T Cells Enhance Tissue Complexity and Function to Study Fibrosis in 3D Skin-Like Tissue Models

Abstract

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 in vivo 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.

Impact Statement

The impact of our research results from incorporating human T cells to enhance the cellular complexity of 3D tissue models to study fibrosis in ways that more closely simulate the fibrotic tissue microenvironment. To our knowledge, this is the first time single-cell analyses have been used to demonstrate the diverse T cell subpopulations sustained in 3D skin-like tissues containing T cells, fibroblasts, and macrophages. The impact of this tissue complexity and cellular heterogeneity supports the broad use of 3D tissues to discover mechanisms of fibrosis and develop new treatments to reverse fibrosis and restore normal tissue and organ function.