Acute cellular rejection (ACR) frequently occurs following lung transplantation (LuTx) and represents a risk factor for the development of chronic lung allograft dysfunction (CLAD) as well as long-term survival. The histopathological diagnosis of ACR carries a burden of interobserver variability. The widespread utilization and cost-effectiveness of immunohistochemistry (IHC) was proven beneficial in diagnosing rejection in human kidney transplantations and LuTx rat models. However, its potential for ACR detection in patients remains unexplored. We analyzed surface markers (CD3, CD4, CD8, CD20, CD68, CD47, PD-1, PD-L1, and CD31/PECAM-1) on lung tissue cryobiopsy samples collected within 6 months post-LuTx from 60 LuTx recipients, 48 of whom were diagnosed with ACR. Additionally, serum samples from 51 patients were analyzed using a multiplex bead-based Luminex assay. The cytokines and markers included PD-L1, IL2, TNFα, IFNγ, and Granzyme B. We observed a significant increase in PD-L1 tissue expression within the rejection group, suggesting a concerted effort to suppress immune responses, especially those mediated by T-cells. Furthermore, we noted significant differences in PECAM-1 levels between ACR/non-ACR. Additionally, peripheral blood C-reactive-protein levels tended to be higher in the ACR group, while Luminex serum analyses did not reveal any significant differences between groups. In conclusion, our findings suggest the potential value of PECAM-1 and PD-L1 markers in diagnosing ACR.

Department of Anesthesiology Resuscitation and Intensive Care Medicine 2nd Faculty of Medicine Charles University and Motol University Hospital Prague Czechia

Department of Immunogenetics Institute for Clinical and Experimental Medicine Prague Czechia

Department of Immunology 2nd Faculty of Medicine Charles University and Motol University Hospital Prague Czechia

Department of Obstetrics and Gynecology 1st Faculty of Medicine Charles University General University Hospital Prague Czechia

Department of Pathology and Molecular Medicine 2nd Faculty of Medicine Charles University and Motol University Hospital Prague Czechia

Department of Public Health and Primary Care Leuven Biostatistics and Statistical Bioinformatics Center KU Leuven Leuven Belgium

Department of Thoracic Surgery University Hospitals Leuven Leuven Belgium

Laboratory of Respiratory Diseases and Thoracic Surgery Department of Chronic Diseases and Metabolism KU Leuven Leuven Belgium

Prague Lung Transplant Program 3rd Department of Surgery 1st Faculty of Medicine Charles University and Motol University Hospital Prague Czechia

Zobrazit více v PubMed

Burton CM, Iversen M, Carlsen J, Mortensen J, Andersen CB, Steinbruchel D, et al. Acute Cellular Rejection Is a Risk Factor for Bronchiolitis Obliterans Syndrome Independent of Post-Transplant Baseline FEV1. J Heart Lung Transpl (2009) 28(9):888–93. 10.1016/j.healun.2009.04.022 PubMed DOI

Clayton PA, McDonald SP, Russ GR, Chadban SJ. Long-Term Outcomes After Acute Rejection in Kidney Transplant Recipients: An ANZDATA Analysis. J Am Soc Nephrol (2019) 30(9):1697–707. 10.1681/ASN.2018111101 PubMed DOI PMC

Reddy KS, Davies D, Ormond D, Tuteja S, Lucas BA, Johnston TD, et al. Impact of Acute Rejection Episodes on Long-Term Graft Survival Following Simultaneous Kidney-Pancreas Transplantation. Am J Transpl (2003) 3(4):439–44. 10.1034/j.1600-6143.2003.00059.x PubMed DOI

Levy L, Huszti E, Tikkanen J, Ghany R, Klement W, Ahmed M, et al. The Impact of First Untreated Subclinical Minimal Acute Rejection on Risk for Chronic Lung Allograft Dysfunction or Death After Lung Transplantation. Am J Transpl (2020) 20(1):241–9. 10.1111/ajt.15561 PubMed DOI

Wu MY, Yiang GT, Liao WT, Tsai AP, Cheng YL, Cheng PW, et al. Current Mechanistic Concepts in Ischemia and Reperfusion Injury. Cell Physiol Biochem (2018) 46(4):1650–67. 10.1159/000489241 PubMed DOI

Gracia-Sancho J, Villarreal G, Jr., Zhang Y, Yu JX, Liu Y, Tullius SG, et al. Flow Cessation Triggers Endothelial Dysfunction During Organ Cold Storage Conditions: Strategies for Pharmacologic Intervention. Transplantation (2010) 90(2):142–9. 10.1097/TP.0b013e3181e228db PubMed DOI PMC

