De-novo donor-specific anti-hla antibodies 30 days after lung transplantation are associated with a worse outcome

http://www.jhltonline.org De-novo donor-specific anti-hla antibodies 30 days after lung transplantation are associated with a worse outcome Jérôme Le Pavec, MD, PhD, a,b,c Caroline Suberbielle, MD, d Lilia Lamrani, a,c Séverine Feuillet, MD, a,b,c Laurent Savale, MD, PhD, a,c,e Peter Dorfmüller, MD, PhD, a,c,f François Stephan, MD, a,c,g Sacha Mussot, MD, a,b,c Olaf Mercier, MD, PhD, a,b,c and Elie Fadel, MD, PhD a,b,c From the a Université Paris-Sud, Faculté de Médecine, Université Paris Saclay, Le Kremlin Bicêtre, France; b Service de Chirurgie Thoracique, Vasculaire et Transplantation Cardio-pulmonaire, Hôpital Marie-Lannelongue, Le Plessis-Robinson, France; c UMR-S 999, Universitaire Paris-Sud, INSERM, Hôpital Marie Lannelongue, Le Plessis Robinson, France; d Laboratoire d histocompatibilité, AP-HP, Hôpital Saint Louis, Paris, France; e AP-HP, Service de Pneumologie, Hôpital de Bicêtre, Le Kremlin Bicêtre, France; f Service d Anatomie Pathologique, Hôpital Marie Lannelongue, Le Plessis-Robinson, France; and the g Service de Réanimation, Hôpital Marie Lannelongue, Le Plessis-Robinson, France. KEYWORDS: lung transplantation; de-novo donor-specific antibodies; antibody mediated rejection; chronic lung allograft dysfunction; survival BACKGROUND: The impact of de-novo donor-specific anti-hla antibodies (DSA) on patient and graft survival after lung transplantation remains controversial. We analyzed DSA that developed at Day 7 and Month (M) 1, M3, M6 and M12 after lung transplantation and evaluated their impact on chronic lung allograft dysfunction (CLAD) development and survival. METHODS: One hundred thirty-four patients who underwent lung transplantation at our institution between November 2007 and August 2013 were included in this study. During the first post-transplant year, 82 (61%) patients developed de novo DSA and 52 (39%) patients did not. Three mean fluorescence intensity (MFI) intervals were used to define scores of anti-hla antibody positivity: score 4 if MFI was 500 to 1,000; score 6 if MFI was 1,000 to 3,000; and score 8 if MFI was Z3,000. Patients records were retrospectively reviewed. RESULTS: DSAwithMFIscoresofZ4 (hazard ratio [HR] 2.21, 95% confidence interval [CI] 1.08 to 4.54, p ¼ 0.03), 6 (HR 2.63, 95% CI 1.27 to 5.20, p o 0.01) and 8 (HR 2.83, 95% CI 1.42 to 5.67, p o 0.01) at M1; female gender (HR 0.49, 95% CI 0.28 to 0.87, P ¼ 0.01); and with post-operative extracorporeal membrane oxygenation (HR 0.09, 95% CI 0.01 to 0.28, p ¼ 0.02) were significantly associated with CLAD. Multivariate analysis identified score 8 at M1 (HR 2.71, 95% CI 1.34 to 5.47, p o 0.01) as an independent risk factor for mortality. Overall, 1-, 3- and 5-year survival rates were 76%, 52% and 41% compared with 84%, 74% and 70% for patients with or without de-novo DSA at M1, respectively (p ¼ 0.02). CONCLUSION: Early de-novo DSA may significantly impact long-term outcomes after lung transplantation and should therefore prompt regular screening. J Heart Lung Transplant 2016;35:1067 1077 r 2016 International Society for Heart and Lung Transplantation. All rights reserved. Reprint requests: Jérôme Le Pavec, MD, PhD, Unité de transplantation pulmonaire, Service de Chirurgie Thoracique, Vasculaire et Transplantation Cardio-pulmonaire, Hôpital Marie-Lannelongue, 133 avenue de la Résistance, 92350 Le Plessis-Robinson, France. Telephone: þ33-1-40-94- 24-30. Fax: þ33-1-40-94-86-82. E-mail address: lepavec@gmail.com 1053-2498/$ - see front matter r 2016 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2016.05.020 See Related Editorial, page 1057 Lung transplantation is well established as a viable option for patients with end-stage lung disease, and longer term survival is now possible because techniques and immunosuppression therapies have improved with

1068 The Journal of Heart and Lung Transplantation, Vol 35, No 9, September 2016 experience. Nevertheless, the current 5-year survival rate for all lung transplantations is only 50.6%. 1 The greatest limitation to long-term survival relates to the development of chronic lung allograft dysfunction (CLAD), mainly manifesting as bronchiolitis obliterans syndrome (BOS). Multiple immunologic and non-immunologic factors are associated with the development of CLAD. 2 6 Growing interest on the impact of anti-human leukocyte antigen (anti-hla) antibody development and antibodymediated rejection (AMR) has emerged. 7 13 In addition, accumulating data suggest that donor-specific antibodies (DSA) may influence long-term graft and patient survival rates. 14 18 However, diagnostic criteria or treatment of AMR remains controversial. Indeed, most existing data are derived from small retrospective studies with limited ability to provide robust conclusions on management and prognosis. Similarly, given the reported observations of patients with post-operative DSA who remain stable over a long period of time, the involvement of DSA in CLAD requires further investigation. 