Ridgway A New Tool for Dissecting Genetic Control of Type 1 Diabetes

The NOD mouse has been a critical tool in the quest to understand the genetic control of type 1 diabetes (T1D), and over 25 murine insulin-dependent diabetes (Idd) loci that modulate the natural history of T1D have been identified (1). Several of the candidate genes identified in NOD mice also play a role in human T1D, suggesting that dysregulated immune pathways in NOD may closely resemble those found in humans, and justifying continuing work on the genetic origin of T1D in NOD (2). The role of specific genes in T1D has been explored by constructing congenic mice (carrying disease-protective Idd loci), by creating transgenic mice, and by knocking out genes. In each case, however, these studies have had a significant potential drawback: the presence of “passenger DNA” that can potentially confound the interpretation of the results (Fig. 1A, top panel ) (3). Knockout mice, for example, have previously been made almost exclusively using non-NOD embryonic stem (ES) cells; the resulting non-NOD mice were backcrossed to NOD mice. The backcrossing process ensures that non-NOD genetic material is bred along with the genetic region of interest (Fig. 1A, top panel ). This is not merely a theoretical concern, as some published studies of transgenic or deleted genes showed an effect on T1D that was later proven to arise from non-NOD passenger DNA (3). In the current issue, Chen et al. (4) have eliminated this passenger DNA problem using NOD ES cells and zincfinger nuclease technology to knock out a candidate gene, Cd137, in NOD mice without introducing non-NOD genetic material. A similar approach was used recently to understand the role of the class II–associated molecule DM, and taking advantage of an existing Balb/c construct in a region of genetic homology to NOD to eliminate DM in NOD ES cells (5). These studies represent breakthroughs for understanding specific genetic effects in NOD mice. The approach used by Chen et al. (4) takes advantage of zinc-finger nucleases targeted to specific genetic regions (in this case, exon 4 of Cd137); the nuclease cleaves the genome at the site of interest, and subsequent genome repair mechanisms excise the damaged region thus knocking out the gene (6). This resulted in a specific CD137 knockout on a 100% NOD genetic background (Fig. 1A, bottom panel ). Cd137 is an interesting candidate gene in the Idd9.3 region, and CD137 has profound effects on the immune system at many levels (7). In T1D, Lyons et al. (8) showed that the NOD Cd137 allele differed from B10 by three exonal single nucleotide polymorphisms. Cannons et al. (9) clearly demonstrated that the NOD CD137 allele was hypofunctional: CD137–mediated stimulation produced significantly less interleukin-2 and proliferation from NOD than NOD.B10 Idd9.3 T cells (expressing the protective B10 CD137 variant). This result, however, was somewhat confusing: how could hypofunctional NOD CD137 contribute to T1D? CD137 has multiple roles throughout the immune system (Fig. 1B); it is critical to T-cell effector function and to the acquisition of CD8 T-cell memory (7). T-regulatory cells (Tregs) also express CD137, and CD137-expressing Tregs were found in the pancreatic islet (10–12). Thus CD137 in T1D might function in both effector T cell (CD8 cells mediating islet cell damage) or in regulation against autoimmunity (on Tregs). Indeed, Irie et al. (11) found that stimulation via CD137 could either diminish or augment T1D depending on the timing of treatment. Kachapati et al. (13) showed that the hypofunctional NOD allele was associated with significantly decreased numbers of CD137 Tregs in NOD mice; that these Tregs produced soluble CD137, which was immunosuppressive; and that CD137 Tregs are functionally superior to other Treg subsets. Thus, a hypofunctional allele could decrease protective immunity, allowing T-cell effector immunity to mediate tissue damage.