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Proteomic Profiling of Extracellular Vesicles during Acute Cellular Rejection
Abstracts
S155
administration of anti-H2k (n=7) nor anti-H2d (n=6) Class I DSA resulted in development of CAV. Conclusion: Donor strain determines if the activating signal provided to NK cells by binding to Class I DSA, coupled with the lack of inhibition from “missing self,” is sufficient to result in NK cell triggering with initial results suggesting that certain donor / recipient MHC combinations demonstrate a permissive phenotype towards NK cell quiescence. 359 Proteomic Profiling of Extracellular Vesicles during Acute Cellular Rejection € O. Gidlöf, I. Sukma Dewi, J. Ohman, M. Evander, T. Laurell and G. Smith. Lund University, Lund, Sweden. Purpose: Non-invasive tests for the diagnosis of acute cellular rejection (ACR) represents an unmet need in the surveillance of cardiac allograft recipients. Extracellular vesicles (EVs) are sub-micron, membraneenclosed particles released into the circulation upon tissue injury or stress, and T-cell derived EVs have been shown to play a role in ACR pathophysiology. The molecular cargo of EVs are thought to reflect the state of their cell of origin and are therefore considered a potential source of biomarkers. The aim of this study was to profile immune-related proteins in isolated EVs from cardiac transplantation patients to identify novel biomarkers for ACR. Methods: Plasma was collected serially from 8 cardiac allograft recipients before, during and after biopsy-proven ACR grade ≥2R (2 [25%] women, mean age 45.2 §21.2). EVs were isolated from plasma using acoustic seed trapping. The levels of 92 low-abundant immune-related proteins were assayed in plasma and EV lysates using a panel of proximity extension assays. Proteins that were dysregulated during ACR were identified by repeated measures ANOVA, adjusting for multiple comparisons using a false discovery rate approach. The presence of EV proteins on leukocyte-, endothelial- and platelet-derived EVs was confirmed by co-staining of isolated EVs with antibodies specific for CD16, CD31 and CD41a, respectively, and analyzed by flow cytometry. Results: 48 proteins were defined as detected (i.e. above the limit of detection in >50% of samples) in the EV lysates. The levels of EV-proteins were overall poorly correlated with those in plasma: only 16.7 % of EVproteins showed a significant correlation between plasma and EV lysates. The mean levels of 20 EV-proteins were numerically elevated during ACR. One of these, TNF-related weak inducer of apoptosis (TWEAK), was significantly increased (q<0.05) during ACR compared to both before and after. In contrast, plasma levels of TWEAK were unaltered during ACR. TWEAK was present on approximately 40% of circulating EVs, and the majority of TWEAK+EVs were of endothelial origin. Conclusion: The EV proteome is perturbed during ACR and differs substantially from that in corresponding plasma samples. The analysis of isolated EVs can provide additional diagnostic and pathophysiological information over plasma alone. TWEAK is a vesicle-associated protein with potential utility as a biomarker for ACR. 360 Recipient CD103+ Classical Dendritic Cells Enhance Acute Rejection in Response to Airway Inflammation after Mouse Lung Transplantation T. Watanabe, T. Martinu, K. Boonstra, J.M. Umana, M. Horie, Z. Guan, D. Hwang, M. Liu, S. Keshavjee and S. Juvet. University Health Network, Toronto, ON, Canada. Purpose: Airway inflammation after lung transplantation (LTX), resulting from infection, pollution, or aspiration, is an important risk factor for chronic lung allograft dysfunction. Classical dendritic cells (cDCs) link innate immunity and adaptive immunity, and exhibit regional and functional specialization in the lung. CD103+ cDCs reside in the airway epithelium. Their role in lung transplantation (LTX) has not been extensively investigated. We hypothesized that CD103+ cDCs augment adaptive immune responses under conditions of lipopolysaccharide (LPS)induced airway inflammation inamouse minor alloantigen-mismatched orthotopic LTX model (C57BL/10 [B10] !C57BL/6 [B6] and vice versa).
