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Linking CLAD in Lung Transplant Recipients after 3 Years Follow Up with Transplant Arteriosclerosis in Humanized Mice
Abstracts significance in this small sample. In 9 subjects with paired measurements, Ang2 increased from a mean of 6.7§3.5 to 11.3§7.6ng/mL (p=0.047) after LVAD implantation. Conclusion: GIADs are already present in the majority of patients undergoing LVAD implantation and appear to be part of the pathophysiology of advanced heart failure. Angiogenic dysregulation predates LVAD implantation but is augmented in the setting of continuous flow LVAD support. Enrollment in this study is ongoing and at the time of abstract presentation, we anticipate a larger sample size.
315 Exosome Proteomics and Machine Learning Identify Novel Biomarkers of Primary Graft Dysfunction N. Giangreco,1 G. Lebreton,2 S. Restaino,3 M. Farr,3 P.C. Colombo,3 E. Zorn,4 N. Tatonetti,1 P. Leprince,2 J. Kobashigawa,5 and B. Fine.3 1Biomedical Informatics, Columbia University, New York, NY; 2 Chirurgie Thoracique et Cardiovasculaire, Pitie-Salp^etriere University Hospital, Paris, France; 3Division of Cardiology, Columbia University, New York, NY; 4Center for Translational Immunology, Columbia University, New York, NY; and the 5Cedars-Sinai Heart Institute, CedarsSinai Medical Center, Los Angeles, CA. Purpose: The etiology of primary graft dysfunction (PGD) is unclear and prediction of PGD remains an area of active research. Exosomes are secreted microvesicles found in numerous body fluids and have protein and RNA content with biomarker potential. Here we investigate pre-transplant serum exosomes using proteomics to identify novel recipient biomarkers of PGD prior to heart transplant. Methods: Pre-transplant serum samples were collected prospectively at Columbia University from June 2014 to December 2015 (n=16). Pre-transplant serum samples were assembled retrospectively from HLA laboratories of Cedars Sinai Medical Center (n=44) and Pitie Salp^etriere (n=29). Exosomes were purified with the Total Exosome Isolation Kit (Life Technologies) and proteins were labelled with TMT10plex isobaric reagent. We performed LC-MS/MS with an Orbitrap Fusion Tribrid mass spectrometer. Proteome Discoverer software was used to search the acquired MS/ MS data against Uniprot human protein database and generate TMT ratios. Conditional logistic regressions with bootstrapping modeled the association of each protein to PGD status controlling for LVAD status and batch. Logistic Regression (LR), Random Forest (RF), Support Vector Machines (SVM), and Gradient Boosting Classifier (GBC) models, with regularization, and 10-fold cross validated recursive feature elimination (RFE) were used to select the most predictive proteins in the dataset. Results: 131 proteins were found in the prospective cohort showing a differential protein expression signature (FDR < 0.05) significantly classifying PGD by conditional logistic regression modeling. After integrating the two retrospective cohorts, recursive feature elimination using four machine learning models selected proteins most predictive of PGD. Classifiers from each machine learning algorithm generated classifiers with receiver operating characteristic curves with C statistics of 0.76 +/¡ 0.16 (LR), 0.78 +/¡ 0.11 (RF), 0.76 +/¡ 0.16 (SVM), and 0.78 +/¡ 0.14 (GBC). In total, 33
S137 proteins showed differential and predictive associations with the development of PGD. Conclusion: Exosome proteomics of serum samples from both prospective and retrospective cohorts followed by machine learning based analysis identified potential biomarkers for PGD within the recipient prior to transplant. 