Sun Jia Hui Angela, Ligi Samantha, Biniazan Felor, Haykal Siba
Latner Thoracic Surgery Laboratories, University Health Network, Toronto General Hospital, Toronto, ON, Canada.
Institute of Medical Science, Temerty Faculty of Medicine, University of Toronto, ON, Canada.
Front Immunol. 2025 Jun 18;16:1595319. doi: 10.3389/fimmu.2025.1595319. eCollection 2025.
Vascularized composite allotransplantation (VCA) offers a promising solution for restoring function in patients with severe tissue trauma, yet acute rejection remains a major hurdle. Acute rejection is driven by an immune response against transplanted tissues, requiring lifelong immunosuppression, increasing risks of infections, malignancies, and organ toxicity. This study aims to develop a mouse hindlimb transplant model to investigate immune responses and the phenotype of infiltrating cells in these grafts.
Syngeneic and allogeneic mouse partial hindlimb models were established to evaluate grafted skin and muscle immunogenicity. Male BABL/c and C57BL/6 mice served as donors, C57BL6.sjl mice were recipients. Hindlimbs were procured, including femur, muscle, and skin, and were heterotopically transplanted into recipient mice. Samples were collected at post-operative days 7 (POD7) and 14 (POD14) for histological analysis (H&E staining, immunohistochemistry for CD8, caspase-3, CD31, and TUNEL) to assess cell infiltration, inflammatory T cell presence, apoptosis, and vascularization. Immune cell populations were characterized through flow cytometry.
Both syngeneic and allogeneic skin and muscle showed increased cellular content at both time points. Allogeneic skin at POD14 exhibited higher cellular content and subclinical rejection, while flow cytometry revealed increased donor-derived and recipient-derived T cells, particularly CD4+ and CD8+ T cells. Additionally, Tr1 cells were more abundant, suggesting a regulatory role in the immune response. Apoptotic markers increased in both grafts at POD14, with more TUNEL-positive cells in allogeneic grafts at POD7. Revascularization, assessed by CD31 expression, was notably present in both syngeneic and allogeneic muscle at POD14.
This study provides valuable insights into acute rejection in VCA using a novel mouse hindlimb transplant model. Findings reveal immune response complexity, with increased CD8+ T cells and Tr1 cells in allogeneic skin, and unexpected vascularization in non-vascularized grafts. The rise in Tr1 cells suggests a potential mechanism for immune regulation, offering potential for tolerance induction strategies. These results emphasize the need for tissue-specific immunosuppressive approaches, where targeting Tr1 cells could reduce dependence on broad immunosuppression, improving long-term graft survival and patient outcomes. This work lays the foundation for refining VCA therapies, with more personalized, less toxic immunosuppressive strategies.
血管化复合组织异体移植（VCA）为严重组织创伤患者恢复功能提供了一种有前景的解决方案，但急性排斥反应仍然是一个主要障碍。急性排斥反应由针对移植组织的免疫反应驱动，需要终身免疫抑制，这增加了感染、恶性肿瘤和器官毒性的风险。本研究旨在建立一种小鼠后肢移植模型，以研究这些移植物中的免疫反应和浸润细胞的表型。
建立同基因和异基因小鼠部分后肢模型，以评估移植皮肤和肌肉的免疫原性。雄性BABL/c和C57BL/6小鼠作为供体，C57BL6.sjl小鼠作为受体。获取后肢，包括股骨、肌肉和皮肤，并将其异位移植到受体小鼠体内。在术后第7天（POD7）和第14天（POD14）收集样本进行组织学分析（苏木精-伊红染色、CD8、半胱天冬酶-3、CD31免疫组化和TUNEL），以评估细胞浸润、炎性T细胞存在、细胞凋亡和血管生成。通过流式细胞术对免疫细胞群体进行表征。
同基因和异基因皮肤及肌肉在两个时间点的细胞含量均增加。POD14时异基因皮肤表现出更高的细胞含量和亚临床排斥反应，而流式细胞术显示供体来源和受体来源的T细胞增加，尤其是CD4+和CD8+T细胞。此外，Tr1细胞更为丰富，表明其在免疫反应中起调节作用。POD14时两个移植物中的凋亡标志物均增加，POD7时异基因移植物中的TUNEL阳性细胞更多。通过CD31表达评估的血管再生在POD14时在同基因和异基因肌肉中均明显存在。
本研究使用一种新型小鼠后肢移植模型，为VCA中的急性排斥反应提供了有价值的见解。研究结果揭示了免疫反应的复杂性，异基因皮肤中CD8+T细胞和Tr1细胞增加，以及非血管化移植物中意外的血管生成。Tr1细胞的增加提示了一种免疫调节的潜在机制，为诱导耐受策略提供了潜力。这些结果强调了组织特异性免疫抑制方法的必要性，靶向Tr1细胞可以减少对广泛免疫抑制的依赖，提高长期移植物存活率和患者预后。这项工作为完善VCA疗法奠定了基础，采用更个性化、毒性更小的免疫抑制策略。