Chen-Yoshikawa TF. Ischemia-Reperfusion Injury in Lung Transplantation. Cells (2021) 10(6):1333. 10.3390/cells10061333 PubMed DOI PMC

Aiello S, Podesta MA, Rodriguez-Ordonez PY, Pezzuto F, Azzollini N, Solini S, et al. Transplantation-Induced Ischemia-Reperfusion Injury Modulates Antigen Presentation by Donor Renal CD11c(+)F4/80(+) Macrophages Through IL-1R8 Regulation. J Am Soc Nephrol (2020) 31(3):517–31. 10.1681/ASN.2019080778 PubMed DOI PMC

Martinu T, Pavlisko EN, Chen DF, Palmer SM. Acute Allograft Rejection: Cellular and Humoral Processes. Clin Chest Med (2011) 32(2):295–310. 10.1016/j.ccm.2011.02.008 PubMed DOI PMC

Loupy A, Lefaucheur C. Antibody-Mediated Rejection of Solid-Organ Allografts. N Engl J Med (2018) 379(12):1150–60. 10.1056/NEJMra1802677 PubMed DOI

Levine DJ, Hachem RR. Lung Allograft Rejection. Thorac Surg Clin (2022) 32(2):221–9. 10.1016/j.thorsurg.2021.12.003 PubMed DOI

Koutsokera A, Levy L, Pal P, Orchanian-Cheff A, Martinu T. Acute Cellular Rejection: Is It Still Relevant? Semin Respir Crit Care Med (2018) 39(2):181–98. 10.1055/s-0037-1617424 PubMed DOI

Martinu T, Howell DN, Palmer SM. Acute Cellular Rejection and Humoral Sensitization in Lung Transplant Recipients. Semin Respir Crit Care Med (2010) 31(2):179–88. 10.1055/s-0030-1249113 PubMed DOI

Martinu T, Chen DF, Palmer SM. Acute Rejection and Humoral Sensitization in Lung Transplant Recipients. Proc Am Thorac Soc (2009) 6(1):54–65. 10.1513/pats.200808-080GO PubMed DOI PMC

Chambers DC, Yusen RD, Cherikh WS, Goldfarb SB, Kucheryavaya AY, Khusch K, et al. The Registry of the International Society for Heart and Lung Transplantation: Thirty-Fourth Adult Lung and Heart-Lung Transplantation Report-2017; Focus Theme: Allograft Ischemic Time. J Heart Lung Transpl (2017) 36(10):1047–59. 10.1016/j.healun.2017.07.016 PubMed DOI

Verleden SE, Ruttens D, Vandermeulen E, Vaneylen A, Dupont LJ, Van Raemdonck DE, et al. Bronchiolitis Obliterans Syndrome and Restrictive Allograft Syndrome: Do Risk Factors Differ? Transplantation (2013) 95(9):1167–72. 10.1097/TP.0b013e318286e076 PubMed DOI

Glanville AR, Aboyoun CL, Havryk A, Plit M, Rainer S, Malouf MA. Severity of Lymphocytic Bronchiolitis Predicts Long-Term Outcome After Lung Transplantation. Am J Respir Crit Care Med (2008) 177(9):1033–40. 10.1164/rccm.200706-951OC PubMed DOI

Girgis RE, Tu I, Berry GJ, Reichenspurner H, Valentine VG, Conte JV, et al. Risk Factors for the Development of Obliterative Bronchiolitis After Lung Transplantation. J Heart Lung Transpl (1996) 15(12):1200–8. PubMed

He X, Xu C. Immune Checkpoint Signaling and Cancer Immunotherapy. Cell Res (2020) 30(8):660–9. 10.1038/s41422-020-0343-4 PubMed DOI PMC

Riella LV, Paterson AM, Sharpe AH, Chandraker A. Role of the PD-1 Pathway in the Immune Response. Am J Transpl (2012) 12(10):2575–87. 10.1111/j.1600-6143.2012.04224.x PubMed DOI PMC

Han Y, Liu D, Li L. PD-1/PD-L1 Pathway: Current Researches in Cancer. Am J Cancer Res (2020) 10(3):727–42. PubMed PMC

Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and Its Ligands in Tolerance and Immunity. Annu Rev Immunol (2008) 26:677–704. 10.1146/annurev.immunol.26.021607.090331 PubMed DOI PMC

Francisco LM, Sage PT, Sharpe AH. The PD-1 Pathway in Tolerance and Autoimmunity. Immunol Rev (2010) 236:219–42. 10.1111/j.1600-065X.2010.00923.x PubMed DOI PMC

