Lymph Node Stromal Cell Regulation of Insulin-specific CD8+ T cells

Background: Insulin is an essential autoantigen in human and murine type 1 diabetes. Thymic negative selection is critical for the removal of self-reactive cells, while peripheral regulation maintains tolerance to self-antigen. As low-affinity autoreactive cells escape central tolerance, peripheral regulation is essential for preventing or reversing insulin-directed autoimmunity. Recently, Lymph Node Stromal Cells (LNSC) have been identified that provide regulatory signals and can directly interact with T cells. The aim of our study was to investigate the role of proinsulin (PI) expression by LNSC subsets in regulating insulin-specific G9Ca-/- TCR transgenic CD8+ T cells.

Methods: LNSC were isolated from NOD.PI2tg (overexpressing PI on MHC class II positive cells), NOD, or C57BL/6 mice. NOD.PI2tg APC-stimulated proliferation of G9Ca-/- CD8+ T cells was evaluated in the presence of LNSC or separated by a trans-well. G9 CD8+ T cells were also subsequently assessed for cytotoxic function after 48 hours co-culture with LNSC.

Results: We have previously reported that LNSC suppressed the proliferation of G9 CD8+ T cells, stimulated by APCs expressing PI2, in an insulin/MHC dependent manner. Separation by trans-wells abrogated this suppressive effect suggesting that contact between the LNSC and T cells and/or APCs is required. To determine if suppressive effects occurred through direct contact with the T cells and if exposure to LNSC could permanently modify T cell function, G9 CD8+ T cells and LNSC were co-cultured for 48 hours. The T cells were collected and cytotoxicity towards peptide-loaded targets was measured. Exposure to NOD or NOD.PI2tg LNSC reduced cytotoxicity by 50%, compared to T cells cultured alone, while prior exposure to MHC mis-matched C57BL/6 LNSC had no effect. These data suggest LNSC presentation of antigen to CD8+ T cells has a direct impact on T cell activation and modulates T cell functionality, shifting the threshold of sensitivity to self-antigen.