316 B Cells Drive iBALT Formation in a Mouse Model of Chronic Lung Allograft Dysfunction N.F. Smirnova,1 T.M. Conlon,2 C. Morrone,2 A.O. Yildirim,2 and O. Eickelberg.1 1PSCCM, University of Colorado - Anschutz Medical Campus, Aurora, CO; and the 2Comprehensive Pneumology Center, Munich, Germany. Purpose: Chronic lung allograft dysfunction (CLAD) is the major limitation to positive outcomes of lung transplantation (LTx). Due to the lack of a reproducible mouse model, our understanding of CLAD pathogenesis and potential therapy remains elusive. We aimed to develop a model of CLAD, after orthotopic LTx in mice. We characterized CLAD-associated iBALT and the molecular determinants of its formation, in order to 1) delineate its similarities with human CLAD and establish its relevance as a preclinical model, 2) gain mechanistic insight into the function of B cells in CLAD; and 3) prevent the onset of CLAD by interfering with iBALT formation. Methods: Left lungs from B6 (syngeneic group) or mice on a B6 background with a single human HLA-A2.1 molecule (mismatched, CLAD group) were orthotopically transplanted into B6 mice. Left lungs from HLA-A2.1 (HLA) mice were transplanted into B cell-deficient (mMT-/-) and EBI2-/- mice to decipher the role of B cells in iBALT formation. Results: Compared with syngeneic grafts, the mismatched HLA grafts presented decreased lung function and histology of lymphocytic bronchiolitis, characteristic of CLAD. The mismatched grafts further exhibited strong iBALT formation, associated with pathological alterations of the bronchial epithelium, notably a selective loss of club cells, one of the reproducible hallmarks of human CLAD. B cells in iBALT were active, as markers CD69, CD80, MHCII, and GL7 were upregulated. The formation of iBALT was disrupted in mismatched grafts transplanted to mMT-/- mice, demonstrating that B cells are necessary for iBALT formation. Histology of mismatched grafts in mMT-/- recipients was avoid of pathological findings, showing that absence of B cells is sufficient to prevent CLAD. Since a systemic depletion of B cells needs to be considered with caution, we focused on iBALT formation as a target for CLAD prevention. The GPCR EBI2 has lately been shown as a key player for B cell organization in secondary and tertiary lymphoid organs. In line with this background, transplantation of mismatched lung grafts into EBI2-/- mice, as well as treatment with a pharmacological EBI2 inhibitor, resulted in decreased iBALT formation and preservation of graft function. Conclusion: In sum, CLAD development is driven by the presence and function of B cells. EBI2 inhibition represents a promising mechanism to prevent CLAD and thus improve outcomes after LTx. 317 Linking CLAD in Lung Transplant Recipients after 3 Years Follow Up with Transplant Arteriosclerosis in Humanized Mice T. Siemeni,1 A. Knöfel,1 F. Ius,1 J. Salman,1 W. Sommer,1 M. Avsar,1 R. Poyanmehr,1 C. K€uhn,1 C. Falk,2 I. Tudorache,1 A. Haverich,1 and G. Warnecke.1 1Hannover HTTG, Hannover, Germany; and the 2Hannover Transplant Immunology, Hannover, Germany. Purpose: Chronic lung allograft dysfunction (CLAD) is a severe complication of lung transplantation (LTX) limiting long term survival. Here, we studied correlations between CLAD after clinical LTX and leukocytemediated development of transplant arteriosclerosis (TA) in a humanized mouse model. Methods: The pericardiophrenic artery was procured from surplus tissue of 27 donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. The experimental mice were then divided into four groups. Negative control
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mice received no human leukocyte reconstitution (neg. co). PBMC group mice received 5 £ 106 allogeneic human peripheral blood mononuclear cells (PBMC) from the respective lung recipients (LR). A further group of animals was reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25high cells (putative regulatory T cells, Treg). Human leukocyte engraftment was monitored by FACS and development of TA was histologically assessed 28 days after PBMC reconstitution. The 27 LR were divided into two groups at least 3 years post transplantation according to their development of chronic rejection. Nine patients (33.3%) developed CLAD 36.2§ 4.16 month after LTX. The remaining eighteen patients (66,6%) did not develop CLAD within 35.9§ 5.1 month after LTX. Results: The neg. co group showed only mild thickening of the intima (6.38§5.70%). In the PBMC CLAD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC CLAD- group (33.19 § 7.02% vs. 13.37 § 2.99%, p= 0.011). Then, intimal