Abstract
Type 1 Diabetes (T1D) is an autoimmune disease in which autoreactive T cells (aTcells) escape the immune system and destroy pancreatic beta cells. aTcell escape is due to a failure in T cell tolerance. A subset of lymph node (LN) stromal cells - Fibroblastic Reticular Cells (FRCs) - which form a network in the LN, has been shown to interact with T cells traveling through the LNs. It has been shown that LN FRCs express peripheral tissue antigens (PTAs), including T1D-relevant antigens, and present them to specific T cells, leading to anergy and/or deletion. Loss of FRCs and/or FRC capability to present and express PTAs to aTcells may contribute to loss of peripheral tolerance. Tumors promote tolerance to the antigens they express and escape immune destruction. Expression of CCL21 in tumors was necessary and sufficient to promote tolerance to tumor antigens and this was mediated by the formation of tertiary lymphoid organs (TLOs), mainly composed of LN FRC-like cells.
The goal of this dissertation is to develop platforms to study the role of FRCs and FRC-containing TLOs in the context of peripheral tolerance in T1D. First, by using the non-obese diabetic (NOD) mouse model of T1D with transgenic expression of CCL21 in the pancreatic beta cells, I showed that CCL21 secretion was sufficient to promote TLO formation in pancreatic islets of NOD mice and in islet grafts within the kidney capsule of recipient mice. CCL21-mediated TLO formation protected the mice from T1D development.
These findings confirmed the importance of FRCs in peripheral tolerance and motivated further characterization of FRC reticula in pancreatic LNs of NOD and diabetes-resistant NOR mice, which served as design criteria to develop a tissue-engineered reticula. The engineered reticula mimicked the delayed reticular remodeling observed between the groups studied and allows tracking of antigen-specific T cells in reticula formed with T1D-relevant antigen expressing FRCs using confocal microscopy. Image processing allowed quantification of T cell interactions with FRCs in 3D reticula as T cell trajectory, speed, and displacement. Flow cytometry analysis allowed quantification of the phenotype of antigen-specific T cells. 3D FRC reticula expressing T1D relevant antigens increased engagement of specific T cells compared to 2D cultures and to FRCs that did not express antigens. Thus, the engineered reticula can be used for conducting mechanistic studies to assess FRC interactions with antigen specific T cells and will help establish a definitive role for FRCs in peripheral tolerance.