Implications for Human Leukocyte Antigen Antibodies After Lung Transplantation. A 10-Year Experience in 441 Patients

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1 CHEST Originl Reserch TRANSPLANTATION Implictions for Humn Leukocyte Antigen Antibodies After Lung Trnsplnttion A 10-Yer Experience in 441 Ptients Lurie D. Snyder, MD, MHS ; Ziwei Wng, BS ; Dong-Feng Chen, PhD ; Nncy L. Reinsmoen, PhD ; C. Ashley Finlen-Copelnd, MS ; W. Austin Dvis, BS ; Dvid W. Zs, MD, MBA ; nd Scott M. Plmer, MD, MHS, FCCP Bckground: Long-term survivl fter lung trnsplnt is limited by the development of chronic nd progressive irflow obstruction, condition known s bronchiolitis obliterns syndrome (BOS). While prior studies strongly implicte cellulr rejection s strong risk fctor for BOS, less is known bout the clinicl significnce of humn leukocyte ntigen (HLA) ntibodies nd donor HLA-specific ntibodies in long-term outcomes. Methods: A single-center cohort of 441 lung trnsplnt recipients, spnning 10-yer period, ws prospectively screened for HLA ntibodies fter trnsplnt using flow cytometry-bsed methods. The prevlence of nd predictors for HLA ntibodies were determined. The impct of HLA ntibodies on survivl fter trnsplnt nd the development of BOS were determined using Cox models. Results: Of the 441 recipients, 139 (32%) hd detectble ntibodies to HLA. Of these 139, 54 (39%) developed ntibodies specific to donor HLA. The detection of posttrnsplnt HLA ntibodies ws ssocited with BOS (HR, 1.54; P 5.04) nd deth (HR, 1.53; P 5.02) in multivrible models. The detection of donor-specific HLA ntibodies ws ssocited with deth (HR, 2.42; P,.0001). The detection of posttrnsplnt HLA ntibodies ws ssocited with pretrnsplnt HLA-ntibody detection, pltelet trnsfusions, nd the development of BOS nd cytomeglovirus pneumonitis. Conclusions: Approximtely one-third of lung trnsplnt recipients hve detectble HLA ntibodies, which re ssocited with worse prognosis regrding grft function nd ptient survivl. CHEST 2013; 144(1): Abbrevitions: ARR 5 cute rejection rtio; BOS 5 bronchiolitis obliterns syndrome; CMV 5 cytomeglovirus; DSA 5 donor humn leukocyte ntigen-specific ntibodies; HLA 5 humn leukocyte ntigen; HR 5 hzrd rtio; IQR 5 interqurtile rnge; ISHLT 5 Interntionl Society for Hert nd Lung Trnsplnt; PGD 5 primry llogrft dysfunction Long-term outcomes fter lung trnsplnt re limited by the development of bronchiolitis obliterns syndrome (BOS), condition of progressive irflow decline. One of the strongest risk fctors for BOS is the number nd severity of cute cellulr rejection episodes mrked by T-cell infiltrtes round blood vessels nd bronchioles in the llogrft. 1 More recently, ntibody-medited, humorl or B-cell, rejection is being recognized s possible risk fctor for poor longterm outcomes in solid-orgn trnsplnttion. Initil reports from renl trnsplnt recipients described endothelil injury tht ws distinctly different from cellulr rejection nd tht corresponded to clinicl decline. 2,3 In ddition, complement split products in tissue smples nd humn leukocyte ntigen (HLA) 226 ntibodies detected in serum corresponded to llogrft dysfunction. 4-6 In lung trnsplnt, centers hve reported widely vrying rtes of ntibody-medited rejection bsed on tissue dignosis. 7-9 The difficulties of tissue dignosis in lung trnsplnt ntibody rejection re evidenced by the inbility of two ntionl conferences on llogrft rejection to crete consensus definition. 10,11 Rther thn focus on tissue, mny centers re using serum HLA ntibodies to identify possible ntibodymedited rejection. Recent dvnces in the determintion of HLA ntibodies by solid-phse technologies hve incresed the sensitivity nd specificity of HLAntibody detection. While likely not the only ntibodies produced in this type of rejection, HLA ntibodies Originl Reserch

