SIÇANLARDA EKTOPİK TİMOGENEZ İLE İMMÜNTOLERANSIN İNDÜKLENMESİ
URALOĞLU M. (Yürütücü), ŞAHİN H., SÖNMEZ M., ERSÖZ Ş., LİVAOĞLU M.
Yükseköğretim Kurumları Destekli Proje, BAP Doktora, 2024 - Devam Ediyor
- Proje Türü: Yükseköğretim Kurumları Destekli Proje
- Destek Programı: BAP Doktora
- Başlama Tarihi: Eylül 2024
- Bitiş Tarihi: Devam ediyor
Proje Özeti
ABSTRACT
Objective: Although vascularized composite tissue allotransplantation (VCA) holds a significant place in the treatment of severe tissue losses, the requirement for lifelong immunosuppression, along with its side effects and costs, remains a major limitation. The aim of this study is to create an ectopic thymus by transplanting thymus tissue into a lymph node in an allogeneic rat model and to investigate its immune tolerance potential through structural and functional characterization.
Materials and Methods: Neonatal Wistar albino rats were used as donors and adult Sprague-Dawley rats as recipients. The subjects were divided into three groups: Sham (Group 1), Inguinal Lymph Node Graft (Group 2), and Mesenteric Lymph Node Graft (Group 3). Donor thymus fragments (1 mm³) were implanted into the lymph nodes of the recipients using microsurgical techniques. All groups were administered Tacrolimus at a dose of 1 mg/kg for 20 days. During the 60-day follow-up period, CD4 and CD8 T cell subsets in the peripheral blood were analyzed by flow cytometry every 10 days. At the end of the study, the thymus-engrafted lymph nodes were excised and evaluated using flow cytometry and immunohistochemical staining [Hematoxylin-Eosin (H&E) and Cytokeratin 5 (CK5) / Cytokeratin 8 (CK8)].
Results: During the 60-day post-operative follow-up period, no signs of graft rejection, systemic toxicity, or Graft-Versus-Host Disease (GVHD) were observed in any of the subjects. Immunohistochemical analyses demonstrated that the minced allogeneic neonatal thymus tissue transplanted into the lymph node parenchyma maintained its viability without undergoing necrosis. On post-operative days 20 and 60, the organization of CK8-positive cortical areas and CK5-positive medullary areas was observed in the graft sites; proving that the cortico-medullary architecture, which is essential for thymocyte selection, was successfully formed within the lymph node. According to flow cytometry findings, the cellular suppression observed in peripheral blood CD4+ and CD8+ T cell levels during the early and mid-terms was replaced by a recovery trend towards day 60. This dynamic response may indicate that the graft releases Recent Thymic Emigrants (RTE) into the peripheral circulation and that active ectopic thymopoiesis occurs. Exploratory lymph node analyses revealed a statistically significant increase in the local CD8+ cell population within the grafted lymph nodes. There was no significant difference between the inguinal and mesenteric lymph node applications in terms of surgical feasibility and graft success.
Conclusion: In an immunocompetent rat model with a major histocompatibility mismatch, intra-lymph node ectopic thymus transplantation was successfully achieved in terms of both structural engraftment and functional thymopoiesis. This study proves that lymph nodes can be utilized as ectopic centers for central tolerance induction, with the ultimate goal of eliminating the dependence on lifelong immunosuppression in vascularized composite tissue allotransplantation recipients. Due to the ease of surgical access and minimal invasiveness, inguinal lymph nodes, in particular, hold strong potential for clinical translation. For the definitive proof of induced tolerance, future studies are recommended to test ectopic thymopoiesis in vivo with simultaneous allografts, confirm AIRE expression in medullary epithelial cells, and map the microenvironment using spatial transcriptomic methods.