Deviation of islet autoreactivity to cryptic epitopes protects NOD mice from diabetes

Abstract

To better understand loss of self-tolerance in diabetes-prone NOD mice, we are generating ICA69 transgenes under control of the tetracycline-regulated tet07 minimal promoter. In vitro pilot studies showed leaky transgene expression, but addition of g -globin genomic insulator flanks prevented leakage and dramatically enhanced transgene expression even in transient transfection, with excellent suppression by Doxycycline. In vivo, the accidental loss of insulator flanks during transgene insertion in one transgenic NOD founder, tet1, re-established leakiness with high level, exclusive ICA69-transgene expression in stromal elements of thymus and spleen. This led to persistent deletion of T cells targeting the immunodominant ICA69 epitope, Tep69, but emergence of T cell pools targeting cryptic ICA69 epitopes not normally generated in sufficient density to select and maintain ICA69-autoreactive T cells. This subtle modification of T cell repertoires reduced insulitis, and protected from diabetes in transgenics and in wild-type mice carrying irradiated tet1 thymus grafts. The low pathogenicity of T cells targeting cryptic epitopes likely reflects the fact that the major ICA69 determinant presented in the islet milieu remains Tep69, while cryptic epitopes are underrepresented. Deviation of T cell autoreactivity from major to cryptic target epitopes in tet1 mice provides a fortuitous model to explain previously observed diabetes protection by immunotherapy or autoantigen transgenes despite apparent failure to achieve tolerance to the full length islet antigens.

Sequence Variation in Promoter of Ica1 Gene, Which Encodes Protein Implicated in Type 1 Diabetes, Causes Transcription Factor Autoimmune Regulator (AIRE) to Increase Its Binding and Down-regulate Expression

“…ICA69 is involved in dense core vesicle signaling and maturation (31) and is recruited to Golgi membranes by activated RAB-2 (32). ICA69 is thought to be a T1D autoantigen based on the following observations: 1) autoantibodies to ICA69 can be detected in both first degree relatives of T1D patients who are followed to overt diabetes and in newly diagnosed diabetic patients (12,14,(33)(34)(35); 2) autoreactive T cells directed to ICA69 can be detected in (a) newly diagnosed diabetic children (ϳ80% of patients with recent onset T1D have either ICA69 autoreactive T cells or autoantibodies against the molecule) (29) and (b) NOD mice (36,37); and 3) T cells specific for the murine ICA69 peptide Tep-69 play a driving role in the acceleration of T cell-mediated islet cell destruction in NOD mice, whereas intraperitoneal injection of the peptide Tep-69 is associated with decreased T1D incidence in NOD mice, apparently as a result of neonatal tolerization of unknown mechanism (38). The latter studies are promising and are based on the generation of a NOD mouse T cell hybridoma that recognizes the murine peptide Tep69.…”

“…The length of these repeats has been directly implicated in the control of the expression levels of insulin mRNA in the thymus (9 -11). In addition to insulin, ICA69 (islet cell autoantigen 69 kDa), a neuroendocrine protein targeted by autoimmmune responses in human T1D and in non-obese diabetic (NOD) mice (12)(13)(14), is also expressed in the thymus, and we considered the likelihood that thymic levels of ICA69 would affect susceptibility to T1D through a mechanism similar to that shown for the insulin VNTRs (2,15). This hypothesis is primarily based on our previous studies indicating that IA-2, GAD65, and ICA69 are transcribed in the human thymus throughout fetal life and childhood (2,10,16).…”

“…This hypothesis is primarily based on our previous studies indicating that IA-2, GAD65, and ICA69 are transcribed in the human thymus throughout fetal life and childhood (2,10,16). The significance of thymic ICA69 expression in T1D susceptibility was reinforced through experiments involving tetracycline-responsive transgene promoter studies in the NOD mouse model, which exogenously overexpressed ICA69 in the thymus and spleen (14). This overexpression of thymic ICA69 resulted in a significant delay of disease progression in this model (14).…”

“…The significance of thymic ICA69 expression in T1D susceptibility was reinforced through experiments involving tetracycline-responsive transgene promoter studies in the NOD mouse model, which exogenously overexpressed ICA69 in the thymus and spleen (14). This overexpression of thymic ICA69 resulted in a significant delay of disease progression in this model (14).…”

The dual role of autoimmune regulator in maintaining normal expression level of tissue-restricted autoantigen in the thymus: A modeling investigation

“…Recent studies of non-obese diabetic (NOD) mice, prone to developing this immunological disorder with 60-80% susceptibility rate [8], have shown that NOD mice have reduced expression level of these proteins in the thymus, when compared to other strains of mice that are T1D-resistant [5, 9, 10]. In fact, exogenously overexpressing some of these autoantigens in the thymus and spleen of NOD mice resulted in a significant delay of disease progression in these mice [11], suggesting that the failure of negative selection may be implicated in disease onset.…”