19 Finally, no study has investigated the impact of systematic as opposed to clinically indicated postoperative DSA screening on outcomes. Only selected studies have investigated the development of de-novo DSA, 11,14 18 and none have analyzed the impact of the timing of de novo DSA on outcomes. In this study, we aimed to investigate the impact on lung function and survival of de-novo DSA according to when DSA are detected. Methods Patients and general management In this prospective, single-center study, conducted during November 2007 to June 2015, we assessed all adult patients undergoing double-lung or heart lung transplantation between November 2007 and December 2013. The minimum period of follow-up was 18 months. Only patients without a DSA before transplantation were included. During this period, 173 patients were transplanted and 134 included in the study. Exclusion criteria were retransplantation, presence of DSA before transplantation and bronchial anastomotic stenosis. Co-primary outcomes were survival and time to CLAD development, as defined in what follows. Date of transplantation was used as the starting point to determine the overall survival duration. All recipients and their donors were HLA typed for HLA-A, -B, -C, -DR and -DQ using a polymerase chain reaction sequencespecific primer (PCR-SSP) at the time of, or before, transplantation. All patients included in the study received a transplant that had a negative cytotoxic donor lymphocyte crossmatch. No patient received immunosuppression as an induction therapy. Post-transplant immunosuppression consisted of triple therapy (calcineurin inhibitors, mycophenolate mofetil and prednisone). During the first year after transplant, trough levels of 10 to 15 μg/liter and 200 to 300 μg/liter were targeted for tacrolimus and cyclosporine, respectively. All patients received ongoing Pneumocystis pneumonia (PCP) prophylaxis with cotrimoxazole plus 6 to 12 months of cytomegalovirus (CMV) prophylaxis with valganciclovir according to recipient-positive (R þ ) and donorpositive/recipient-negative (D þ /R - ) status, respectively. At our institution, surveillance transbronchial biopsies are performed at the end of the first month, and if clinically indicated. Regarding acute cellular rejection, an acute rejection score was calculated by adding the sum of grades of each rejection episode divided by the number of surveillance and clinically indicated biopsies during first year post-transplant. Patients who developed allograft dysfunction were investigated to rule-out acute cellular rejection, lymphocytic bronchiolitis/ neutrophilic reversible allograft dysfunction, gastroesophageal reflux or airway injury caused by infection/colonization. Our institutional review board approved this retrospective study and waived the need for informed consent. DSA detection and therapy Recipients were screened for anti-hla antibodies immediately before lung transplantation at Day 7 and at Months 1, 3, 6 and 12 post-transplantation, and upon any indication, using Luminex (LABScreen Single Antigen;One-Lambda, Inc., Canoga Park, CA) assays. 20 Three mean fluorescence intensity (MFI) intervals were used to define scores of anti-hla antibody positivity: a score of 4 if MFI was 500 to 1,000; a score of 6 if MFI was 1,000 to 3,000; and a score of 8 if MFI was Z3,000. The highest MFI value was considered in patients who showed DSA against more 41 antigen. During the first month post-transplant, treatment of DSA was usually performed irrespective of the presence of graft dysfunction. Patients with scores 4 or 6 were treated with IgM-enriched intravenous immunoglobulins (IVIg) at an initial 1-g/kg body weight; the dose was then loaded by additional courses of 1 g/kg IVIg every 4 weeks until DSA clearance. For patients with an MFI score of 8, treatment was enhancedwith3to5sessionsofplasmaexchange,1g/kgivig,plusa single dose of rituximab (375 mg/m 2 ), when possible. After discharge, DSA were then re-assessed at regular outpatient visits according to our standard evaluation timing or if clinically indicated. Diagnosis of CLAD CLAD was diagnosed according to international criteria as previously described 21,22 at routine outpatient assessments of 1- to 3-month intervals after a minimum of 3 months post-transplantation. CLAD was suspected if forced expiratory volume in 1 second (FEV 1 ) and/or forced vital capacity (FVC) decreased by r90% from baseline for Z3 weeks. Extended pulmonary function tests, including spirometry and lung volumes, high-resolution computed tomography (HRCT) of the thorax and bronchoscopy with bronchoalveolar lavage and transbronchial biopsy specimens, were performed to exclude causes of acute lung allograft dysfunction, including persistent acute rejection, azithromycinresponsive allograft dysfunction, infection, anastomotic stricture or disease recurrence. CLAD was defined when the FEV 1 and/or FVC declined to r80% of the post-operative best values, despite treatment or without identifying a clear cause. CLAD encompasses a restrictive phenotype (restrictive allograft syndrome, RAS) if FVC was r80% at baseline and when CLAD is diagnosed for Z3 weeks with interstitial infiltrates on thoracic HRCT, as an obstructive phenotype (bronchiolitis obliterans syndrome, BOS) if FEV 1 was r80% at baseline for Z3 weeks without interstitial infiltrates on a thoracic HRCT, or a mixed phenotype (BOS and RAS) if FEV 1 was r80% at baseline for Z3 weeks with interstitial infiltrates on thoracic HRCT. 22 Statistical analyses Baseline demographics and characteristics were compared between the 2 groups using Student s t-test or chi-square analysis, as appropriate. Normally distributed data were compared using Student s t-test and non-normally distributed data were compared

