Gonzalez Badillo Freddy, Zisi Tegou Flavia, Masina Riccardo, Wright Shane, Scully Mackenzie, Harwell Laura, Lupp Michael, Postigo-Fernandez Jorge, Creusot Remi J, Tomei Alice A
Department of Biomedical Engineering, University of Miami, 1251 Memorial Dr, Coral Gables, FL 33146 USA.
Diabetes Research Institute, University of Miami Miller School of Medicine, 1450 NW 10th Ave, Miami, FL 33136 USA.
Cell Mol Bioeng. 2020 Jun 26;13(5):419-434. doi: 10.1007/s12195-020-00627-y. eCollection 2020 Oct.
Fibroblastic reticular cells (FRCs) support and remodel the lymph node (LN), express and present self-antigens to T cells to promote tolerance. In Type 1 diabetes (T1D), decrease in FRC frequency and in their expression of T1D-related self-antigens may hinder tolerogenic engagement of autoreactive T cells. FRC reticular organization in LNs is critical for adaptive immunity. Thus, we engineered LN-like FRC reticula to determine if FRC reticular properties were altered in T1D and to study engagement of autoreactive T cells .
We characterized FRC networks in pancreatic and skin-draining LNs of 4- and 12-week old non-obese diabetic (NOD) and diabetes resistant NOR mice by immunofluorescence. Murine FRCs isolated from NOR, NOD or human pancreatic LNs were cultured in collagen sponges for up to 21 days before immunofluorescence and flow cytometry analysis. NOD FRCs expressing T1D antigens were co-cultured with CellTrace-labeled specific T cells in 2D or in scaffolds. T cell engagement was quantified by CD25 upregulation, CellTrace dilution and by T cell tracking.
FRC networks in both 4- and 12-week old NOD LNs displayed larger reticular pores than NOR controls. NOD FRCs had delayed scaffold remodeling compared to NOR FRCs. Expression of the gp38 FRC marker in NOD FRCs was lower than in NOR but improved in 3D. FRC reticula expressing T1D antigens promoted higher engagement of specific T cells than 2D.
We engineered LN-like FRC reticula that recapitulate FRC organization and phenotype of T1D LNs for studying tolerogenic autoreactive T cell engagement in T1D.
成纤维网状细胞（FRCs）支持并重塑淋巴结（LN），表达自身抗原并将其呈递给T细胞以促进免疫耐受。在1型糖尿病（T1D）中，FRC频率及其与T1D相关自身抗原的表达降低可能会阻碍自身反应性T细胞的耐受性参与。LN中的FRC网状组织对适应性免疫至关重要。因此，我们构建了类似LN的FRC网状结构，以确定T1D中FRC网状特性是否改变，并研究自身反应性T细胞的参与情况。
我们通过免疫荧光对4周龄和12周龄非肥胖糖尿病（NOD）小鼠及糖尿病抗性NOR小鼠的胰腺引流淋巴结和皮肤引流淋巴结中的FRC网络进行了表征。从NOR、NOD或人胰腺淋巴结中分离出的小鼠FRC在胶原海绵中培养长达21天，然后进行免疫荧光和流式细胞术分析。将表达T1D抗原的NOD FRC与CellTrace标记的特异性T细胞在二维或支架中共同培养。通过CD25上调、CellTrace稀释和T细胞追踪对T细胞参与情况进行定量。
4周龄和12周龄NOD淋巴结中的FRC网络显示出比NOR对照更大的网状孔隙。与NOR FRC相比，NOD FRC的支架重塑延迟。NOD FRC中gp38 FRC标志物的表达低于NOR，但在三维环境中有所改善。表达T1D抗原的FRC网状结构比二维环境更能促进特异性T细胞的参与。
我们构建了类似LN的FRC网状结构，其概括了T1D淋巴结的FRC组织和表型，用于研究T1D中耐受性自身反应性T细胞的参与情况。
