Highlights d IRE1a deletion in NOD b cells before insulitis causes their transient dedifferentiation d Dedifferentiated b cells show diminished expression of b cell autoantigens d Knockout mice exhibit impaired T cell diabetogenic activity d IRE1a-deficient NOD mice are protected from autoimmune destruction and diabetes

Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4–1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1−/− recipients, even when CD25+ cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4+CD25+ regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains.

Author contributions: A.E.C. and D.M.S. designed experiments, performed experimental work, analyzed results, prepared figures, and wrote the manuscript. B.F. performed experimental work and provided intellectual contribution. G.P. provided critical cell sorting expertise. M.Y.K. analyzed single-cell RNA sequencing (scRNA-seq) data and revised the manuscript. C.-W.L. analyzed the scRNA-seq data. R.B. generated gene regulatory network models. W.R.D. provided reagents and intellectual contribution. W.C. and Y.-G.C. conceived the study, supervised the project, and revised the manuscript.The scRNA-seq data presented in this article have been submitted to the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE155595) under accession number GSE155595.

SUMMARY Human genetic studies implicate interleukin-27 (IL-27) in the pathogenesis of type 1 diabetes (T1D), but the underlying mechanisms remain largely unexplored. To further define the role of IL-27 in T1D, we generated non-obese diabetic (NOD) mice deficient in IL-27 or IL-27Rα. In contrast to wild-type NOD mice, both NOD.Il27−/− and NOD.Il27ra−/− strains are completely resistant to T1D. IL-27 from myeloid cells and IL-27 signaling in T cells are critical for T1D development. IL-27 directly alters the balance of regulatory T cells (Tregs) and T helper 1 (Th1) cells in pancreatic islets, which in turn modulates the diabetogenic activity of CD8 T cells. IL-27 also directly enhances the effector function of CD8 T cells within pancreatic islets. In addition to T1D, IL-27 signaling in T cells is also required for lacrimal and salivary gland inflammation in NOD mice. Our study reveals that IL-27 contributes to autoimmunity in NOD mice through multiple mechanisms and provides substantial evidence to support its pathogenic role in human T1D.

We previously reported that CD137 (encoded by Tnfrsf9) deficiency suppressed type 1 diabetes (T1D) progression in nonobese diabetic (NOD) mice. We also demonstrated that soluble CD137 produced by regulatory T cells contributed to their autoimmune suppressive function in this model. These results suggest that CD137 can either promote or suppress T1D development in NOD mice depending on where it is expressed. Here, we show that NOD.Tnfrsf9−/− CD8 T cells had significantly reduced diabetogenic capacity, while absence of CD137 in non-T and non-B cells had a limited impact on T1D progression. In contrast, NOD.Tnfrsf9−/− CD4 T cells highly promoted T1D development. We further demonstrated that CD137 was important for the accumulation of β-cell autoreactive CD8 T cells but was dispensable for their activation in pancreatic lymph nodes. The frequency of islet infiltrating CD8 T cells was reduced in NOD.Tnfrsf9−/− mice in part due to their decreased proliferation. Furthermore, CD137 deficiency did not suppress T1D development in NOD mice expressing the transgenic NY8.3 CD8 T cell receptor. This suggests that increased precursor frequency of β-cell autoreactive CD8 T cells in NY8.3 mice obviated a role for CD137 in diabetogenesis. Finally, blocking CD137-CD137L interaction significantly delayed T1D onset in NOD mice. Collectively, our results indicate that one important diabetogenic function of CD137 is to promote the expansion and accumulation of β-cell autoreactive CD8 T cells, and in the absence of CD137 or its interaction with CD137L, T1D progression is suppressed.