Le Pavec et al. Impact of Early De-novo DSA on Outcome After Lung Transplantation 1069 using Wilcoxon s rank-sum test and the Mann Whitney U-test, as appropriate. Survival analyses were performed using the Kaplan Meier method. Comparisons between groups were assessed by the log-rank test. Cox s proportional hazards analysis was used for uniand multivariate analyses for both patient survival and time to CLAD development after the proportionality assumption was tested and confirmed. Multivariate analyses determined whether any of the potential confounding variables were independent predictors of poor patient survival or CLAD development. Variables tested in these analyses were those that showed trends in the univariate analyses (i.e., p o 0.1). Variables found that were not independently predictive of survival or development of CLAD were dropped. A 2-tailed p-value was regarded as significant. All analyses were performed using STATA (version 11) statistical software (StataCorp, College Station, TX) Results Patients characteristics Between November 2007 and December 2013, 173 patients underwent a double-lung or heart lung transplantation, among whom 39 were excluded for the following reasons: retransplantation, n ¼ 15; no DSA testing, n ¼ 12; a positive DSA test before transplantation, n ¼ 7; or anastomotic stenosis, n ¼ 5. A total of 134 patients with a mean population age of 43.5 13.3 years were analyzed. The underlying lung pathologies were as follows: pulmonary hypertension (PH) in 80 (60%), including pulmonary arterial hypertension (n ¼ 74) and chronic thromboembolic PH (n ¼ 6); chronic obstructive pulmonary disease (COPD) in 23 (17%); interstitial lung disease in 22 (16%); and 9 (7%) with other diagnoses (Table 1). Post-transplant immunology, antibody distribution and therapy A total of 402 sera from the 134 patients (mean 3 1.3 samples per patient) included in the study were tested for the presence of DSA at routine clinic visits and additional samples were collected at times of clinical deterioration; 121 (90%) patients were found to have developed HLA-specific antibodies in the first year post-transplant. Of these, 82 (61%) had a DSA and 106 (79%) had non-donor-specific HLA antibodies. Of those with a DSA, 22 (27%) had antibodies directed against donor HLA Class I antigens and 60 (73%) had antibodies directed against donor HLA Class II antigens (Table 2). In 45 (56%) of the 60 patients with Class II-directed DSA, the antibodies were directed against donor HLA-DQ specificities. A trend toward a higher HLA- DR mismatching score was observed in DSA-positive patients (p ¼ 0.06; Table 2). However, acute rejection score did not differ between the positive and negative DSA groups (Table 2). DSA therapies are summarized in Table 2. Only 21 patients received DSA treatment, which was administered as follows: plasma exchange and IVIg for 11; IVIg alone for 4; plasma exchange alone for 3; and plasma exchange with rituximab and IVIg for 3. Factors associated with CLAD development Tables 3 and 4 show the univariate analyses relating CLAD development to DSA MFI scores and the selected variables during the first post-transplantation year. Interestingly, only DSA with an MFI score of Z4 (hazard ratio [HR] 2.21, 95% confidence interval [CI] 1.08 to 4.54, p ¼ 0.03), 6 (HR 2.63, 95%CI1.27to5.20,p o 0.01) and 8 (HR 2.83, 95% CI 1.42 to 5.67, p o 0.01) at Month 1; female gender status (HR 0.49, 95% CI 0.28 to 0.87, p ¼ 0.01); and post-operative extracorporeal membrane oxygenation (HR 0.09, 95% CI 0.01 to 0.28, p ¼ 0.02) were significantly associated with CLAD development. Accordingly, 3 successive multivariate analyses models were set and included female gender, chronic obstructive pulmonary disease (COPD), post-operative extracorporeal membrane oxygenation (ECMO) and the 3 DSA MFI intervals at Month 1 (i.e., scores Z4, Z6 and 8). Variables found to be independent predictors of CLAD development were DSA with a MFI score of Z4 (HR 2.4, 95% CI 1.16 to 4.98, p ¼ 0.02), Z6 (HR 2.6, 95% CI 1.27 to 5.25, p o 0.01) and 8 (HR 2.7, 95% CI 1.34 to 5.46, p o 0.01) at Month 1 (results not shown). Rates of freedom of CLAD were 73%, 53% and 43% at 1, 3 and 5 years, respectively, in patients with DSA, and 88%, 71% and 71% at 1, 3 and 5 years, respectively, in patients without DSA (Figure 1). Survival analyses The mean follow-up period of the study was 56.8 19.6 (range 17.9 to 91.5) months, including a minimum period of follow-up of 18 months. At the end of the follow-up period, 77 patients were alive, 56 patients died and 1 was lost to follow-up. Overall, 1-, 3- and 5-year survival rates were 80.5%, 63.6% and 51.2%, respectively (Figure 2). The results from the univariate analyses, including DSA MFI scores and selected variables in the first post-transplantation year, are shown in Tables 5 and 6, respectively. DSA with MFI scores at Month 1 of Z4, Z6 and 8; recipient s age; dialysis; and longer ventilation time during ICU stay were significantly associated with a worse outcome. Of note, although not significant, there were trends toward increased mortality rates associated with DSA with an MFI score of Z4 at Month 3 (p ¼ 0.09), lung transplantation for pulmonary hypertension (p ¼ 0.09) or COPD (p ¼ 0.09), and CMV infection within the first year post-transplant (p ¼ 0.07). In the multivariate model, recipient s age, dialysis during ICUstayandDSAwithaMFIscoreof8atMonth1werethe only significant risk factors for mortality (Table 7 and Figure 3). Causes of death Fifty-six patients died during follow-up. The majority of deaths were attributed to acute or end-stage respiratory failure (n ¼ 17) and multiorgan failure (n ¼ 17). The remaining causes of death were as follows: cancer, n ¼ 6; bronchial complications, n ¼ 4; pneumonia, n ¼ 4; hemoptysis, n ¼ 3; and other, n ¼ 5 (i.e., stroke in 1, invasive aspergillosis in 1, thrombotic microangiopathy in 1, pulmonary embolism in 1, unknown in 1).