thickening was significantly inhibited in the PBMC+Treg CLAD+ group as compared to the PBMC CLAD+ group (0.39 § 3.69% vs 33.19 § 7.02%, p=0.012). In the experiments using PBMC from lung recipients without CLAD, enriching Treg also further suppressed the development of transplant arteriosclerosis (13.37 § 2.99% PBMC CLAD- vs. 0.69 § 5.88% PBMC+Treg CLAD-, p=0.007). Conclusion: Lung transplant recipients, who later developed CLAD, had peripheral leukocytes already at the time of transplant that transfered proinflammatory properties leading to TA into a humanized mouse model. TA remained sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention and treatment of CLAD after LTX. 318 Pulmonary Antibody-Mediated Rejection (AMR) Accelerates AgingEvidence from Whole Genome DNA Methylation Sequencing S. Agbor-Enoh,1 F. Seifuddin,1 M. Pirooznia,2 M. Jang,1 and H. Valantine.1 1Genomic Research Alliance for Transplantation (GRAfT), Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, MD; and the 2Genomic Research Alliance for Transplantation (GRAfT), Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, MD. Purpose: AMR show strikingly similar molecular mechanisms (chronic inflammation, fibrogenesis, and others processes) to conditions that accelerate lung aging. Clinically, both AMR and age acceleration manifest as progressive loss of lung function leading us to hypothesize that AMR is associated with age acceleration. Age acceleration triggers signature methylation changes at cytosine-guanosine (CpGs) motifs of “aging” genes. Thus, to test this hypothesis, we used unbiased bisulfite sequencing. Methods: Six lung transplant patients with AMR (positive allograft dysfunction, DSA and histopathology and/or C4d) were identified. Two bronchoalveolar (BAL) samples were collected from each patient, one pre-AMR control (no rejection, no infection) and one at AMR diagnosis. BAL cells were used for DNA isolation, bisulfite treatment, and sequencing. Sequences were analyzed via a custom-built computation workflow (bismark, bsseq, bumphunter) that assigns CpGs to DNA regions, normalizes data for cell composition, performs paired analysis to identify differentially methylated regions (DMRs), and maps the DMRs to genes for pathway enrichment analysis. Thresholds for CpG coverage (6X) and DMR mean difference (10%) were arbitrarily selected. P-values were corrected for multiple analyses. Results: We identified 900K DMRs between AMR and control. Average DMR length was 238 base pairs with median 4.7 CpGs per DMR (range 3-64). 38.8K DMRs showed 10% or higher methylation difference between AMR and control, with 67% of the DMRs mapped to genes within 1 Killobase. “Aging” genes were overrepresented (q=2.880 £ 10¡13): genes that promote lung age acceleration (n=370) were predominantly hypo-methylated in AMR, while genes that inhibit lung age acceleration (n=454) were predominantly hyper-methylated. Antibody-mediated pathways such as macrophage activation, Fc-gamma-mediated cell-death, and cytotoxic T-cell activation were also overrepresented (q=0.0007 - 0.0040). Conclusion: Both antibody-mediated pathways and “aging” genes were differentially methylated in AMR suggesting epigenetic regulation of these processes and a strong association between AMR and age acceleration. Future studies are needed to validate these findings and assess whether age
acceleration contribute to the progressive loss of lung function often observed with AMR. 319 Increased NKG2C+ Natural Killer Cells in Bronchoalveolar Lavage Precede CMV Viremia and Are Associated with CLAD or Death in Lung Transplant Recipients D.R. Calabrese,1 T. Chong,1 A.S. Wang,1 J.P. Singer,1 M. Gottschall,2 S.R. Hays,1 J.A. Golden,1 J. Kukreja,3 Q. Tang,3 and J.R. Greenland.4 1Department of Medicine, University of California San Francisco, San Francisco, CA; 2Department of Clinical Lab Medicine, University of California San Francisco, San Francisco, CA; 3Department of Surgery, University of California San Francisco, San Francisco, CA; and the 4Medical Service, VA Health Care System, San Francisco, CA. Purpose: Cytomegalovirus (CMV) mediates clinical outcomes in lung allograft