2 provide mrker for B-cell ctivtion. To our knowledge, our group ws one of the first to report tht lung trnsplnt recipients who develop donor-specific HLA ntibodies (DSA) hve higher risk of developing BOS nd of worse posttrnsplnt survivl compred with individuls who did not develop DSA. 12 Subsequent studies hve confirmed tht pretrnsplnt presence of HLA ntibodies is ssocited with worse survivl, nd in smll series, HLA ntibodies detected posttrnsplnt re ssocited with rejection nd llogrft dysfunction More recently, prospective study t single center noted tht recipients with DSA who received tretment did not hve n incresed risk for cute cellulr rejection, lymphocytic bronchiolitis, BOS, or worse survivl. 16 Given the diverse reports on the incidence of HLA ntibodies nd ssocition with llogrft dysfunction, we sought to review our lrge recipient cohort with extended longitudinl follow-up for HLA ntibodies nd to outline the risk fctors for nd incidence nd implictions of detection of HLA ntibodies fter lung trnsplnt. Since 2000, we hve used prospective screening protocol for HLA ntibodies. We specificlly focused on HLA ntibodies, given the lck of consensus regrding histologic definition of ntibody rejection. Study Cohort Mterils nd Methods Adults ( 18 yers old) receiving first, cdveric lung trnsplnt t Duke University Medicl Center between Jnury 1, 2000, nd October 1, 2008, with t lest 30-dy survivl were eligible for this study. Multiorgn, living lobr, nd retrnsplnt recipients were excluded. All recipients received stndrdized immunosuppression, pulmonry function tests, nd trnsbronchil biopsies s described in the supplementl mteril (e-appendix 1). 17 The study ws pproved through the Duke University institutionl review bord (IRB# ). Mnuscript received Mrch 2, 2012; revision ccepted December 9, Affilitions: From the Deprtment of Medicine (Drs Snyder, Zs, nd Plmer; Mss Wng nd Finlen-Copelnd; nd Mr Dvis), nd Deprtment of Pthology (Dr Chen), Duke University, Durhm, NC; nd Deprtment of Peditrics nd Medicl Genetics Reserch Institute (Dr Reinsmoen), Cedrs-Sini Helth Systems, Los Angeles, CA. Funding/Support: This work ws supported by the Ntionl Institutes of Helth (NIH) [grnt KL2RR024127] nd the Americn Society of Trnsplnttion Clinicl Fculty Development Awrd (Dr Snyder), nd by the NIH/Ntionl Hert Lung nd Blood Institute SCCOR [grnts 1P50-HL nd K ] (Dr Plmer). Correspondence to: Lurie D. Snyder, MD, MHS, Deprtment of Medicine, Duke University, DUMC , Durhm, NC 27710; e-mil: lurie.snyder@dm.duke.edu 2013 Americn College of Chest Physicins. Reproduction of this rticle is prohibited without written permission from the Americn College of Chest Physicins. See online for more detils. DOI: /chest HLA Antibody Determintion nd Screening Protocol Prior to trnsplnt nd serilly fter trnsplnt, ll recipients re screened for the presence nd specificity of HLA ntibodies. Routine screening is done to coincide with surveillnce bronchoscopies t 1, 3, 6, 9, nd 12 months posttrnsplnt. Additionl HLA ntibody screens re performed in the setting of clinicl decline. Dt collection for this nlysis ended April 1, Allogrft Assessments Acute rejection ws defined s perivsculr infiltrtes detected on trnsbronchil biopsies s described by Interntionl Society for Hert nd Lung Trnsplnt (ISHLT) guidelines. 11 We used time-dependent cute rejection rtio (ARR), where the sum of the ISHLT grde A scores ws divided by the totl number of trnsbronchil biopsies nd considered s time-dependent predictor. BOS ws defined s progressive irflow obstruction ccording to the ISHLT guidelines. 