Ozkaynak E, Wang L, Goodearl A, McDonald K, Qin S, O’Keefe T, et al. Programmed Death-1 Targeting Can Promote Allograft Survival. J Immunol (2002) 169(11):6546–53. 10.4049/jimmunol.169.11.6546 PubMed DOI

Morita M, Fujino M, Jiang G, Kitazawa Y, Xie L, Azuma M, et al. PD-1/B7-H1 Interaction Contribute to the Spontaneous Acceptance of Mouse Liver Allograft. Am J Transpl (2010) 10(1):40–6. 10.1111/j.1600-6143.2009.02859.x PubMed DOI PMC

Riella LV, Watanabe T, Sage PT, Yang J, Yeung M, Azzi J, et al. Essential Role of PDL1 Expression on Nonhematopoietic Donor Cells in Acquired Tolerance to Vascularized Cardiac Allografts. Am J Transpl (2011) 11(4):832–40. 10.1111/j.1600-6143.2011.03451.x PubMed DOI

Strizova Z, Bartunkova J, Smrz D. The Challenges of Adoptive Cell Transfer in the Treatment of Human Renal Cell Carcinoma. Cancer Immunol Immunother (2019) 68(11):1831–8. 10.1007/s00262-019-02359-z PubMed DOI PMC

Park J, Byun SW, Yu E, Park SK, Han DJ, Cho YM. Immunohistochemical Profile of Acute Cellular Rejection in Renal Allograft. J Pathol Translational Med (2007) 41(1):15–20.

Coons AH, Creech HJ, Jones RN. Immunological Properties of an Antibody Containing a Fluorescent Group. Exp Biol Med (1941) 47(2):200–2. 10.3181/00379727-47-13084p DOI

Kohler G, Milstein C. Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity. Nature (1975) 256(5517):495–7. 10.1038/256495a0 PubMed DOI

Cheng L, Guo H, Qiao X, Liu Q, Nie J, Li J, et al. T Cell Immunohistochemistry Refines Lung Transplant Acute Rejection Diagnosis and Grading. Diagn Pathol (2013) 8:168. 10.1186/1746-1596-8-168 PubMed DOI PMC

Stewart S, Fishbein MC, Snell GI, Berry GJ, Boehler A, Burke MM, et al. Revision of the 1996 Working Formulation for the Standardization of Nomenclature in the Diagnosis of Lung Rejection. J Heart Lung Transpl (2007) 26(12):1229–42. 10.1016/j.healun.2007.10.017 PubMed DOI

Zhang P, Wang Y, Miao Q, Chen Y. The Therapeutic Potential of PD-1/pd-L1 Pathway on Immune-Related Diseases: Based on the Innate and Adaptive Immune Components. Biomed Pharmacother (2023) 167:115569. 10.1016/j.biopha.2023.115569 PubMed DOI

Wang C, Feng Y, Patel D, Xie H, Lv Y, Zhao H. The Role of CD47 in Non-Neoplastic Diseases. Heliyon (2023) 9(12):e22905. 10.1016/j.heliyon.2023.e22905 PubMed DOI PMC

Muller WA, Weigl SA, Deng X, Phillips DM. PECAM-1 Is Required for Transendothelial Migration of Leukocytes. J Exp Med (1993) 178(2):449–60. 10.1084/jem.178.2.449 PubMed DOI PMC

Vos R, Vanaudenaerde BM, De Vleeschauwer SI, Van Raemdonck DE, Dupont LJ, Verleden GM. Plasma C-Reactive Protein Levels Correlate With Markers of Airway Inflammation After Lung Transplantation: A Role for Systemic Inflammation in Bronchiolitis Obliterans Syndrome? Transpl Proc (2009) 41(2):595–8. 10.1016/j.transproceed.2008.12.024 PubMed DOI

Vos R, Vanaudenaerde BM, De Vleeschauwer SI, Willems-Widyastuti A, Scheers H, Van Raemdonck DE, et al. Circulating and Intrapulmonary C-Reactive Protein: A Predictor of Bronchiolitis Obliterans Syndrome and Pulmonary Allograft Outcome. J Heart Lung Transpl (2009) 28(8):799–807. 10.1016/j.healun.2009.05.011 PubMed DOI

van Gelder T, Balk AH, Zondervan PE, Maat AW, Mochtar B, van der Meer P, et al. C-Reactive Protein in the Monitoring of Acute Rejection After Heart Transplantation. Transpl Int (1998) 11(5):361–4. 10.1007/s001470050158 PubMed DOI

Gouin JP, Glaser R, Malarkey WB, Beversdorf D, Kiecolt-Glaser J. Chronic Stress, Daily Stressors, and Circulating Inflammatory Markers. Health Psychol (2012) 31(2):264–8. 10.1037/a0025536 PubMed DOI PMC

