Table_1_Adaptation of Imaging Mass Cytometry to Explore the Single Cell Alloimmune Landscape of Liver Transplant Rejection.docx

Rejection continues to be an important cause of graft loss in solid organ transplantation, but deep exploration of intragraft alloimmunity has been limited by the scarcity of clinical biopsy specimens. Emerging single cell immunoprofiling technologies have shown promise in discerning mechanisms of autoimmunity and cancer immunobiology. Within these applications, Imaging Mass Cytometry (IMC) has been shown to enable highly multiplexed, single cell analysis of immune phenotypes within fixed tissue specimens. In this study, an IMC panel of 10 validated markers was developed to explore the feasibility of IMC in characterizing the immune landscape of chronic rejection (CR) in clinical tissue samples obtained from liver transplant recipients. IMC staining was highly specific and comparable to traditional immunohistochemistry. A single cell segmentation analysis pipeline was developed that enabled detailed visualization and quantification of 109,245 discrete cells, including 30,646 immune cells. Dimensionality reduction identified 11 unique immune subpopulations in CR specimens. Most immune subpopulations were increased and spatially related in CR, including two populations of CD45+/CD3+/CD8+ cytotoxic T-cells and a discrete CD68+ macrophage population, which were not observed in liver with no rejection (NR). Modeling via principal component analysis and logistic regression revealed that single cell data can be utilized to construct statistical models with high consistency (Wilcoxon Rank Sum test, p=0.000036). This study highlights the power of IMC to investigate the alloimmune microenvironment at a single cell resolution during clinical rejection episodes. Further validation of IMC has the potential to detect new biomarkers, identify therapeutic targets, and generate patient-specific predictive models of clinical outcomes in solid organ transplantation.

移植物排斥持续是实体器官移植中移植物丢失的重要病因，然而，由于临床活检标本的稀缺，对移植物内同种免疫的深入探索受到了限制。新兴的单细胞免疫分析技术显示出在揭示自身免疫和癌症免疫生物学机制方面的潜力。在这些应用中，成像流式细胞术（IMC）已被证明能够实现对固定组织样本中免疫表型的多参数、单细胞分析。在本研究中，开发了一个包含10个验证标记物的IMC面板，以探索IMC在表征肝移植受者临床组织样本中慢性排斥（CR）免疫景观的可行性。IMC染色具有高度特异性，与传统免疫组化相当。开发了一套单细胞分割分析流程，该流程能够详细可视化并量化109,245个独立细胞，包括30,646个免疫细胞。降维分析在CR样本中识别出11个独特的免疫亚群。大多数免疫亚群在CR中均增加且空间相关，包括两个CD45+/CD3+/CD8+细胞毒性T细胞群体和一个离散的CD68+巨噬细胞群体，这些群体在无排斥（NR）的肝脏中并未观察到。通过主成分分析和逻辑回归建模，揭示了单细胞数据可用于构建高一致性统计模型（威尔科克斯秩和检验，p=0.000036）。本研究突出了IMC在临床排斥期间以单细胞分辨率研究同种免疫微环境的能力。进一步验证IMC具有检测新生物标志物、识别治疗靶点以及生成针对实体器官移植临床结果的患者特异性预测模型的潜力。