18 In ddition, to be eligible for our BOS nlysis, recipients hd posttrnsplnt survivl of 90 dys nd hd undergone t lest five pulmonry function tests. Primry llogrft dysfunction (PGD) grde 3 ws defined by P o 2 /Fio 2 rtio, 200 t 72 h posttrnsplnt nd the presence of rdiogrphic infiltrtes. 19 Recipients who were on extrcorporel membrne oxygention t 72 h posttrnsplnt were considered to hve PGD grde 3. Sttisticl Anlysis Demogrphic vribles were compred between individuls with nd without HLA ntibodies using x 2, Fisher, or Wilcoxon rnk-sum tests s pproprite. Cox proportionl hzrds models were used to evlute the impct of both time-dependent nd timeindependent covrites on llogrft survivl, BOS, nd detection of posttrnsplnt HLA ntibodies nd DSA. The first episodes of cytomeglovirus (CMV) pneumonitis, ARR, detection of HLA ntibodies, nd DSA were ll limited to occurrence prior to BOS onset nd were considered time-dependent covrites. The selection of predictor vribles ws bsed upon sttisticl differences between ptients with nd without HLA ntibodies nd prior published risk fctors. In ddition, the development of BOS nd detection of HLA ntibodies nd DSA were lso considered timedependent covrites in the survivl model; HLA ntibodies nd DSA lso were considered time-dependent covrites in the BOS model. Ech predictor vrible ws first entered into univrite model; those meeting significnce level.05 were included in the multivrible regression model. Anlyses were performed using SAS version 9.2 (SAS Institute Inc). Results Study Cohort Demogrphics nd HLA Evlution There were 460 lung trnsplnt recipients who met initil inclusion criteri nd were eligible for nlysis. A totl of 5,813 individul serum smples were evluted for HLA ntibodies, including 2,119 pretrnsplnt smples nd 3,694 posttrnsplnt smples. Of these 460 recipients, 19 were subsequently excluded from the nlysis becuse they hd no posttrnsplnt HLA-ntibody tests. Of the remining 441 subjects, 139 (32%) hd detectble HLA ntibodies; these composed the positive HLA-ntibody cohort. The remining 302 (71%) subjects composed the negtive HLA-ntibody cohort. HLA ntibody-positive recipients were more likely to be femle; non-white ; journl.publictions.chestnet.org CHEST / 144 / 1 / JULY

3 hve detectble, pretrnsplnt HLA ntibodies; hve received pltelets or cryoprecipitte during the first 30 dys fter trnsplnt; be dischrged on mycophenolte mofetil; nd to hve hd retrospective positive crossmtch t the time of trnsplnt ( Tble 1 ). Of the 139 recipients with positive HLA ntibodies posttrnsplnt, 108 (78%) hd their first detectble HLA ntibody within the first yer fter trnsplnt, nd 54 (39%) developed DSA. The medin time to DSA development ws 52 dys posttrnsplnt (interqurtile rnge [IQR], ). Posttrnsplnt HLA Antibodies Are Associted With BOS Of the 441 subjects in the cohort, 415 were eligible for BOS ssessment. Of these, 181 (44%) developed BOS during the nlysis study period (medin follow-up time, 4.68 yers). The detection of HLA ntibodies ws risk fctor for BOS in univrite nlysis (hzrd rtio [HR], 1.57; 95% CI, ). The detection of DSA ws not significnt predictor for BOS (HR, 1.45; 95% CI, ; P 5.19). In the univrite nlysis, pretrnsplnt ntibodies, type of trnsplnt, PGD grde 3, nd ARR were significntly ssocited with the development of BOS ( Tble 2 ). In our cohort, community-cquired virl infections, pthogenic Aspergillus pulmonry infections, nd Pseudomons eruginos pulmonry infection or coloniztion prior to the development of BOS were not different between those with nd without BOS, nd these infections were not ssocited with BOS in our cohort. In multivrible regression model, the detection of HLA ntibodies remined significnt predictor for BOS when controlling for pretrnsplnt ntibodies, type of trnsplnt, PGD grde 3, nd ARR (HR, 1.54; 95% CI, ; P 5.04) ( Tble 3 ). Posttrnsplnt HLA Antibodies Are Associted With