1070 Table 1 Demographics and Post-operative Data of the Patient Cohort The Journal of Heart and Lung Transplantation, Vol 35, No 9, September 2016 Overall population (n ¼ 134) DSA positive (n ¼ 82) DSA negative (n ¼ 52) p Female gender [n (%)] 82 (61) 49 (36) 33 (25) 0.40 Recipient age (years) 43.5 13.3 40.3 13.0 48.5 12.1 0.01 Blood group [n (%)] 0.89 O 61 (45) 35 (26) 26 (19) A 58 (43) 37 (28) 21 (15) B 11 (8) 7 (5) 4 (3) AB 3 (4) 1 (2) 1 (2) CMV risk [n (%)] 0.06 Low 32 (24) 24 (18) 8 (6) Intermediate 73 (54) 45 (34) 28 (20) High 29 (22) 16 (12) 13 (10) Transplantation indication [n (%)] 0.05 PH 80 (60) 56 (42) 24 (18) COPD 23 (17) 9 (7) 14 (10) Interstitial lung disease 22 (16) 12 (9) 10 (7) Other 9 (7) 5(4) 4 (3) FEV 1 (% predicted) 64 27 58 27 47 26 0.02 Pre-operative intensive care unit [n (%)] 39 (29) 29 (22) 10 (7) 0.05 High-emergency transplantation program [n (%)] 46 (34) 34 (25) 12 (9) 0.04 Pre-operative ECMO [n (%)] 10 (7) 7 (5) 3 (2) 0.74 Donor Age (years) 44 14 43 14 46 15 0.30 PO 2 (100% mm Hg) 440 71 435 74 445 69 0.44 Lung transplant procedure [n (%)] 0.18 Double lung 98 (73) 56 (42) 42 (31) Heart-lung 33 (25) 23 (17) 10 (8) Unilateral 3 (2) 3 (2) 0 (0) Cardiopulmonary bypass [n (%)] 83 (71) 57 (42) 26 (29) 0.01 Intra-operative ECMO [n (%)] 15 (11) 11 (8) 4 (3) 0.40 Post-operative ECMO [n (%)] 21 (16) 16 (12) 5 (4) 0.15 Lung volume reduction [n (%)] 20 (15) 16 (12) 4 (3) 0.08 Ischemic time (min) Right 219 67 229 71 206 61 0.10 Left 303 64 312 64 292 63 0.14 Heart-lung 222 32 229 29 205 36 0.06 Post transplant thoracotomy for bleeding [n (%)] 43 (32) 31 (23) 12 (9) 0.12 Dialysis during ICU stay [n (%)] 19 (14) 15 (11) 4 (3) 0.12 PGD score Grade 3 at 72 hours [n (%)] 39 (30) 30 (22) 9 (8) 0.02 Post-operative tracheotomy [n (%)] 44 (32) 32 (24) 11 (8) 0.04 Ventilation time during ICU stay [median (IQR)] 5 (2 to 14) 7 (4 to 19) 2 (1 to 6) 0.01 In-hospital mortality [n (%)] 20 (15) 13 (10) 7 (5) 0.81 Pneumonia within first year [n (%)] 93 (74) 58 (43) 46 (31) 0.83 CMV infection within first year [n (%)] 23 (17) 18 (13) 5 (4) 0.10 CLAD [n (%)] 49 (37) 31 (23) 18 (14) 0.33 Obstructive CLAD 38 (28) 24 (18) 14 (10) Restrictive CLAD 5 (4) 4 (3) 1 (1) Mixed CLAD 6 (5) 2 (3) 2 (2) CLAD, chronic lung allograft dysfunction; CMV, cytomegalovirus; COPD, chronic obstructive pulmonary disease; DSA, donor-specific antibodies; ECMO, extracorporeal membrane oxygenation; FEV 1, forced expiratory volume in 1 second; ICU, intensive care unit; PGD, primary graft dysfunction; PH, pulmonary hypertension; PO 2, partial pressure of oxygen. Discussion In this study we have presented a longitudinal analysis of a double-lung and heart lung transplant cohort where denovo production of DSA was an independent risk factor for CLAD development and a poor outcome. More importantly, by investigating the impact of DSA according to standardized timed detection points, we showed that the most important effect on lung function and outcome was associated with DSA formed within the first month posttransplantation. Therefore, in accordance with previous studies, 14,33 we report on the potential benefit of a standardized strategy of post-operative DSA screening. In addition to timing, our study showed that the intensity of

Le Pavec et al. Impact of Early De-novo DSA on Outcome After Lung Transplantation 1071 Table 2 Post-transplant Immunology, Antibody Distribution and Therapy Overall population (n ¼ 134) DSA positive (n ¼ 82) DSA negative (n ¼ 52) p HLA-A mismatches 0 / 1 / 2 4 / 39 / 91 1 / 25 / 55 3 / 14 / 36 0.33 HLA-B mismatches 0 / 1/ 2 0 / 35 / 99 0 / 21 / 60 0 / 14 / 39 1.00 HLA-DR mismatches 0/1/2 2/44/88 0/23/58 2/21/30 0.06 HLA-DQ mismatches 0 /1/2 a 3/ 35 / 51 1 / 22 / 37 2 / 13 / 14 0.25 HLA antibodies (not DSA) 0.43 HLA I 34 (25) 19 (14) 15 (11) HLA II 14 (10) 8 (6) 6 (4) HLA I and II 58 (43) 40 (30) 32 (13) DSA anti-hla I HLA-A 13 (16) HLA-B 9 (11) DSA anti-hla II HLA-DQ 45 (56) HLA-DR 15 (17) DSA therapy None 61 (74) IVIg 4 (5) Plasma exchange 3 (4) Plasma exchange þ IVIg 11 (13) Plasma exchange þ IVIg þ rituximab 3 (4) Acute rejection score b 0.48 0.44 0.50 0.43 0.46 0.47 0.61 DSA, donor-specific antibodies; HLA, human leukocyte antigen; IVIg, intravenous immunoglobulin. a Tested in 89 patients. b The acute rejection score was calculated by adding the sum of grades of each rejection episode divided by the number of surveillance and clinically indicated biopsies. Table 3 Univariate Analysis Relating CLAD development to DSA (Y/N) According to Mean Fluorescence Intensity Scores a Variables b Hazard ratio 95% confidence interval p At day 7 Overall 0.65 0.31 to 1.35 0.25 DSA MFI Z score 4 0.83 0.40 to 1.72 0.62 DSA MFI Z score 6 0.66 0.26 to 1.70 0.39 DSA MFI score 8 0.81 0.25 to 2.65 0.73 At Month 1 Overall 1.87 0.90 to 3.88 0.09 DSA MFI Z score 4 2.21 1.08 to 4.54 0.03 DSA MFI Z score 6 2.63 1.27 to 5.20 o0.01 DSA MFI score 8 2.83 1.42 to 5.67 o0.01 At Month 3 Overall 1.30 0.62 to 2.73 0.49 DSA MFI Z score 4 1.27 0.61 to 2.66 0.51 DSA MFI Z score 6 1.48 0.71 to 3.08 0.29 DSA MFI score 8 1.65 0.75 to 3.63 0.21 At Month 6 Overall 1.60 0.79 to 3.24 0.19 DSA MFI Z score 4 1.58 0.77 to 3.22 0.21 DSA MFI Z score 6 1.90 0.90 to 4.05 0.09 DSA MFI score 8 2.13 0.81 to 5.60 0.12 At Month 12 Overall 0.82 0.41 to 1.64 0.57 DSA MFI Z score 4 0.82 0.41 to 1.64 0.60 DSA MFI Z score 6 0.81 0.37 to 1.82 0.62 DSA MFI score 8 1.79 0.63 to 5.11 0.27 DSA, donor-specific antibodies; MFI, mean fluorescence intensity. a MFI scores defined as follows: score 4 ¼ 500 r MFI o 1,000; score 6 ¼ 1,000 r MFI o 3,000; score 8 ¼ MFI Z 3,000. b DSA at Day 7 and months 1, 3, 6 and 12 were tested at 4.5 2.4 days, 1.3 0.7, 3.2 1.5, 6.2 2.4 and 12.9 5.5 months after transplantation in 112, 93, 73, 66 and 65 patients, respectively.