Landry A, Docherty P, Ouellette S, Cartier LJ. Causes and Outcomes of Markedly Elevated C-Reactive Protein Levels. Can Fam Physician (2017) 63(6):e316–23. PubMed PMC

Wang L, Han R, Hancock WW. Programmed Cell Death 1 (PD-1) and Its Ligand PD-L1 Are Required for Allograft Tolerance. Eur J Immunol (2007) 37(10):2983–90. 10.1002/eji.200737583 PubMed DOI

Tanaka K, Albin MJ, Yuan X, Yamaura K, Habicht A, Murayama T, et al. PDL1 Is Required for Peripheral Transplantation Tolerance and Protection From Chronic Allograft Rejection. J Immunol (2007) 179(8):5204–10. 10.4049/jimmunol.179.8.5204 PubMed DOI PMC

Choudhary A, Brinkley DM, Besharati S, Meijers WC, Atkinson JB, Amancherla K, et al. PD-L1 (Programmed Death Ligand 1) as a Marker of Acute Cellular Rejection After Heart Transplantation. Circ Heart Fail (2021) 14(10):e008563. 10.1161/CIRCHEARTFAILURE.121.008563 PubMed DOI PMC

Righi I, Vaira V, Morlacchi LC, Croci GA, Rossetti V, Blasi F, et al. PD-1 Expression in Transbronchial Biopsies of Lung Transplant Recipients Is a Possible Early Predictor of Rejection. Front Immunol (2022) 13:1024021. 10.3389/fimmu.2022.1024021 PubMed DOI PMC

Kaiho T, Suzuki H, Hata A, Matsumoto H, Tanaka K, Sakairi Y, et al. Targeting PD-1/PD-L1 Inhibits Rejection in a Heterotopic Tracheal Allograft Model of Lung Transplantation. Front Pharmacol (2023) 14:1298085. 10.3389/fphar.2023.1298085 PubMed DOI PMC

Hudson K, Cross N, Jordan-Mahy N, Leyland R. The Extrinsic and Intrinsic Roles of PD-L1 and Its Receptor PD-1: Implications for Immunotherapy Treatment. Front Immunol (2020) 11:568931. 10.3389/fimmu.2020.568931 PubMed DOI PMC

Xiao Y, Li ZZ, Zhong NN, Cao LM, Liu B, Bu LL. Charting New Frontiers: Co-Inhibitory Immune Checkpoint Proteins in Therapeutics, Biomarkers, and Drug Delivery Systems in Cancer Care. Transl Oncol (2023) 38:101794. 10.1016/j.tranon.2023.101794 PubMed DOI PMC

Khan MA, Shamma T, Altuhami A, Ahmed HA, Assiri AM, Broering DC. CTLA4-Ig Mediated Immunosuppression Favors Immunotolerance and Restores Graft in Mouse Airway Transplants. Pharmacol Res (2022) 178:106147. 10.1016/j.phrs.2022.106147 PubMed DOI

Yeung MY, McGrath M, Najafian N. The Emerging Role of the TIM Molecules in Transplantation. Am J Transpl (2011) 11(10):2012–9. 10.1111/j.1600-6143.2011.03727.x PubMed DOI PMC

He W, Fang Z, Wang F, Wu K, Xu Y, Zhou H, et al. Galectin-9 Significantly Prolongs the Survival of Fully Mismatched Cardiac Allografts in Mice. Transplantation (2009) 88(6):782–90. 10.1097/TP.0b013e3181b47f25 PubMed DOI

Wang F, He W, Yuan J, Wu K, Zhou H, Zhang W, et al. Activation of Tim-3-Galectin-9 Pathway Improves Survival of Fully Allogeneic Skin Grafts. Transpl Immunol (2008) 19(1):12–9. 10.1016/j.trim.2008.01.008 PubMed DOI

Seki M, Oomizu S, Sakata KM, Sakata A, Arikawa T, Watanabe K, et al. Galectin-9 Suppresses the Generation of Th17, Promotes the Induction of Regulatory T Cells, and Regulates Experimental Autoimmune Arthritis. Clin Immunol (2008) 127(1):78–88. 10.1016/j.clim.2008.01.006 PubMed DOI

Cui X, Yan C, Xu Y, Li D, Guo M, Sun L, et al. Allograft Rejection Following Immune Checkpoint Inhibitors in Solid Organ Transplant Recipients: A Safety Analysis From a Literature Review and a Pharmacovigilance System. Cancer Med (2023) 12(5):5181–94. 10.1002/cam4.5394 PubMed DOI PMC