recipients. The NKG2C receptor on natural killer (NK) cells is encoded by the KLRC2 gene and recognizes CMV with memory-like capabilities. We hypothesized that NKG2C+ NK cells in bronchoalveolar lavage (BAL) would be associated with CMV burden and decreased chronic lung allograft dysfunction (CLAD)-free survival. Methods: BAL cells were prospectively collected from 130 lung transplant recipients at a single center, with a median 391 days follow up time. NKG2C+ and - NK cell populations in BAL were compared for markers of maturation (NKG2A, CD16, KIR), propagation (Ki67), and KLRC2 genotype (rs2734561) by Student’s t-test or ANOVA. CMV viremia was defined as negative, low (<1000 copies/mL) or high (>1000 copies/mL). NKG2C+ NK cell association with CMV viremia was determined using generalized linear modeling, and association with CLAD-free survival was determined by Cox proportional hazards models adjusted for transplant characteristics, KLRC2genotype, CMV viremia, and CMV serostatus. Results: BAL NKG2C+ NK cells were more mature (NKG2A-CD16+, 25.8% vs 18.4%, p< 0.0001) and proliferative (Ki67+, 12.6% vs 8.1%, p<0.001) than NKG2C- NK cells. NKG2C+ NK cell frequency was decreased in subjects with null genotypes compared to wildtype genotypes (p = 0.05). BAL NKG2C+ NK cells increased prior to high level CMV viremia (Figure 1A, p < 0.0001). There were 23 episodes of CLAD or death. Subjects with higher than the median frequency of NKG2C+ NK cells had a nearly 6-fold higher risk of CLAD or death (Figure 1B, HR 5.7, 95% CI 1.6 - 20.3, p = 0.008). Conclusion: BAL NKG2C+ NK cells were more mature than NKG2C- NK cells and expression differed between KLRC2 genotypes. NKG2C+ NK cells increased prior to viremia and higher NKG2C+ NK cell frequencies were associated with worse CLAD-free survival. Measurement of NKG2C + NK cells in BAL may be a useful tool for assessing CMV disease burden in this population.
320 Development of a New Local Therapeutic Approach for BOS: Efficacy of Imatinib Loaded -antiCD44 Coated Gold Nanoparticles In Vitro and In Vivo F. Meloni,1 R. Di Paola,2 P. Morbini,3 L. Pandolfi,4 V. Frangipane,4 E. Gugliandolo,2 E. Cova,5 M. Colombo,6 D. Prosperi,6 P. Vitulo,7 G. Ferrario,8 R. Fusco,2 C. Pacini,6 R. Siracusa,2 and S. Cuzzocrea.2 1Internal Medicine, University of Pavia, Pavia, Italy; 2 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; 3Molecular Medicine,
315 Exosome Proteomics and Machine Learning Identify Novel Biomarkers of Primary Graft Dysfunction N. Giangreco,1 G. Lebreton,2 S. Restaino,3 M. Farr,3 P.C. Colombo,3 E. Zorn,4 N. Tatonetti,1 P. Leprince,2 J. Kobashigawa,5 and B. Fine.3 1Biomedical Informatics, Columbia University, New York, NY; 2 Chirurgie Thoracique et Cardiovasculaire, Pitie-Salp^etriere University Hospital, Paris, France; 3Division of Cardiology, Columbia University, New York, NY; 4Center for Translational Immunology, Columbia University, New York, NY; and the 5Cedars-Sinai Heart Institute, CedarsSinai Medical Center, Los Angeles, CA. Purpose: The etiology of primary graft dysfunction (PGD) is unclear and prediction of PGD remains an area of active research. Exosomes are secreted microvesicles found in numerous body fluids and have protein and RNA content with biomarker potential. Here we investigate pre-transplant serum exosomes using proteomics to identify novel recipient biomarkers of PGD prior to heart transplant. Methods: Pre-transplant serum samples were collected prospectively at Columbia University from June 2014 to December 2015 (n=16). Pre-transplant serum samples were assembled retrospectively from HLA laboratories of Cedars Sinai Medical Center (n=44) and Pitie Salp^etriere (n=29). Exosomes were purified with the Total Exosome Isolation Kit (Life Technologies) and proteins were labelled with TMT10plex isobaric reagent. We performed LC-MS/MS with an Orbitrap Fusion Tribrid mass spectrometer. Proteome Discoverer software was used to search the acquired MS/ MS data against Uniprot human protein database and generate TMT ratios. Conditional logistic regressions with bootstrapping modeled the association of each protein to PGD status controlling for LVAD status and