1072 Table 4 Univariate Analysis Relating CLAD Development to Selected Variables The Journal of Heart and Lung Transplantation, Vol 35, No 9, September 2016 Variables Hazard ratio 95% confidence interval p Female gender 0.49 0.28 to 0.87 0.01 Recipient age 1.04 0.78 to 1.39 0.76 Blood Group O 1.05 0.61 to 1.91 0.79 Blood Group A 0.76 0.43 to 1.35 0.36 CMV risk Low 0.94 0.48 to 1.84 0.85 Intermediate 1.04 0.59 to 1.84 0.87 High 1.00 0.51 to 1.96 0.99 Transplant indication PH 0.74 0.42 to 1.31 0.31 COPD 1.81 0.94 to 3.51 0.07 Interstitial lung disease 1.16 0.54 to 2.48 0.70 FEV 1 0.76 0.53 to 1.08 0.13 Pre-operative intensive care unit 1.06 0.56 to 2.00 0.85 High-emergency transplant program 1.12 0.62 to 2.02 0.70 Pre-operative ECMO 1.09 0.43 to 2.77 0.84 Lung transplant procedure Double lung 0.69 0.38 to 1.24 0.21 Heart-lung 1.35 0.74 to 2.45 0.32 Cardiopulmonary bypass 1.18 0.61 to 2.29 0.61 Intra-operative ECMO 1.20 0.43 to 3.37 0.72 Post-operative ECMO 0.09 0.01 to 0.67 0.02 Lung volume reduction 1.59 0.77 to 3.30 0.21 Ischemic time Right 1.12 0.83 to 1.54 0.44 Left 1.14 0.84 to 1.55 0.40 Posttransplant thoracotomy for bleeding 0.84 0.45 to 1.56 0.59 Dialysis during ICU stay 0.82 0.30 to 2.30 0.71 PGD score grade 3 at 72 hours 1.05 0.57 to 1.96 0.87 Post-operative tracheotomy 1.07 0.59 to 1.93 0.81 Ventilation time during ICU stay 0.93 0.68 to1.27 0.66 Pneumonia within first year 0.91 0.48 to 1.74 0.79 CMV infection within first year 0.91 0.38 to 2.13 0.81 HLA mismatch 0.68 0.11 to 4.59 0.69 Acute rejection score a 0.83 0.44 to 1.56 0.56 DSA therapy 1.19 0.40 to 3.84 0.77 Hazard ratios and 95% confidence intervals for continuous variables are displayed per each unit increase in the standard deviation. CMV, cytomegalovirus; COPD, chronic obstructive pulmonary disease; ECMO, extracorporeal membrane oxygenation; FEV 1, forced expiratory volume in 1 second; HLA, human leukocyte antigen; ICU, intensive care unit; PGD, primary graft dysfunction; PH, pulmonary hypertension. a The acute rejection score was calculated by adding the sum of grades of each rejection episode divided by the number of surveillance and clinically indicated biopsies. DSA assessed by MFI was associated with outcome, as the risk of death or CLAD development occurred only if the DSA MFI score was Z4. Solid-phase assays, particularly Luminex-based assays, can detect HLA antibodies in more patients than previously possible and, based on MFI, can help evaluate the intensities. It is likely that patients can produce de-novo DSA after transplantation and it is important to understand the clinical relevance and long-term effects of DSA after lung transplantation. Therefore, patients found to have a preformed DSA before transplantation were excluded to specifically determine the effects of de-novo DSA on outcome after transplantation. According to the time of detection, de-novo DSA were identified in 82 patients during first year post-transplantation. Of note, only DSA with an MFI score of Z4 at Month 1 were found to be associated with development of CLAD and poor patient survival in the univariate analyses. In multivariate analyses, de-novo DSA and MFIscore Z 4 at Month 1 were found to be independent predictors of CLAD development and poor patient survival. These findings are similar to those seen in both renal 23,24 and cardiac 25,26 transplant studies. In a large study of renal allografts, Lachmann et al used Luminex single-antigen bead assays to show that DSA detected post-transplant were associated with significantly reduced graft survival, 23 whereas Smith et al previously found that the occurrence of de-novo DSA after cardiac transplantation was an independent predictor of poor patient survival. 25 Previous studies have also suggested a role for HLA antibodies after lung transplantation. 8,27 31 Some of these studies 8,27 have been enzyme-linked immunoassays and thus have probably

Le Pavec et al. Impact of Early De-novo DSA on Outcome After Lung Transplantation 1073 Figure 1 Kaplan-Meier freedom from CLAD estimates from date of transplantation according to presence or absence of DSA at Month 1. Rates of freedom of CLAD were 73%, 53% and 43% at 1, 3 and 5 years, respectively, in patients with DSA, and 88%, 71% and 71% at 1, 3 and 5 years, respectively, in patients without DSA (p ¼ 0.08, log-rank test). underestimated the level of HLA antibody production. Other early studies 30,32 utilized complement-dependent cytotoxicity assays to detect antibodies and did not assign donor reactivity to the antibodies detected. However, recent studies have provided indirect evidence of a role for denovo DSA in the development of BOS and outcomes after lung transplantation. Hachem et al 14 showed that patients with persistent DSA, despite antibody-targeted therapies (including rituximab and IVIg), more frequently developed BOS compared with patients who were clear of DSA. In the study by Witt and colleagues, lung transplant recipients with an HLA antibody response, and who progressed to AMR, developed severe allograft dysfunction, which led to a high mortality rate despite aggressive therapeutic measures. 15 In addition, 2 recent studies further suggested a strong association between de-novo DSA and post-transplant outcomes. 17,33 In the study by Safavi et al, de-novo DSA detected in 38 (26%) of 148 lung transplant recipients during follow-up were significantly associated with shorter Figure 2 Kaplan-Meier survival estimates from date of transplantation in the overall population of 134 patients. Survival rates were 80.5%, 63.6% and 51.2% at 1, 3 and 5 years, respectively. delay of occurrence and a higher grade of BOS, and also a higher risk of death. 17 In keeping with these results, Morrell et al found that presence of de-novo DSA detected at the time of surveillance bronchoscopies in 58 (13%) of 445 lung transplant recipients was significantly associated with BOS development and death. 