Worse Survivl During the study period, 187 of the 441 subjects (42%) died (medin survivl, 4.36 yers; IQR, ). Detection of HLA ntibodies posttrnsplnt ws ssocited with worse survivl in univrite nlysis (HR, 2.43; 95% CI, ; P,.0001). Severl vribles were considered in seprte univrite survivl nlyses ( Tble 4 ). Older ge, ntive lung disese (cystic or restrictive), positive crossmtch, pretrnsplnt HLAntibody sttus, PGD grde 3, CMV pneumonitis, nd BOS were significntly ssocited with worse survivl in the univrite nlysis nd therefore were included in the multivrible nlysis ( Tble 5 ). The detection of HLA ntibodies posttrnsplnt remined significnt predictor of worse survivl in the multivrite survivl model (HR, 1.53; 95% CI, ; P 5.02). Tble 1 Demogrphics of 441 Lung Trnsplnt Recipients Chrcteristics Not HLA Positive Posttrnsplnt (n 5 302) HLA Positive Posttrnsplnt (n 5 139)P Vlue Mle sex 183 (61) 67 (48).01 Rce.01 White 278 (92) 114 (82) Blck 24 (7) 22 (16) Other 2 (1) 3 (2) HLA-positive ntibody pretrnsplnt 22 (7) 63 (45),.0001 Bilterl trnsplnt 291 (96) 138 (99).11 Ntive disese.08 Cystic 60 (20) 14 (10) Obstructive 120 (40) 65 (47) Restrictive 114 (38) 56 (40) Vsculr 8 (3) 4 (3) Age t trnsplnt, medin (IQR), y 57 (44-63) 53 (47-61).76 PGD grde 3 t 72 h fter trnsplnt 34 (11) 19 (14).48 Positive crossmtch 0 (0) 3 (2).01 Recipients receiving blood products in first 30 d fter trnsplnt RBCs 282 (93) 134 (96).20 Pltelets 104 (34) 71 (51).0009 Plsm 196 (65) 94 (68).58 Cryoprecipitte 32 (11) 32 (23).0006 Study dys, medin (IQR) 1,752 (1,140-2,558) 1,447 (861-19,52).003 Died during study period 114 (38) 73 (53).004 Reched BOS sttus during study period 117 (41) 64 (50).06 Posttrnsplnt HLA tests per ptient, medin (IQR), No. 6 (4-9) 11 (8-16),.0001 Dt given s No. (%) unless otherwise indicted. BOS 5 bronchiolitis obliterns syndrome; HLA 5 humn leukocyte ntigen; IQR 5 interqurtile rnge; PGD 5 primry grft dysfunction. Bsed on 415 eligible recipients. 228 Originl Reserch

4 Tble 2 Univrite Anlysis of Risk Fctors for BOS Risk Fctors HR (95% CI) P Vlue Age. 56 y 1.06 ( ).72 Pretrnsplnt HLA-ntibody 1.59 ( ).01 positive Bilterl trnsplnt 0.40 ( ).005 Positive crossmtch 3.59 ( ).21 PGD grde 3 t 72 h 1.54 ( ).04 CMV pneumonitis b 1.21 ( ).30 ARR b 1.80 ( ).014 Detection of HLA ntibodies b 1.57 ( ).007 Detection of DSA b 1.45 ( ).19 ARR 5 cute rejection rtio; CMV 5 cytomeglovirus; DSA 5 donor humn leukocyte ntigen-specific ntibodies; HR 5 hzrd rtio. See Tble 1 legend for expnsion of other bbrevitions. Limited to 356 recipients with repet rteril blood gs vlues in first 72 h. b Events considered before the development of BOS nd in timedependent mnner. Posttrnsplnt DSA Is Associted With Worse Survivl In the univrite nlysis, the first detection of DSA s time-dependent predictor ws ssocited with worse survivl (HR, 3.43; 95% CI, ; P.0001). DSA clss I or DSA clss II modeled s time-dependent predictors in seprte univrite nlyses were lso ssocited with worse survivl (clss I HR, 3.49; 95% CI, ; P.0001; clss II HR, 4.23; 95% CI, ; P.0001). The development of ny DSA, DSA clss I, nd DSA clss II were considered in seprte multivrible models tht included ge, pretrnsplnt HLA-ntibody sttus, ntive lung disese, PGD grde 3, CMV pneumonitis, nd BOS. Controlling for these other vribles, the detection of ny DSA ws significnt predictor for worse survivl (HR, 2.39; 95% CI, ; P ) ( Tble 6 ). In seprte multivrible survivl nlysis, both the detection of DSA clss I (HR, 2.43; 95% CI, ; P 5.003) nd the detection of DSA clss II (HR, 3.12; 95% CI, ; P.0001) lso predicted worse survivl fter lung trnsplnt ( Tble 7 ). Tble 3 Multivrible Anlysis of Risk Fctors for BOS Risk Fctors HR (95% CI) P Vlue Pretrnsplnt HLA-ntibody positive 1.26 ( ).30 Bilterl trnsplnt 0.43 ( ).01 PGD grde 3 t 72 h 1.49 ( ).13 ARR 1.92 ( ).007 Detection of HLA ntibodies 1.54 ( ).04 Events considered before the development of BOS nd in timedependent mnner. Tble 4 Univrite Anlysis of Risk Fctors for Deth Risk Fctors HR (95% CI) P Vlue Age. 56 y t trnsplnt 1.37 ( ).03 Sex 0.99 ( ).96 White rce 0.79 ( ).30 Pretrnsplnt HLA-ntibody positive 1.92 ( ),.0001 Positive crossmtch 4.71 ( ).03 Ntive lung disese (reference group: ll others) Cystic 0.53 ( ).005 Obstructive 1.03 ( ).84 Vsculr 0.96 ( ).96 Restrictive 1.39 ( ).03 Bilterl trnsplnt 0.72 ( ).34 PGD grde 3 t 72 h 1.56 ( ).05 Development of CMV pneumonitis 1.65 ( ).003 Detection of HLA ntibodies 2.43 ( ),.0001 Development of BOS 12.9 ( ),.0001 Development of DSA 3.43 ( ),.0001 Development of DSA clss I 3.49 ( ),.0001 Development of DSA clss II 4.23 ( ),.0001 Time-dependent covrite. Predictors for HLA Detection After Trnsplnt In univrite Cox regression nlyses, sex, rce, positive crossmtch, pltelet trnsfusion, cryoprecipitte trnsfusion, pretrnsplnt HLA ntibodies, CMV pneumonitis (prior to HLA ntibody detection), nd the development of BOS (prior to HLA ntibody detection) were ssocited with posttrnsplnt HLA ntibodies ( Tble 8 ) nd, therefore, included in the multivrible model. Of note, ll of the recipients hd t lest one posttrnsplnt HLA-ntibody test prior to the development of CMV or BOS. In the multivrible model, pltelet trnsfusion (HR, 1.56; 95% CI, ; P 5.03), pretrnsplnt HLA ntibodies (HR, 6.19; 95% CI, ; P.0001), CMV pneumonitis (HR, 2.03; 95% CI, ; P 5.03), nd the development of BOS (HR, 9.53; 95% CI, ; P.0001) remined significnt predictors for HLA ntibodies fter trnsplnt ( Tble 9 ). Tble 5 Multivrible Model of HLA Antibodies nd Risk Fctors for Deth Risk Fctors HR (95% CI) P Vlue Age. 56 y t trnsplnt 1.24 ( ).21 Cystic 1.01 ( ).97 Restrictive 1.21 ( ).27 Positive crossmtch 1.98 ( ).37 Pretrnsplnt HLA-ntibody positive 1.26 ( ).28 PGD grde 3 t 72 h 1.25 ( ).41 Development of CMV pneumonitis 1.38 ( ).07 Development of BOS ( ),.0001 Detection of HLA ntibodies 1.53 ( ).02 Time-dependent covrite. journl.publictions.chestnet.org CHEST / 144 / 1 / JULY

5 Tble 6 Multivrible Model of DSA nd Risk Fctors for Deth Risk Fctors HR (95% CI) P Vlue Age. 56 y t trnsplnt 1.28 ( ).16 Cystic 1.10 ( ).74 Restrictive 1.13 ( ).48 Positive crossmtch 1.24 ( ).79 Pretrnsplnt HLA-ntibody positive 1.31 ( ).16 PGD grde 3 t 72 h 1.30 ( ).34 Development of CMV pneumonitis 1.45 ( ).04 Development of BOS ( ),.0001 Development of DSA 2.39 ( ).0002 Time-dependent covrite. Predictors for DSA Detection After Trnsplnt Given the reltively few DSA events in our study cohort, we limited our nlysis to three predictors tht were the strongest predictors for HLA ntibodies fter lung trnsplnt: pretrnsplnt HLA ntibodies, CMV pneumonitis (prior to DSA detection), nd the development of BOS (prior to DSA detection). In seprte univrite nlyses, pretrnsplnt HLA ntibodies nd the development of BOS were significnt nd included in the multivrible model ( Tble 10 ). Both pretrnsplnt HLA ntibodies (HR, 3.68; 95% CI, ; P.0001) nd the development of BOS (HR, 5.65; 95% CI, ; P ) remined significnt predictors for the development of DSA fter lung trnsplnt ( Tble 11 ). Discussion In this lrge cohort of 441 lung trnsplnt recipients, we hve found strong ssocition between posttrnsplnt HLA ntibodies nd the subsequent development of BOS nd worse survivl. Furthermore, the detection of DSA, prticulrly DSA clss II, ws ssocited with significntly worse survivl thn simply the presence of HLA ntibodies. Although somewht Tble 7 Multivrible Model of DSA Clss I nd Clss II nd Risk Fctors for Deth Risk Fctors HR (95% CI) P Vlue Age. 56 y t trnsplnt 1.24 ( ).22 Cystic 1.06 ( ).85 Restrictive 1.16 ( ).40 PGD grde 