batch. Logistic Regression (LR), Random Forest (RF), Support Vector Machines (SVM), and Gradient Boosting Classifier (GBC) models, with regularization, and 10-fold cross validated recursive feature elimination (RFE) were used to select the most predictive proteins in the dataset. Results: 131 proteins were found in the prospective cohort showing a differential protein expression signature (FDR < 0.05) significantly classifying PGD by conditional logistic regression modeling. After integrating the two retrospective cohorts, recursive feature elimination using four machine learning models selected proteins most predictive of PGD. Classifiers from each machine learning algorithm generated classifiers with receiver operating characteristic curves with C statistics of 0.76 +/¡ 0.16 (LR), 0.78 +/¡ 0.11 (RF), 0.76 +/¡ 0.16 (SVM), and 0.78 +/¡ 0.14 (GBC). In total, 33
S137 proteins showed differential and predictive associations with the development of PGD. Conclusion: Exosome proteomics of serum samples from both prospective and retrospective cohorts followed by machine learning based analysis identified potential biomarkers for PGD within the recipient prior to transplant. 316 B Cells Drive iBALT Formation in a Mouse Model of Chronic Lung Allograft Dysfunction N.F. Smirnova,1 T.M. Conlon,2 C. Morrone,2 A.O. Yildirim,2 and O. Eickelberg.1 1PSCCM, University of Colorado - Anschutz Medical Campus, Aurora, CO; and the 2Comprehensive Pneumology Center, Munich, Germany. Purpose: Chronic lung allograft dysfunction (CLAD) is the major limitation to positive outcomes of lung transplantation (LTx). Due to the lack of a reproducible mouse model, our understanding of CLAD pathogenesis and potential therapy remains elusive. We aimed to develop a model of CLAD, after orthotopic LTx in mice. We characterized CLAD-associated iBALT and the molecular determinants of its formation, in order to 1) delineate its similarities with human CLAD and establish its relevance as a preclinical model, 2) gain mechanistic insight into the function of B cells in CLAD; and 3) prevent the onset of CLAD by interfering with iBALT formation. Methods: Left lungs from B6 (syngeneic group) or mice on a B6 background with a single human HLA-A2.1 molecule (mismatched, CLAD group) were orthotopically transplanted into B6 mice. Left lungs from HLA-A2.1 (HLA) mice were transplanted into B cell-deficient (mMT-/-) and EBI2-/- mice to decipher the role of B cells in iBALT formation. Results: Compared with syngeneic grafts, the mismatched HLA grafts presented decreased lung function and histology of lymphocytic bronchiolitis, characteristic of CLAD. The mismatched grafts further exhibited strong iBALT formation, associated with pathological alterations of the bronchial epithelium, notably a selective loss of club cells, one of the reproducible hallmarks of human CLAD. B cells in iBALT were active, as markers CD69, CD80, MHCII, and GL7 were upregulated. The formation of iBALT was disrupted in mismatched grafts transplanted to mMT-/- mice, demonstrating that B cells are necessary for iBALT formation. Histology of mismatched grafts in mMT-/- recipients was avoid of pathological findings, showing that absence of B cells is sufficient to prevent CLAD. Since a systemic depletion of B cells needs to be considered with caution, we focused on iBALT formation as a target for CLAD prevention. The GPCR EBI2 has lately been shown as a key player for B cell organization in secondary and tertiary lymphoid organs. In line with this background, transplantation of mismatched lung grafts into EBI2-/- mice, as well as treatment with a pharmacological EBI2 inhibitor, resulted in decreased iBALT formation and preservation of graft function. Conclusion: In sum, CLAD development is driven by the presence and function of B cells. EBI2 inhibition represents a promising mechanism to prevent CLAD and thus improve outcomes after LTx. 317 Linking CLAD in Lung Transplant Recipients after 3 Years Follow Up with Transplant Arteriosclerosis in Humanized Mice T. Siemeni,1 A. Knöfel,1 F. Ius,1 J. Salman,1 W. Sommer,1 M. Avsar,1 R. Poyanmehr,1 C. K€uhn,1 C. Falk,2 I. Tudorache,1 A. Haverich,1 and G. Warnecke.1 1Hannover HTTG, Hannover, Germany; and the 2Hannover Transplant Immunology, Hannover, Germany. Purpose: Chronic lung allograft dysfunction (CLAD) is a severe complication of lung transplantation (LTX) limiting long term survival. Here, we studied correlations between CLAD after clinical LTX and leukocytemediated development of transplant arteriosclerosis (TA) in a humanized mouse model. Methods: The pericardiophrenic artery was procured from surplus tissue of 27 donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. The experimental mice were then divided into four groups. Negative control