33 Although recent studies 14,17,33 addressed the impact of post-operative DSA based on a standardized screening protocol, none identified any relevant time-points to detect outcomes. In that context, our study also showed that DSA detected at 1 month post-transplantation were already significantly associated with outcome, suggesting that this early post-operative time-point may be particularly relevant for DSA screening. Therefore, implementation of routine surveillance for DSA is warranted and has been encouraged in a recent summary statement from the International Society for Heart and Lung Transplantation. 20 The basis of CLAD development is undoubtedly multifactorial and includes immunologic factors. However, the mechanisms that precisely determine the effect of DSA on the allograft are currently unknown. The reason why only early DSA seem to have a greater impact on outcome may be related to the fact that the initial post-transplantation period is usually more prone to inflammatory conditions during which HLA exposure of the allograft may be increased. Therefore, we hypothesize that, under inflammatory conditions, including pneumonia or primary graft dysfunction (PGD), production of pro-inflammatory cytokines may be increased and induce pulmonary endothelial cells to upregulate and express Class II HLA molecules, as described elsewhere. 34 In light of this increasing evidence for poor clinical outcomes in patients with a de-novo DSA, Hachem and colleagues showed that patients with a de-novo DSA, but who had been removed by a targeted therapy, were less likely to develop BOS and had better survival than those who had persistent DSA. 14 However, in our study we did not specifically investigate whether clearance of de-novo DSA could improve outcomes, as the number of treated patients was limited. One striking result from our study involves the particularly high incidence (i.e., 61%) of de-novo DSA identified during first year post-transplant. First, DSA detection was exclusively based on Luminex assays, which can detect HLA antibodies in more patients than previously possible. Also of interest is that the primary indication for transplantation in our study was PH, which may account for the higher number of female recipients, particularly in the de-novo DSA group (60%). Therefore, female recipients may be more likely to develop DSA as a result of alloimmune reactivity caused by anamnestic responses to previous sensitizing events, such as pregnancy. In addition, mainly due to severe PH, 7% of the lung recipients in our study were bridged to transplantation with ECMO, which has recently been reported to be associated with development of anti-hla sensitization. 35 Last, the relationship between PGD and development of anti-hla Class II antibodies has already been demonstrated. 13,16 Thus, PGD, which was significantly overexpressed in the sensitized

1074 Table 5 The Journal of Heart and Lung Transplantation, Vol 35, No 9, September 2016 Univariate Analysis Relating Survival to DSA (Y/N) According to Mean Fluorescence Intensity Scores a Variables b Hazard ratio 95% confidence interval p At Day 7 Overall 0.89 0.47 to 1.67 0.72 DSA MFI Z score 4 0.81 0.41 to 1.59 0.54 DSA MFI Z score 6 0.87 0.39 to 1.96 0.75 DSA MFI score 8 0.97 0.35 to 2.71 0.96 At Month 1 Overall 1.24 0.65 to 2.40 0.51 DSA MFI Z score 4 1.96 1.02 to 3.64 0.04 DSA MFI Z score 6 2.23 1.09 to 4.58 0.03 DSA MFI score 8 2.61 1.30 to 5.25 o0.01 At Month 3 Overall 1.81 0.74 to 4.44 0.19 DSA MFI Z score 4 2.16 0.88 to 5.31 0.09 DSA MFI Z score 6 1.83 0.79 to 4.24 0.16 DSA MFI score 8 1.92 0.81 to 4.59 0.14 At Month 6 Overall 1.49 0.64 to 3.45 0.19 DSA MFI Z score 4 1.24 0.52 to 2.98 0.61 DSA MFI Z score 6 1.60 0.65 to 3.93 0.31 DSA MFI score 8 1.53 0.45 to 5.17 0.50 At Month 12 Overall 0.90 0.34 to 2.34 0.82 DSA MFI Z score 4 0.90 0.34 to 2.35 0.83 DSA MFI Z score 6 0.70 0.22 to 2.19 0.54 DSA MFI score 8 1.42 0.32 to 6.23 0.64 DSA, donor-specific antibodies; MFI, mean fluorescence intensity. a MFI scores defined as follows: score 4 ¼ 500 r MFI o 1,000; score 6 ¼ 1,000 r MFI o 3,000; score 8 ¼ MFI Z 3,000. b DSA Day 7 and Months 1, 3, 6 and 12 were tested at 4.5 2.4 days, 1.3 0.7, 3.2 1.5, 6.2 2.4 and 12.9 5.5 months post-transplantation in 112, 93, 73, 66 and 65 patients, respectively. group, may also account for subsequent development of denovo DSA. In keeping with other studies, 18,25,36 the majority of DSA detected in our study were directed against HLA Class II antigens, particularly HLA-DQ. Of the 60 patients with Class II-directed DSA, 45 (75%) were directed against donor HLA-DQ specificities. HLA Class II molecules are normally expressed in lung tissue, and are upregulated in patients with obliterative bronchiolitis after lung transplantation. 37,38 Although the physiologic hallmark of chronic rejection has been a persistent fall in FEV 1 associated with an obstructive ventilatory defect, for which the term BOS has been defined to allow a uniformity of description and grading of severity throughout the world, a restrictive form of chronic rejection has recently been described. 39 Thus, the concept of CLAD, rather than BOS itself, was chosen by the authors to best describe chronic graft dysfunction. However, no difference was observed in terms of risk factors for chronic graft dysfunction when analyses were switched from CLAD to BOS (results not shown). There are several limitations to our study that could influence the generalizability of the results. First, these results are from a single transplant center and, despite the significant number of patients relative to other studies that addressed the effects of de-novo DSA, our sample size limits the possibility of multivariate statistical analyses. In addition, the number of patients tested for DSA decreased with time, which may have underpowered the analyses. Another limitation is the lack of information regarding histologically defined AMR. Indeed, at the time of routine or clinically oriented transbronchial biopsies, neither C4d staining nor peri-capillary neutrophilic infiltration was analyzed. This will be addressed in an upcoming investigation. A