Berman ME, Xie Y, Muller WA. Roles of Platelet/endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) in Natural Killer Cell Transendothelial Migration and Beta 2 Integrin Activation. J Immunol (1996) 156(4):1515–24. 10.4049/jimmunol.156.4.1515 PubMed DOI

Tran-Dinh A, Laurent Q, Even G, Tanaka S, Lortat-Jacob B, Castier Y, et al. Personalized Risk Predictor for Acute Cellular Rejection in Lung Transplant Using Soluble CD31. Sci Rep (2022) 12(1):17628. 10.1038/s41598-022-21070-1 PubMed DOI PMC

Fang J, Lu Y, Zheng J, Jiang X, Shen H, Shang X, et al. Exploring the Crosstalk Between Endothelial Cells, Immune Cells, and Immune Checkpoints in the Tumor Microenvironment: New Insights and Therapeutic Implications. Cell Death Dis (2023) 14(9):586. 10.1038/s41419-023-06119-x PubMed DOI PMC

Woodfin A, Voisin MB, Nourshargh S. PECAM-1: A Multi-Functional Molecule in Inflammation and Vascular Biology. Arterioscler Thromb Vasc Biol (2007) 27(12):2514–23. 10.1161/ATVBAHA.107.151456 PubMed DOI

Yan HC, Baldwin HS, Sun J, Buck CA, Albelda SM, DeLisser HM. Alternative Splicing of a Specific Cytoplasmic Exon Alters the Binding Characteristics of Murine Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1). J Biol Chem (1995) 270(40):23672–80. 10.1074/jbc.270.40.23672 PubMed DOI

Baldwin HS, Shen HM, Yan HC, DeLisser HM, Chung A, Mickanin C, et al. Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31): Alternatively Spliced, Functionally Distinct Isoforms Expressed During Mammalian Cardiovascular Development. Development (1994) 120(9):2539–53. 10.1242/dev.120.9.2539 PubMed DOI

Privratsky JR, Newman DK, Newman PJ. PECAM-1: Conflicts of Interest in Inflammation. Life Sci (2010) 87(3-4):69–82. 10.1016/j.lfs.2010.06.001 PubMed DOI PMC

Strizova Z, Benesova I, Bartolini R, Novysedlak R, Cecrdlova E, Foley LK, et al. M1/M2 Macrophages and Their Overlaps - Myth or Reality? Clin Sci (Lond) (2023) 137(15):1067–93. 10.1042/CS20220531 PubMed DOI PMC

Arcasoy SM, Berry G, Marboe CC, Tazelaar HD, Zamora MR, Wolters HJ, et al. Pathologic Interpretation of Transbronchial Biopsy for Acute Rejection of Lung Allograft Is Highly Variable. Am J Transpl (2011) 11(2):320–8. 10.1111/j.1600-6143.2010.03382.x PubMed DOI

Yamada Y, Nguyen TT, Impellizzieri D, Mineura K, Shibuya R, Gomariz A, et al. Biased IL-2 Signals Induce Foxp3-Rich Pulmonary Lymphoid Structures and Facilitate Long-Term Lung Allograft Acceptance in Mice. Nat Commun (2023) 14(1):1383. 10.1038/s41467-023-36924-z PubMed DOI PMC

Yamada Y, Impellizzieri D, Jang J, Nguyen T, Karakus U, Inci I, et al. Regulatory T Cells Induce Persistent Acceptance by IL-2 Complexes After Mouse Lung Transplantation. J Heart Lung Transplant (2018) 37(4):S124–5. 10.1016/j.healun.2018.01.297 DOI

Sweet SC, De La Morena MT, Shapiro SD, Mendeloff EN, Huddleston CB. Interleukin-2-Receptor Blockade With Daclizumab Decreases the Incidence of Acute Rejection in Pediatric Lung Transplant Recipients. J Heart Lung Transplant (2001) 20(2):221–2. 10.1016/s1053-2498(00)00486-1 PubMed DOI

Jordan SC, Marchevski A, Ross D, Toyoda M, Waters PF. Serum Interleukin-2 Levels in Lung Transplant Recipients: Correlation With Findings on Transbronchial Biopsy. J Heart Lung Transpl (1992) 11(5):1001–4. PubMed

Steinack C, Gaspert A, Gautschi F, Hage R, Vrugt B, Soltermann A, et al. Transbronchial Cryobiopsy Compared to Forceps Biopsy for Diagnosis of Acute Cellular Rejection in Lung Transplants: Analysis of 63 Consecutive Procedures. Life (Basel) (2022) 12(6):898. 10.3390/life12060898 PubMed DOI PMC