Generation and maintenance of autoantigen-specific CD8(+) T cell clones isolated from NOD mice

The non-obese diabetic (NOD) mouse develops insulin dependent diabetes mellitus (IDDM) spontaneously with a higher incidence in females than in males. There are many similarities to the human disease, making it an ideal model. Our group is examining the role that CD4(+) and CD8(+) T cells play in IDDM in the NOD mouse, as it is known that both T cell subsets are required for onset of disease. Although IDDM has an autoimmune etiology, the initial triggering event is unknown and the autoantigen involved has not been identified. This investigation focussed on one of the potential autoantigens involved, the enzyme glutamic acid decarboxylase (GAD). We raised GAD peptide-specific CD8(+) T cells by immunising NOD mice with the GAD peptide alongside an irrelevant peptide that induced a CD4(+) T cell response. In order to maintain these peptide specific T cells in vitro and generate clones, it was found that antibodies specific to CD4(+) and MHC class II molecules needed to be included in the culture medium. This paper outlines the methods we employed to generate and maintain these CD8(+) T cells in vitro.