Methods: BATF3-/- mice (B6 background), lacking CD103+ cDCs, were used. Three comparisons were made: 1) B10 ! 6 vs. B10 ! ATF3-/-, 2) B10 !B6 vs. B10 ! ATF3-/- with 6 doses of intra-tracheal (i.t.) LPS (5mg in 50mlPBS)on serial postoperative days (POD), 3)B6 ! 10 and BATF3-/- ! 10 with 8 doses of i.t. LPS on serial PODs. The grafts were assessed on POD 28. Results: Acute rejection (ISHLT criteria) did not differ significantly between B10 ! 6 and B10 ! ATF3-/- without LPS, or between B6 ! 10 and BATF3-/- ! 10 with LPS. In contrast, with LPS exposure, acute rejection scores were lower in B10aBATF3-/- compared to B10 aB6 (Figure A). Surprisingly, lung cell analysis by flow cytometry showed that monocytes and neutrophils were significantly higher in BATF3-/- recipients than wild-type recipients under repeated airway LPS exposure (Figure B). Conclusion: Our data show that recipient, but not donor, CD103+ cDCs contribute to the augmentation of acute rejection after LTx in the setting of ongoing airway inflammation. As CD103+ cDCs can promote immune regulation, they may be an important therapeutic target after LTX.
361 Development of Novel Murine Antibody Mediated Rejection Model after Orthotopic Lung Transplant Y. Shiina, H. Suzuki, T. Kaiho, A. Hata, T. Yamamoto, J. Morimoto, Y. Sakairi, H. Wada, T. Nakajima and I. Yoshino Department of General Thoracic Surgery, Chiba University Graduate School of Medicine, Chiba, Japan. Purpose: Antibody mediated rejection (AMR) has been playing an important role in organ transplantation inducing acute or chronic graft failure, which directly influence the survival of graft. There is little knowledge about AMR in lung transplant compared with other organ. Animal disease model is warranted to elucidate mechanism and development of novel drug for AMR, hence our purpose of this study is to establish murine AMR model of lung transplant.
S155
administration of anti-H2k (n=7) nor anti-H2d (n=6) Class I DSA resulted in development of CAV. Conclusion: Donor strain determines if the activating signal provided to NK cells by binding to Class I DSA, coupled with the lack of inhibition from “missing self,” is sufficient to result in NK cell triggering with initial results suggesting that certain donor / recipient MHC combinations demonstrate a permissive phenotype towards NK cell quiescence. 359 Proteomic Profiling of Extracellular Vesicles during Acute Cellular Rejection € O. Gidlöf, I. Sukma Dewi, J. Ohman, M. Evander, T. Laurell and G. Smith. Lund University, Lund, Sweden. Purpose: Non-invasive tests for the diagnosis of acute cellular rejection (ACR) represents an unmet need in the surveillance of cardiac allograft recipients. Extracellular vesicles (EVs) are sub-micron, membraneenclosed particles released into the circulation upon tissue injury or stress, and T-cell derived EVs have been shown to play a role in ACR pathophysiology. The molecular cargo of EVs are thought to reflect the state of their cell of origin and are therefore considered a potential source of biomarkers. The aim of this study was to profile immune-related proteins in isolated EVs from cardiac transplantation patients to identify novel biomarkers for ACR. Methods: Plasma was collected serially from 8 cardiac allograft recipients before, during and after biopsy-proven ACR grade ≥2R (2 [25%] women, mean age 45.2 §21.2). EVs were isolated from plasma using acoustic seed trapping. The levels of 92 low-abundant immune-related proteins were assayed in plasma and EV lysates using a panel of proximity extension assays. Proteins that were dysregulated during ACR were identified by repeated measures ANOVA, adjusting for multiple comparisons using a false discovery rate approach. The presence of EV proteins on leukocyte-, endothelial- and platelet-derived EVs was confirmed by co-staining of isolated EVs with antibodies specific for CD16, CD31 and CD41a, respectively, and