Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice

Abstract

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.

Figure 1

Serum anti-insulin autoantibody levels. a, Insulin autoantibodies fail to occur in transgene⁺ mice in the absence of native insulin 1 and insulin 2 genes. b, Mice with insulin 1 gene (insulin 1^+/-, insulin 2^-/-, transgene⁺ or transgene^-) produce insulin autoantibodies (IAA). Lines connect results for individual mice. mIAA, micro-IAA assay.

Figure 2

Histology of non-diabetic native insulin-negative mice (insulin 1^-/-, insulin 2^-/-, transgene⁺). Histology of pancreas and salivary gland of NOD mice lacking both native insulin 1 and 2 genes, with mutated (B16:alanine) preproinsulin transgene. a–c, Sections from a mouse killed at 26 weeks, showing islets stained for insulin (a), islets stained for glucagon (b) and salivary gland stained with haematoxylin and eosin (c). d, Islets from a mouse killed at 23 weeks, stained for insulin.

Figure 3

Histology of native insulin-positive mice. Histology showing marked insulitis of NOD mice with one or more copies of the insulin 1 or insulin 2 gene, with or without the mutated preproinsulin (B16:alanine) transgene. a, Insulin 1^+/+, insulin 2^-/-, transgene^-, killed at 14 weeks, diabetic at 14 weeks. b, Insulin 1^+/+, insulin 2^-/-, transgene^-, killed at 10.5 weeks, diabetic at 10.5 weeks. c, Insulin 1^+/-, insulin 2^-/-, transgene⁺, killed at 15 weeks, diabetic at 15 weeks. d, Insulin 1^-/-, insulin 2^+/+, transgene^-, killed at 20 weeks, not diabetic. e, Insulin 1^-/-, insulin 2^+/+, transgene⁺, killed at 21 weeks, not diabetic. f, Insulin 1^-/-, insulin 2^+/-, transgene⁺, killed at 35 weeks, not diabetic.

Figure 4

Life tables of diabetes development. a, Lack of progression to diabetes of NOD mice lacking both insulin native genes. b, c, Lack of difference in progression to diabetes for speed congenic female NOD mice with control regions from the 129 mouse surrounding the insulin genes (insulin 1 region (b); insulin 2 region (c)). d, Progression to diabetes of the four transgenic founder strains with the mutated preproinsulin gene on NOD background (insulin 1^+/+, insulin 2^+/+, transgene⁺). Founder strain F was significantly different (P < 0.01) from the others. e, NOD.SCID recipients of splenocytes from mice lacking both native insulin genes had delayed progression to diabetes compared with insulin 1⁺, insulin 2⁺ mice (P < 0.02). ins, insulin; Tg, transgene.