recognizes IgG2 subclasses, thus suggesting that the capacity to sense IgG2 antibodies may lead to impairment of pathogen surveillance in patients that lack the H allele. This SNP has been shown to play a role in the susceptibility to bacterial infections as FCGR2A [131H/H] individuals have greater potential to mediate IgG2-dependent bacterial phagocytosis than patients genotyped as FCGR2A [131R/R]. A distinct SNP (rs12746613) within the FCGR2A gene was previously associated with a higher risk of respiratory infections and mortality after LTx, but this variant was not associated with the risk of developing CLAD (34). Unlike other studies, our analysis of the present LTR cohort did not reveal any significant association between FCGR2A [131R/H] and occurrence of respiratory infections or the number of infectionrelated deaths. This discrepancy may relate to preventive and curative treatment of bacterial infections that differ between the transplantation centers. It may also reflect a complex interaction of these FCGR genotypes that encourages the overall survival of LTR for patients that have better capacities to thwart infections and overcome early acute rejection events. The observed finding of a inverse link between the FCGR2A [131R/R] susceptibility genotype and presence of the FCGR3A-V allele encoding the In addition to this protective role against pathogens, the SNP dependent affinity of CD32 for the Fc fragment of IgG and/or CRP ligands was also identified as promoting inflammation. CD32-dependent triggering of immune cells is in part conditioned by the polymorphism and the expression profile of this functional receptor at the surface of immune cells. As is expressed by most leukocytes/macrophages that infiltrate the lung graft, FCGR2A [131R/H] could also influence the acquisition of an inflammatory-activated profile that favors tissue recruitment of activated lymphocytes to the lung (66). Interestingly, the FCGR2A [131R/R] susceptible genotype identified in this study has been associated with higher CRP binding avidity for the CD32 receptor expressed at the surface of monocytes and neutrophils.
Considering the growing evidence of the key role of CRP as an inflammatory mediator involved in the development of atherosclerosis and endothelial dysfunction, it is expected that CRP may be more powerful in triggering the proinflammatory function of CD32-expressing cell subsets such as platelets, endothelial cells, monocytes, and leukocytes in FCGR2A [131R/R] LTRs.
In conclusion, these data highlight that FCGR2A and FCGR3A polymorphisms constitute predisposing factors that are associated with the outcome of lung allografts. This study suggests that the combined assessment of the FCGR genotype and CRP or IgG ligands is thus an intriguing prospect to further decipher the complex mechanisms that shape the alloimmune and inflammatory responses in response to infectious and humoral threats. As shown in other organ transplant settings, our study indicates FCGR genotyping may favor early stratification of patients at risk and may create new perspectives to adapt personalized preventive and therapeutic approaches to prevent adverse outcomes of lung transplants.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
All patients from the French cohort (COLT, Cohort in Lung Transplantation, l'Institut du Thorax, INSERM UMR1087/CNRS UMR 6291, CNIL 911142) were recruited in this study and gave their written informed consent to participate to the study in accordance with the Declaration of Helsinki. A group of 184 healthy unrelated of volunteer French bone marrow donors were also recruited to constitute a control cohort allowing analysis of FcγR genotype. Blood donations were collected in the Etablissement Francais du Sang, in accordance with BSL-2 practices. A medical interview was carried out prior to blood donation to exclude donors with medical contraindications. This study was carried out in accordance with the French Public Health Code (art L1221-1), approved by institutional ethics committee and conducted in compliance with the Good Clinical Practice Guidelines, declaration of Helsinki and Istanbul.