analyzed by flow cytometry. Results: 48 proteins were defined as detected (i.e. above the limit of detection in >50% of samples) in the EV lysates. The levels of EV-proteins were overall poorly correlated with those in plasma: only 16.7 % of EVproteins showed a significant correlation between plasma and EV lysates. The mean levels of 20 EV-proteins were numerically elevated during ACR. One of these, TNF-related weak inducer of apoptosis (TWEAK), was significantly increased (q<0.05) during ACR compared to both before and after. In contrast, plasma levels of TWEAK were unaltered during ACR. TWEAK was present on approximately 40% of circulating EVs, and the majority of TWEAK+EVs were of endothelial origin. Conclusion: The EV proteome is perturbed during ACR and differs substantially from that in corresponding plasma samples. The analysis of isolated EVs can provide additional diagnostic and pathophysiological information over plasma alone. TWEAK is a vesicle-associated protein with potential utility as a biomarker for ACR. 360 Recipient CD103+ Classical Dendritic Cells Enhance Acute Rejection in Response to Airway Inflammation after Mouse Lung Transplantation T. Watanabe, T. Martinu, K. Boonstra, J.M. Umana, M. Horie, Z. Guan, D. Hwang, M. Liu, S. Keshavjee and S. Juvet. University Health Network, Toronto, ON, Canada. Purpose: Airway inflammation after lung transplantation (LTX), resulting from infection, pollution, or aspiration, is an important risk factor for chronic lung allograft dysfunction. Classical dendritic cells (cDCs) link innate immunity and adaptive immunity, and exhibit regional and functional specialization in the lung. CD103+ cDCs reside in the airway epithelium. Their role in lung transplantation (LTX) has not been extensively investigated. We hypothesized that CD103+ cDCs augment adaptive immune responses under conditions of lipopolysaccharide (LPS)induced airway inflammation inamouse minor alloantigen-mismatched orthotopic LTX model (C57BL/10 [B10] !C57BL/6 [B6] and vice versa).
Methods: BATF3-/- mice (B6 background), lacking CD103+ cDCs, were used. Three comparisons were made: 1) B10 ! 6 vs. B10 ! ATF3-/-, 2) B10 !B6 vs. B10 ! ATF3-/- with 6 doses of intra-tracheal (i.t.) LPS (5mg in 50mlPBS)on serial postoperative days (POD), 3)B6 ! 10 and BATF3-/- ! 10 with 8 doses of i.t. LPS on serial PODs. The grafts were assessed on POD 28. Results: Acute rejection (ISHLT criteria) did not differ significantly between B10 ! 6 and B10 ! ATF3-/- without LPS, or between B6 ! 10 and BATF3-/- ! 10 with LPS. In contrast, with LPS exposure, acute rejection scores were lower in B10aBATF3-/- compared to B10 aB6 (Figure A). Surprisingly, lung cell analysis by flow cytometry showed that monocytes and neutrophils were significantly higher in BATF3-/- recipients than wild-type recipients under repeated airway LPS exposure (Figure B). Conclusion: Our data show that recipient, but not donor, CD103+ cDCs contribute to the augmentation of acute rejection after LTx in the setting of ongoing airway inflammation. As CD103+ cDCs can promote immune regulation, they may be an important therapeutic target after LTX.
361 Development of Novel Murine Antibody Mediated Rejection Model after Orthotopic Lung Transplant Y. Shiina, H. Suzuki, T. Kaiho, A. Hata, T. Yamamoto, J. Morimoto, Y. Sakairi, H. Wada, T. Nakajima and I. Yoshino Department of General Thoracic Surgery, Chiba University Graduate School of Medicine, Chiba, Japan. Purpose: Antibody mediated rejection (AMR) has been playing an important role in organ transplantation inducing acute or chronic graft failure, which directly influence the survival of graft. There is little knowledge about AMR in lung transplant compared with other organ. Animal disease model is warranted to elucidate mechanism and development of novel drug for AMR, hence our purpose of this study is to establish murine AMR model of lung transplant.