significant proportion of the lung transplant candidates referred to our center required inclusion in a highemergency allocation program and pre-operative lifeassistance support, mainly because of PH; thus, it is likely our selection was biased toward inclusion of patients who were particularly ill. Further, although evaluation of the efficacy of DSA treatment was not our focus of concern, the number of treated patients was limited and many data regarding the evolution of DSA after antibody-specific therapies are missing, leading to an underpowered ability to evaluate the impact of these therapeutic approaches. Thus, only the impact of treatment as a confounding factor on the end-points was evaluated. In conclusion, the present results suggest that denovo production of DSA is an independent risk factor for CLAD development and poor post-transplantation outcome. More importantly, our study, by investigating the impact of de-novo DSA from standardized timepoints for detection, showed that the greatest effect on

Le Pavec et al. Impact of Early De-novo DSA on Outcome After Lung Transplantation 1075 Table 6 Univariate Analysis Relating Survival Time to Selected Variables Variables Hazard ratio 95% confidence interval p Female gender 0.66 0.39 to 1.11 0.12 Recipient age 1.45 1.09 to 1.94 0.01 Blood Group O 1.30 0.77 to 2.21 0.32 Blood Group A 0.64 0.37 to 1.10 0.10 CMV risk Low 0.86 0.45 to 1.62 0.63 Intermediate 1.11 0.65 to 1.89 0.69 High 1.01 0.52 to 1.90 0.99 Transplant indication PH 0.64 0.38 to 1.07 0.09 COPD 1.69 0.92 to 3.11 0.09 Interstitial lung disease 1.12 0.56 to 2.22 0.74 FEV 1 1.01 0.81 to 1.46 0.57 Pre-operative intensive care unit 1.31 0.74 to 2.32 0.35 High-emergency transplant program 0.98 0.56 to 1.72 0.95 Pre-operative ECMO 0.58 0.18 to 1.87 0.37 Lung transplant procedure Double lung 0.92 0.52 to 1.65 0.79 Heart-lung 0.85 0.46 to 1.59 0.62 Cardiopulmonary bypass 1.00 0.54 to 1.85 0.99 Intra-operative ECMO 1.59 0.67 to 3.77 0.29 Post-operative ECMO 0.77 0.34 to 1.70 0.52 Lung volume reduction 1.61 0.83 to 3.10 0.16 Ischemic time Right 1.02 0.76 to 1.39 0.87 Left 1.07 0.81 to 1.41 0.65 Post-transplant thoracotomy for bleeding 1.09 0.63 to 1.89 0.76 Dialysis during ICU stay 2.10 1.04 to 4.21 0.04 PGD score Grade 3 at 72 hours 1.33 0.75 to 2.36 0.33 Post-operative tracheotomy 1.04 0.59 to 1.82 0.89 Ventilation time during ICU stay 1.29 1.03 to 1.61 0.02 Pneumonia within first year 1.12 0.60 to 2.10 0.72 CMV infection within first year 1.79 0.96 to 3.37 0.07 HLA mismatch 1.79 0.96 to 3.37 0.07 Acute rejection score a 1.19 0.40 to 3.84 0.77 DSA therapy 1.04 0.32 to 3.34 0.94 Hazard ratios and 95% confidence intervals for continuous variables are displayed per each unit increase in the standard deviation. CMV, cytomegalovirus; COPD, chronic obstructive pulmonary disease; ECMO, extracorporeal membrane oxygenation; FEV 1, forced expiratory volume in 1 second; HLA, human leukocyte antigen; ICU, intensive care unit; PGD, primary graft dysfunction; PH, pulmonary hypertension. a The acute rejection score was calculated by adding the sum of grades of each rejection episode divided by the number of surveillance and clinically indicated biopsies. lung function and outcome was associated with DSA formation by 1 month post-transplantation, thus indicating the need for regular screening. Randomized, controlled trials should be undertaken to determine whether use of therapies that can remove DSA will improve outcomes. Disclosure statement The authors have no conflicts of interest to disclose. This study was supported in part by the Département Hospitalo Universitaire Thorax Innovation and the Laboratoire d Excellence en Recherche sur le Médicament et l Innovation Thérapeutique. Table 7 Multivariate Analysis Relating Survival Time to Selected Variables Variables Hazard ratio 95% confidence interval p Recipient age 1.75 1.18 to 2.61 o0.01 Dialysis during ICU stay 3.13 1.30 to 7.53 0.02 Ventilation time during ICU stay 1.08 0.81 to1.44 0.59 DSA MFI score 8 at Month 1 4.00 1.67 to 9.60 o0.01 Hazard ratios and 95% confidence intervals for continuous variables are displayed per each unit increase in the standard deviation. DSA, donor-specific antibodies; ICU, intensive care unit. MFI, mean fluorescence intensity.

1076 The Journal of Heart and Lung Transplantation, Vol 35, No 9, September 2016 Figure 3 Kaplan-Meier survival estimates from date of transplantation according to presence or absence of DSA at Month 1. Survival rates were 76%, 52% and 41% at 1, 3 and 5 years, respectively, in patients with DSA, and 84%, 74% and 70% at 1, 3 and 5 years, respectively, in patients without DSA (p ¼ 0.02, logrank test). The authors thank Professor Bruno Falissard, Professor Gabriel Thabut and Dr. Amina Zemitti for assistance with biostatistics. References 1. Christie JD, Edwards LB, Kucheryavaya AY, et al. The Registry of the International Society for Heart and Lung Transplantation: 29th adult lung and heart-lung transplant report 2012. J Heart Lung Transplant 2012;31:1073-86. 2. Nath DS, Basha HI, Mohanakumar T. Antihuman leukocyte antigen antibody-induced autoimmunity: role in chronic rejection. Curr Opin Organ Transplant 2010;15:16-20. 3. Angaswamy N, Saini D, Ramachandran S, et al. Development of antibodies to human leukocyte antigen precedes development of antibodies to major histocompatibility class I-related chain A and are significantly associated with development of chronic rejection after human lung transplantation. Hum Immunol 2010;71:560-5. 4. Angaswamy N, Tiriveedhi V, Sarma NJ, et al. Interplay between immune responses to HLA and non-hla self-antigens in allograft rejection. Hum Immunol 2013;74:1478-85. 5. Martinu T, Howell DN, Palmer SM. Acute cellular rejection and humoral sensitization in lung transplant recipients. Semin Respir Crit Care Med 2010;31:179-88. 6. McManigle W, Pavlisko EN, Martinu T. Acute cellular and antibodymediated allograft rejection. Semin Respir Crit Care Med 2013;34: 320-35. 