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The Journal of Heart and Lung Transplantation, Vol 38, No 4S, April 2019
mice received no human leukocyte reconstitution (neg. co). PBMC group mice received 5 £ 106 allogeneic human peripheral blood mononuclear cells (PBMC) from the respective lung recipients (LR). A further group of animals was reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25high cells (putative regulatory T cells, Treg). Human leukocyte engraftment was monitored by FACS and development of TA was histologically assessed 28 days after PBMC reconstitution. The 27 LR were divided into two groups at least 3 years post transplantation according to their development of chronic rejection. Nine patients (33.3%) developed CLAD 36.2§ 4.16 month after LTX. The remaining eighteen patients (66,6%) did not develop CLAD within 35.9§ 5.1 month after LTX. Results: The neg. co group showed only mild thickening of the intima (6.38§5.70%). In the PBMC CLAD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC CLAD- group (33.19 § 7.02% vs. 13.37 § 2.99%, p= 0.011). Then, intimal thickening was significantly inhibited in the PBMC+Treg CLAD+ group as compared to the PBMC CLAD+ group (0.39 § 3.69% vs 33.19 § 7.02%, p=0.012). In the experiments using PBMC from lung recipients without CLAD, enriching Treg also further suppressed the development of transplant arteriosclerosis (13.37 § 2.99% PBMC CLAD- vs. 0.69 § 5.88% PBMC+Treg CLAD-, p=0.007). Conclusion: Lung transplant recipients, who later developed CLAD, had peripheral leukocytes already at the time of transplant that transfered proinflammatory properties leading to TA into a humanized mouse model. TA remained sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention and treatment of CLAD after LTX. 318 Pulmonary Antibody-Mediated Rejection (AMR) Accelerates AgingEvidence from Whole Genome DNA Methylation Sequencing S. Agbor-Enoh,1 F. Seifuddin,1 M. Pirooznia,2 M. Jang,1 and H. Valantine.1 1Genomic Research Alliance for Transplantation (GRAfT), Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, MD; and the 2Genomic Research Alliance for Transplantation (GRAfT), Division of Intramural Research, National Heart, Lung and Blood Institute, Bethesda, MD. Purpose: AMR show strikingly similar molecular mechanisms (chronic inflammation, fibrogenesis, and others processes) to conditions that accelerate lung aging. Clinically, both AMR and age acceleration manifest as progressive loss of lung function leading us to hypothesize that AMR is associated with age acceleration. Age acceleration triggers signature methylation changes at cytosine-guanosine (CpGs) motifs of “aging” genes. Thus, to test this hypothesis, we used unbiased bisulfite sequencing. Methods: Six lung transplant patients with AMR (positive allograft dysfunction, DSA and histopathology and/or C4d) were identified. Two bronchoalveolar (BAL) samples were collected from each patient, one pre-AMR control (no rejection, no infection) and one at AMR diagnosis. BAL cells were used for DNA isolation, bisulfite treatment, and sequencing. Sequences were analyzed via a custom-built computation workflow (bismark, bsseq, bumphunter) that assigns CpGs to DNA regions, normalizes data for cell composition, performs paired analysis to identify differentially methylated regions (DMRs), and maps the DMRs to genes for pathway enrichment analysis. Thresholds for CpG coverage (6X) and DMR mean difference (10%) were arbitrarily selected. P-values were corrected for multiple analyses. Results: We identified 900K DMRs between AMR and control. Average DMR length was 238 base pairs with median 4.7 CpGs per DMR (range 3-64). 