3 t 72 h 1.21 ( ).48 Development of CMV pneumonitis 1.54 ( ).02 Development of BOS ( ),.0001 Development of DSA clss I 2.43 ( ).003 Development of DSA clss II 3.12 ( ),.0001 Time-dependent covrite. Tble 8 Univrite Anlysis of Predictors for HLA Antibodies After Trnsplnt Predictor HR (95% CI) P Vlue Femle sex 1.59 ( ).006 White rce 0.48 ( ).001 Positive crossmtch 5.34 ( ).004 Pltelet trnsfusion within 1.79 ( ) d of trnsplnt Cryoprecipitte trnsfusion within 1.89 ( ) d of trnsplnt Pretrnsplnt HLA-ntibody positive 6.86 ( ),.0001 Development of BOS 3.77 ( ).0003 Development of CMV pneumonitis 2.04 ( ).02 Time-dependent covrite occurring before the detection of HLA ntibodies. surprising, DSA ws not ssocited with BOS. Our results dd to the growing interest in the clinicl significnce of HLA ntibodies by providing nlysis of lrge cohort, mnged by protocol screening nd with extended follow-up, which llowed for ssessment of llogrft function nd survivl. There re severl possible mechnisms by which ntibodies my contribute to llogrft dysfunction. Antibodies my promote complement deposition, directly dmge the epithelium, or stimulte growth fctors. Severl reports hve noted complement deposition in the llogrft of recipients with circulting HLA ntibodies. 7,20 Another potentil mechnism, supported by in vitro work, suggests humn epithelil irwy cells exposed to nti-hla ntibodies led to incresed levels of defensins, which, in turn, induce growth fc tor production nd epithelil prolifertion in mnner similr to oblitertive bronchiolitis. 21 Similrly, in vitro nd niml fibrosis models indicte tht HLA ntibodies cn promote profibrotic growth fctors. 22,23 Our findings tht HLA-ntibody detection is ssocited with the development of BOS is consistent Tble 9 Multivrible Anlysis of Predictors for HLA Antibodies After Trnsplnt Predictor HR (95% CI) P Vlue Femle sex 1.30 ( White rce 0.69 ( ).69 Positive crossmtch 1.13 ( ).84 Pltelet trnsfusion within 1.56 ( ) d of trnsplnt Cryoprecipitte trnsfusion within 1.17 ( ) d of trnsplnt Pretrnsplnt HLA-ntibody positive 6.19 ( ),.0001 Development of BOS 9.53 ( ),.0001 Development of CMV pneumonitis 2.03 ( ).03 Time-dependent covrite occurring before the detection of HLA ntibodies. 230 Originl Reserch

6 Tble 10 Univrite Anlysis of Predictors for DSA After Trnsplnt Predictor HR (95% CI) P Vlue Pretrnsplnt detectble 4.14 ( ),.0001 HLA ntibody Development of BOS 5.96 ( ).0002 Development of CMV pneumonitis 1.12 ( ).83 Time-dependent covrite nd prior to the detection of DSA. with other, smller studies. 12,15 Surprisingly, our nlysis did not find tht DSA ws predictor for BOS, lthough it ws ssocited with worse survivl. This my reflect the competing risk for deth nd BOS in this group. We did consider the possibility tht the incresed risk of mortlity ws relted to DSA tretment. However, only two of the 33 deths were both within 1 yer fter incresed immunosuppression nd relted to infection. Alterntively, this my indicte tht our study is underpowered for BOS in the DSA cohort, given the reltively low incidence of DSA. Importntly, our incidence of DSA is similr to tht of bdominl solid-orgn trnsplnt reports but notbly lower thn nother study of 122 lung trnsplnt recipients in which over one-hlf the cohort hd DSA. 16,24,25 In ddition, tht prticulr study did not find n increse in BOS or worse survivl with DSA; however, the follow-up period ws considerbly shorter thn our nlysis. Leverging lrge number of smples, lung trnsplnt recipients, nd outcome events, we were ble to identify specific risk fctors for posttrnsplnt HLA ntibodies. Our finding of sex nd pretrnsplnt presence of HLA ntibodies s risk fctors for posttrnsplnt HLA ntibodies is not surprising. Prior reports noted more complictions nd worse survivl in lung trnsplnt recipients with pretrnsplnt HLA ntibodies. 