Data Sheet 1_Evaluating the immunogenicity of a mouse partial hindlimb for composite allotransplantation.docx

BackgroundVascularized 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.
Materials and methodsSyngeneic 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.
ResultsBoth 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.
DiscussionThis 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.

背景：血管化复合组织异体移植（vascularized composite allotransplantation, VCA）为重症组织创伤患者的功能重建提供了颇具前景的解决方案，但急性排斥反应仍是其主要临床障碍。急性排斥反应由针对移植组织的免疫应答所介导，需终身免疫抑制治疗，进而增加感染、恶性肿瘤及器官毒性的发生风险。本研究旨在构建小鼠后肢移植模型，以探究移植组织中的免疫应答及浸润细胞的表型特征。
材料与方法：构建同基因与异基因小鼠部分后肢移植模型，以评估移植皮肤与肌肉的免疫原性。以雄性BALB/c及C57BL/6小鼠作为供体，C57BL/6.SJL小鼠作为受体。获取包含股骨、肌肉及皮肤的后肢组织，将其异位移植至受体小鼠体内。分别于术后第7天（post-operative day 7, POD7）及第14天（post-operative day 14, POD14）采集样本，行组织学分析（苏木精-伊红（hematoxylin-eosin, H&E）染色、CD8免疫组织化学染色、半胱天冬酶-3（caspase-3）免疫组织化学染色、CD31免疫组织化学染色及TUNEL染色），以评估细胞浸润、炎性T细胞浸润、细胞凋亡及血管生成情况。通过流式细胞术（flow cytometry）对免疫细胞群进行表型鉴定。
结果：同基因与异基因移植的皮肤及肌肉在两个时间点均可见细胞含量升高。异基因移植皮肤在POD14时细胞含量更高，且出现亚临床排斥反应；流式细胞术结果显示供体来源及受体来源的T细胞数量增多，尤以CD4+及CD8+ T细胞最为显著。此外，1型调节性T细胞（Type 1 regulatory T cell, Tr1）的丰度更高，提示其在免疫应答中发挥调控作用。术后POD14时，两组移植组织的凋亡标志物水平均升高；且POD7时异基因移植组织中的TUNEL阳性细胞数量更多。通过CD31表达评估的血管重建情况显示，术后POD14时同基因与异基因移植的肌肉组织均出现明显的血管重建。
讨论：本研究通过构建新型小鼠后肢移植模型，为血管化复合组织异体移植（VCA）中的急性排斥反应研究提供了重要见解。研究结果揭示了免疫应答的复杂性：异基因移植皮肤中CD8+ T细胞及1型调节性T细胞数量增多，而非血管化移植组织中出现了意料之外的血管重建。1型调节性T细胞的增多提示了一种潜在的免疫调控机制，为免疫耐受诱导策略提供了新的思路。本研究结果凸显了组织特异性免疫抑制策略的必要性：靶向1型调节性T细胞可减少患者对广谱免疫抑制治疗的依赖，进而改善移植组织的长期存活及患者预后。本研究为优化血管化复合组织异体移植治疗方案奠定了基础，有望推动更具个性化、更低毒性的免疫抑制策略的发展。