38.8K DMRs showed 10% or higher methylation difference between AMR and control, with 67% of the DMRs mapped to genes within 1 Killobase. “Aging” genes were overrepresented (q=2.880 £ 10¡13): genes that promote lung age acceleration (n=370) were predominantly hypo-methylated in AMR, while genes that inhibit lung age acceleration (n=454) were predominantly hyper-methylated. Antibody-mediated pathways such as macrophage activation, Fc-gamma-mediated cell-death, and cytotoxic T-cell activation were also overrepresented (q=0.0007 - 0.0040). Conclusion: Both antibody-mediated pathways and “aging” genes were differentially methylated in AMR suggesting epigenetic regulation of these processes and a strong association between AMR and age acceleration. Future studies are needed to validate these findings and assess whether age
acceleration contribute to the progressive loss of lung function often observed with AMR. 319 Increased NKG2C+ Natural Killer Cells in Bronchoalveolar Lavage Precede CMV Viremia and Are Associated with CLAD or Death in Lung Transplant Recipients D.R. Calabrese,1 T. Chong,1 A.S. Wang,1 J.P. Singer,1 M. Gottschall,2 S.R. Hays,1 J.A. Golden,1 J. Kukreja,3 Q. Tang,3 and J.R. Greenland.4 1Department of Medicine, University of California San Francisco, San Francisco, CA; 2Department of Clinical Lab Medicine, University of California San Francisco, San Francisco, CA; 3Department of Surgery, University of California San Francisco, San Francisco, CA; and the 4Medical Service, VA Health Care System, San Francisco, CA. Purpose: Cytomegalovirus (CMV) mediates clinical outcomes in lung allograft recipients. The NKG2C receptor on natural killer (NK) cells is encoded by the KLRC2 gene and recognizes CMV with memory-like capabilities. We hypothesized that NKG2C+ NK cells in bronchoalveolar lavage (BAL) would be associated with CMV burden and decreased chronic lung allograft dysfunction (CLAD)-free survival. Methods: BAL cells were prospectively collected from 130 lung transplant recipients at a single center, with a median 391 days follow up time. NKG2C+ and - NK cell populations in BAL were compared for markers of maturation (NKG2A, CD16, KIR), propagation (Ki67), and KLRC2 genotype (rs2734561) by Student’s t-test or ANOVA. CMV viremia was defined as negative, low (<1000 copies/mL) or high (>1000 copies/mL). NKG2C+ NK cell association with CMV viremia was determined using generalized linear modeling, and association with CLAD-free survival was determined by Cox proportional hazards models adjusted for transplant characteristics, KLRC2genotype, CMV viremia, and CMV serostatus. Results: BAL NKG2C+ NK cells were more mature (NKG2A-CD16+, 25.8% vs 18.4%, p< 0.0001) and proliferative (Ki67+, 12.6% vs 8.1%, p<0.001) than NKG2C- NK cells. NKG2C+ NK cell frequency was decreased in subjects with null genotypes compared to wildtype genotypes (p = 0.05). BAL NKG2C+ NK cells increased prior to high level CMV viremia (Figure 1A, p < 0.0001). There were 23 episodes of CLAD or death. Subjects with higher than the median frequency of NKG2C+ NK cells had a nearly 6-fold higher risk of CLAD or death (Figure 1B, HR 5.7, 95% CI 1.6 - 20.3, p = 0.008). Conclusion: BAL NKG2C+ NK cells were more mature than NKG2C- NK cells and expression differed between KLRC2 genotypes. NKG2C+ NK cells increased prior to viremia and higher NKG2C+ NK cell frequencies were associated with worse CLAD-free survival. Measurement of NKG2C + NK cells in BAL may be a useful tool for assessing CMV disease burden in this population.
320 Development of a New Local Therapeutic Approach for BOS: Efficacy of Imatinib Loaded -antiCD44 Coated Gold Nanoparticles In Vitro and In Vivo F. Meloni,1 R. Di Paola,2 P. Morbini,3 L. Pandolfi,4 V. Frangipane,4 E. Gugliandolo,2 E. Cova,5 M. Colombo,6 D. Prosperi,6 P. Vitulo,7 G. Ferrario,8 R. Fusco,2 C. Pacini,6 R. Siracusa,2 and S. Cuzzocrea.2 1Internal Medicine, University of Pavia, Pavia, Italy; 2 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina, Italy; 3Molecular Medicine,