7. Hadjiliadis D, Chaparro C, Reinsmoen NL. Pre-transplant panel reactive antibody in lung transplant recipients is associated with significantly worse post-transplant survival in a multicenter study. J Heart Lung Transplant 2005;24(suppl):S249-54. 8. Girnita AL, Duquesnoy R, Yousem SA. HLA-specific antibodies are risk factors for lymphocytic bronchiolitis and chronic lung allograft dysfunction. Am J Transplant 2005;5:131-8. 9. Mangi AA, Mason DP, Nowicki ER, et al. Predictors of acute rejection after lung transplantation. Ann Thorac Surg 2011;91:1754-62. 10. Saini D, Weber J, Ramachandran S. Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts. J Heart Lung Transpl 2011;30: 624-31. 11. Snyder LD, Wang Z, Chen D-F, et al. Implications for human leukocyte antigen antibodies after lung transplantation: a 10-year experience in 441 patients. Chest 2013;144:226-33. 12. Brugière O, Suberbielle C, Thabut G, et al. Lung transplantation in patients with pretransplantation donor-specific antibodies detected by Luminex assay. Transplantation 2013;95:761-5. 13. Bharat A, Kuo E, Steward N, et al. Immunological link between primary graft dysfunction and chronic lung allograft rejection. Ann Thorac Surg 2008;86:189-95. 14. Hachem RR, Yusen RD, Meyers BF, et al. Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation. J Heart Lung Transplant 2010;29:973-80. 15. Witt CA, Gaut JP, Yusen RD, et al. Acute antibody-mediated rejection after lung transplantation. J Heart Lung Transplant 2013;32:1034-40. 16. Ius F, Sommer W, Tudorache I, et al. Early donor-specific antibodies in lung transplantation: risk factors and impact on survival. J Heart Lung Transplant 2014;33:1255-63. 17. Safavi S, Robinson DR, Soresi S, et al. De novo donor HLAspecific antibodies predict development of bronchiolitis obliterans syndrome after lung transplantation. J Heart Lung Transplant 2014;33:1273-81. 18. Lobo LJ, Aris RM, Schmitz J, et al. Donor-specific antibodies are associated with antibody-mediated rejection, acute cellular rejection, bronchiolitis obliterans syndrome, and cystic fibrosis after lung transplantation. J Heart Lung Transplant 2013;32:70-7. 19. Glanville AR. Antibody-mediated rejection in lung transplantation: myth or reality? J Heart Lung Transplant 2010;29:395-400. 20. Berry G, Burke M, Andersen C, et al. Pathology of pulmonary antibody-mediated rejection: 2012 update from the Pathology Council of the ISHLT. J Heart Lung Transplant 2013;32:14-21. 21. Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 2002;21:297-310. 22. Verleden GM, Raghu G, Meyer KC, et al. A new classification system for chronic lung allograft dysfunction. J Heart Lung Transplant 2014; 33:127-33. 23. Lachmann N, Terasaki PI, Budde K, et al. Anti-human leukocyte antigen and donor-specific antibodies detected by Luminex posttransplant serve as biomarkers for chronic rejection of renal allografts. Transplantation 2009;87:1505-13. 24. Worthington JE, Martin S, Al-Husseini DM, et al. Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome. Transplantation 2003;75:1034-40. 25. Smith JD, Banner NR, Hamour IM, et al. De novo donor HLA-specific antibodies after heart transplantation are an independent predictor of poor patient survival. Am J Transplant 2011;11:312-9. 26. Tambur AR, Pamboukian SV, Costanzo M-R, et al. The presence of HLA-directed antibodies after heart transplantation is associated with poor allograft outcome. Transplantation 2005;80:1019-25. 27. Girnita AL, McCurry KR, Iacono AT, et al. HLA-specific antibodies are associated with high-grade and persistent-recurrent lung allograft acute rejection. J Heart Lung Transplant 2004;23:1135-41. 28. Aubert O, Bories M-C, Suberbielle C, et al. Risk of antibody-mediated rejection in kidney transplant recipients with anti-hla-c donorspecific antibodies. Am J Transplant 2014;14:1439-45. 29. Palmer SM, Davis RD, Hadjiliadis D, et al. Development of an antibody specific to major histocompatibility antigens detectable by flow cytometry after lung transplant is associated with bronchiolitis obliterans syndrome. Transplantation 2002;74:799-804. 30. Smith MA, Sundaresan S, Mohanakumar T, et al. Effect of development of antibodies to HLA and cytomegalovirus mismatch on lung transplantation survival and development of bronchiolitis obliterans syndrome. J Thorac Cardiovasc Surg 1998;116:812-20. 31. Jaramillo A, Smith MA, Phelan D, et al. Development of ELISAdetected anti-hla antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation. Transplantation 1999; 67:1155-61. 32. Sundaresan S, Mohanakumar T, Smith MA, et al. HLA-A locus mismatches and development of antibodies to HLA after lung transplantation correlate with the development of bronchiolitis obliterans syndrome. Transplantation 1998;65:648-53. 33. Morrell MR, Pilewski JM, Gries CJ, et al. De novo donor-specific HLA antibodies are associated with early and high-grade bronchiolitis obliterans syndrome and death after lung transplantation. J Heart Lung Transplant 2014;33:1288-94.

Le Pavec et al. Impact of Early De-novo DSA on Outcome After Lung Transplantation 1077 34. Cunningham AC, Zhang JG, Moy JV, et al. A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells. Immunology 1997;91:458-63. 35. Hayes D, Preston TJ, Kirkby S, et al. Human leukocyte antigen sensitization in lung transplant candidates supported by extracorporeal membrane oxygenation. Am J Respir Crit Care Med 2013;188:627-8. 36. Kaczmarek I, Deutsch M-A, Kauke T, et al. Donor-specific HLA alloantibodies: long-term impact on cardiac allograft vasculopathy and mortality after heart transplant. Exp Clin Transplant 2008;6:229-35. 37. Yousem SA, Curley JM, Dauber J, et al. HLA-class II antigen expression in human heart-lung allografts. Transplantation 1990;49: 991-5. 38. Taylor PM, Rose ML, Yacoub MH. Expression of MHC antigens in normal human lungs and transplanted lungs with obliterative bronchiolitis. Transplantation 1989;48:506-10. 39. Sato M, Waddell TK, Wagnetz U, et al. Restrictive allograft syndrome (RAS): a novel form of chronic lung allograft dysfunction. J Heart Lung Transplant 2011;30:735-42.