13,26,27 In retrospect, this my hve reflected posttrnsplnt HLA-ntibody presence, s our nlysis found strong reltionship between pretrnsplnt nd posttrnsplnt HLA ntibodies. In considering HLA exposure, we ssessed blood product trnsfusions within 30 dys of trnsplnt s risk fctors for HLAntibody development. We noted tht pltelets nd cryoprecipitte trnsfusions were strong risk fctors for the detection of HLA ntibodies fter trnsplnt, Tble 11 Multivrible Anlysis of Predictors for DSA After Trnsplnt Predictor HR (95% CI) P Vlue Pretrnsplnt detectble 3.68 ( ),.0001 HLA ntibody Development of BOS 5.65 ( ).0004 Time-dependent covrite nd prior to the detection of DSA. likely reflecting the potentil for HLA exposure of pooled donor blood products. Surprisingly, PGD grde 3 ws not ssocited with pretrnsplnt or posttrnsplnt HLA-ntibody detection. As PGD is ssocited with BOS nd worse survivl, we initilly thought there my be link to HLA ntibodies. Our nlysis did not confirm tht, though we re limited by the reltively low incidence of PGD grde 3 in our cohort. However, our finding tht the development of BOS nd virl pneumonitis prior to HLA-ntibody detection re risk fctors for ntibody detection is novel nd highlights our limited understnding of the triggers for ntibody production. This finding opens new venues for understnding the mechnism of ntibody-medited rejection. However, the mjority of the recipients hd detectble HLA ntibodies before they developed BOS or CMV pneumonitis. This clls for further explortion in other cohorts to vlidte the reltionship. One limittion of our study is the exclusive focus on HLA ntibodies to define possible humorl rejection. This is widely ccepted nd reproducible mesure of humorl immunity, nd, therefore, our study is pplicble to the mjority of lung trnsplnt recipients. However, ntibodies to other llogrft ntigens, including endothelil ntigens, collgen type V, nd K- tubulin, hve been implicted in humorl rejection Recent reports in renl nd hert trnsplnt noted tht complement-fixing HLA ntibodies my offer more specificity in risk of grft dysfunction thn just HLAntibody mesurements or men fluorescence intensity. 31,32 Although controversil, pthology my lso offer n lterntive pproch to define humorl rejection. The optiml ntibody nd ssy for humorl rejection my still be evolving. It is importnt to note tht the technology of ntibody detection chnged nd improved over the course of this study, most notbly in 2005 with the ddition of single-ntigen bed ssys, s described in e-appendix 1. Potentilly, there were erlier-er smples with HLA ntibodies nd/or DSA present but not detected in the older technology. This could hve bised our nlysis ginst finding difference, nd thus strengthens our finding of n ssocition with ny detectble HLA ntibodies nd worse outcomes. Another limittion is the focus on the first HLA-ntibody detection fter trnsplnt regrdless of clinicl sttus. Although we followed specific protocol for HLA-ntibody testing, we did not prospectively record the clinicl events tht my hve prompted dditionl testing. Despite these limittions, this nlysis represents the most comprehensive, systemtic nlysis of HLA ntibodies fter lung trnsplnt to dte. We confirmed tht HLA ntibodies fter lung trnsplnt re ssocited with n incresed risk for BOS nd worse survivl, nd tht DSA development is ssocited with worse survivl. HLA ntibodies pper to be n integrl journl.publictions.chestnet.org CHEST / 144 / 1 / JULY

