Singh Isha, Morrisson Madeline, Shenk Sasha, Jarnagin Helen, Hauer Johanna, Lobo Ayesha, Brown Lev, Houck Tamara, Altman-Sagan Liora, Pioli Patricia A, Whitfield Michael L, Garlick Jonathan A
Basic & Clinical Translational Sciences, Tufts University School of Dental Medicine, Boston, Massachusetts, USA.
Department of Biomedical Data Science, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
Tissue Eng Part C Methods. 2025 Jul;31(7):248-260. doi: 10.1177/19373341251360742.
Fibrosis causes altered tissue structure and function in multiple organs due to a complex interplay between inflammatory cells, myofibroblasts, and extracellular matrix (ECM) components. While it is known that T cells play a role in tissue fibrosis, it remains unclear how they modulate cellular interactions to activate fibrogenesis. Since conventional monolayer cell cultures do not mimic the tissue complexity and cellular heterogeneity in the fibrotic tissue environment, there is a need to bridge the gap between monolayer cultures and animal studies of fibrosis by providing a more predictive 3D model for preclinical drug screening and mechanistic studies of fibrotic diseases. We have developed 3D skin-like tissues harboring blood-derived human T cells that offer a model to better understand the role these cells play in the pathogenesis of tissue fibrosis. In the current study, we constructed skin-like tissues harboring T cells, fibroblasts, macrophages, and keratinocytes and analyzed them using tissue analysis and single-cell RNA sequencing (scRNA-seq). Skin-like tissues constructed with fully autologous cells (donor-matched fibroblasts and T cells) or nonautologous cells (mismatched fibroblasts and T cells) derived from patients with scleroderma (SSc) demonstrated normal distribution of tissue markers of epithelial differentiation and proliferation. T cells in these tissues were viable and functional as seen by elevated IL-6 production by enzyme-linked immunosorbent assay, expression of alpha smooth muscle actin in fibroblasts, and scRNA-seq. We used scRNA-seq to identify five distinct T cell subpopulations: CD8 T cells (identified by KLRK1 and CD8A), proliferating CD4 T cells (identified by PCNA, MKI67, and CD4), activated CD4 T cells (identified by IL2RA, RORA, and CD4), naïve CD4 T cells (identified by CCR7 and CD4), and Th17 CD4 T cells (identified by KLRB1, RORA, IL2RA, and CD4). Fabrication of complex 3D tissues are an important step toward establishing tissue engineering approaches to study fibrosis in multiple diseases, including SSc, idiopathic pulmonary fibrosis, as well as liver and kidney fibrosis. Understanding the roles of T cells in the ECM environment and their interactions with fibroblasts will support the development of novel treatments to reverse fibrosis and restore normal tissue and organ function.
纤维化由于炎症细胞、肌成纤维细胞和细胞外基质（ECM）成分之间复杂的相互作用，导致多个器官的组织结构和功能发生改变。虽然已知T细胞在组织纤维化中起作用，但它们如何调节细胞间相互作用以激活纤维化形成仍不清楚。由于传统的单层细胞培养无法模拟纤维化组织环境中的组织复杂性和细胞异质性，因此需要通过提供一个更具预测性的三维模型来弥合单层培养与纤维化动物研究之间的差距，用于纤维化疾病的临床前药物筛选和机制研究。我们开发了含有血液来源的人T细胞的三维皮肤样组织，为更好地理解这些细胞在组织纤维化发病机制中所起的作用提供了一个模型。在本研究中，我们构建了含有T细胞、成纤维细胞、巨噬细胞和角质形成细胞的皮肤样组织，并使用组织分析和单细胞RNA测序（scRNA-seq）对其进行分析。用硬皮病（SSc）患者来源的完全自体细胞（供体匹配的成纤维细胞和T细胞）或非自体细胞（不匹配的成纤维细胞和T细胞）构建的皮肤样组织显示上皮分化和增殖的组织标志物分布正常。通过酶联免疫吸附测定法检测到的IL-6产生增加、成纤维细胞中α平滑肌肌动蛋白的表达以及scRNA-seq，可见这些组织中的T细胞是有活力且有功能的。我们使用scRNA-seq鉴定出五个不同的T细胞亚群：CD8 T细胞（由KLRK1和CD8A鉴定）、增殖性CD4 T细胞（由PCNA、MKI67和CD4鉴定）、活化的CD4 T细胞（由IL2RA、RORA和CD4鉴定）、幼稚CD4 T细胞（由CCR7和CD4鉴定）以及Th17 CD4 T细胞（由KLRB1、RORA、IL2RA和CD4鉴定）。构建复杂的三维组织是朝着建立组织工程方法来研究多种疾病（包括SSc、特发性肺纤维化以及肝和肾纤维化）中的纤维化迈出的重要一步。了解T细胞在ECM环境中的作用及其与成纤维细胞的相互作用将有助于开发新的治疗方法来逆转纤维化并恢复正常组织和器官功能。