7 prt of the immune response to the llogrft both preceding grft dysfunction nd fter llogrft dysfunction. A key question to ddress in further nlysis is if the decrese or elimintion of these ntibodies correltes with improved outcomes. Acknowledgments Author contributions: Dr Snyder hd full ccess to ll of the dt in the study nd tkes responsibility for the integrity of the dt nd the ccurcy of the dt nlysis. Dr Snyder: contributed to study design, dt nlysis, mnuscript preprtion, nd served s principl uthor. Ms Wng: contributed to dt collection nd nlysis nd review of the mnuscript. Dr Chen: contributed to mnuscript preprtion nd review. Dr Reinsmoen: contributed to mnuscript preprtion nd review. Ms Finlen-Copelnd: contributed to mnuscript preprtion nd review. Mr Dvis: contributed to study design, dt collection nd nlysis, nd review of the mnuscript. Dr Zs: contributed to study design nd mnuscript review. Dr Plmer: contributed to study design, dt nlysis, nd mnuscript review. Finncil/nonfinncil disclosures: The uthors hve reported to CHEST tht no potentil conflicts of interest exist with ny compnies/orgniztions whose products or services my be discussed in this rticle. Role of sponsors: The sponsors hd no role in the design of the study, the collection nd nlysis of the dt, or in the preprtion of the mnuscript. Other contributions : The uthors would like to cknowledge the contributions of Mry Lee Cmpbell, MT; Anthony Cstleberry, MD; Mtthew G. Hrtwig, MD; Scott Shofer, MD, PhD; Jmie Todd, MD; nd Wyne Tsung, MD, to this study. Additionl informtion: The e-appendix cn be found in the Supplementl Mterils re of the online rticle. References 1. Estenne M, Hertz MI. Bronchiolitis obliterns fter humn lung trnsplnttion. Am J Respir Crit Cre Med ;166(4): Hllorn PF, Schlut J, Solez K, Srinivs NS. The significnce of the nti-clss I response. II. Clinicl nd pthologic fetures of renl trnsplnts with nti-clss I-like ntibody. Trnsplnttion ;53(3): Hllorn PF, Wdgymr A, Ritchie S, Flk J, Solez K, Srinivs NS. The significnce of the nti-clss I ntibody response. I. Clin icl nd pthologic fetures of nti-clss I-medited rejection. Trnsplnttion ;49(1): Crespo M, Pscul M, Tolkoff-Rubin N, et l. Acute humorl rejection in renl llogrft recipients: I. Incidence, serology nd clinicl chrcteristics. Trnsplnttion ; 71 ( 5 ): de Gouvei RH, Vitorino E, Rmos S, et l. C4d-the witness of humorl rejection. Trnsplnt Proc ;41(3): Collins AB, Schneeberger EE, Pscul MA, et l. Complement ctivtion in cute humorl renl llogrft rejection: dignostic significnce of C4d deposits in peritubulr cpillries. J Am Soc Nephrol ;10(10): Ionescu DN, Girnit AL, Zeevi A, et l. C4d deposition in lung llogrfts is ssocited with circulting nti-hla llontibody. Trnspl Immunol ;15(1): Mgro CM, Deng A, Pope-Hrmn A, et l. Humorlly medited posttrnsplnttion septl cpillry injury syndrome s common form of pulmonry llogrft rejection: hypothesis. 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HLA-specific ntibodies re ssocited with high-grde nd persistent-recurrent lung llogrft cute rejection. J Hert Lung Trnsplnt ; 23 (10 ): Girnit AL, Duquesnoy R, Yousem SA, et l. HLA-specific ntibodies re risk fctors for lymphocytic bronchiolitis nd chronic lung llogrft dysfunction. Am J Trnsplnt ;5 (1 ): Hchem RR, Yusen RD, Meyers BF, et l. Anti-humn leukocyte ntigen ntibodies nd preemptive ntibody-directed therpy fter lung trnsplnttion. J Hert Lung Trnsplnt ;29 (9 ): Hrtwig MG, Snyder LD, Finlen-Copelnd A, et l. Lung trnsplnttion t Duke University. Clin Trnspl ; Estenne M, Murer JR, Boehler A, et l. Bronchiolitis obliterns syndrome 2001: n updte of the dignostic criteri. J Hert Lung Trnsplnt ;21 (3 ): Christie JD, Crby M, Bg R, Corris P, Hertz M, Weill D ; ISHLT Working Group on Primry Lung Grft Dysfunction. Report of the ISHLT Working Group on Primry Lung Grft Dysfunction prt II: definition. A consensus sttement of the Interntionl Society for Hert nd Lung Trnsplnttion. 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