B-lymphocytes play a key role in type 1 diabetes (T1D) development by serving as a subset of APC preferentially supporting expansion of autoreactive pathogenic T-cells. As a result of their pathogenic importance, B-lymphocyte-targeted therapies have received considerable interest as potential T1D interventions. Unfortunately, B-lymphocyte-directed T1D interventions tested to date failed to halt β-cell demise. IgG autoantibodies marking humans at future T1D risk indicate B-lymphocytes producing them have undergone the affinity maturation processes of class switch recombination (CSR) and possibly somatic hypermutation (SHM). This study found that CRISPR/Cas9-mediated ablation of the Aicda gene required for CSR/SHM induction, inhibits T1D development in the NOD mouse model. The Aicda encoded AID molecule induces genome-wide DNA breaks that, if not repaired through RAD51-mediated homologous recombination (HR), result in B-lymphocyte death. Treatment with the RAD51 inhibitor 4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS) also strongly inhibited T1D development in NOD mice. Both the genetic and small molecule-targeting approaches expanded CD73+ B-lymphocytes exerting regulatory activity suppressing diabetogenic T-cell responses. Hence, an initial CRISPR/Cas9 mediated genetic modification approach has identified the AID/RAD51 axis as a target for a potentially clinically translatable pharmacological approach that can block T1D development by converting B-lymphocytes to a disease inhibitory CD73+ regulatory state.

Although the autoimmune destruction of pancreatic β-cells underlying type 1 diabetes (T1D) development is ultimately mediated by T cells in NOD mice and also likely in humans, B cells play an additional key pathogenic role. It appears that the expression of plasma membrane–bound Ig molecules that efficiently capture β-cell antigens allows autoreactive B cells that bypass normal tolerance induction processes to be the subset of antigen-presenting cells most efficiently activating diabetogenic T cells. NOD mice transgenically expressing Ig molecules recognizing antigens that are (insulin) or are not (hen egg lysozyme [HEL]) expressed by β-cells have proven useful in dissecting the developmental basis of diabetogenic B cells. However, these transgenic Ig specificities were originally selected for their ability to recognize insulin or HEL as foreign, rather than autoantigens. Thus, we generated and characterized NOD mice transgenically expressing an Ig molecule representative of a large proportion of naturally occurring islet-infiltrating B cells in NOD mice recognizing the neuronal antigen peripherin. Transgenic peripherin-autoreactive B cells infiltrate NOD pancreatic islets, acquire an activated proliferative phenotype, and potently support accelerated T1D development. These results support the concept of neuronal autoimmunity as a pathogenic feature of T1D, and targeting such responses could ultimately provide an effective disease intervention approach.

In NOD mice and also likely humans, B-lymphocytes play an important role as APC expanding autoreactive T-cell responses ultimately causing type 1 diabetes (T1D). Currently, humans at high future T1D risk can only be identified at late prodromal stages of disease indicated by markers such as insulin autoantibodies (IAA). When commenced in already IAA+ NOD mice, continuous BAFFR-Fc treatment alone or in combination with anti-CD20 (designated combo therapy) inhibited T1D development. Despite eliciting broader B-lymphocyte depletion, continuous combo therapy afforded no greater T1D protection than BAFFR-Fc alone. As previously observed, late disease stage initiated anti-CD20 mono-therapy did not inhibit T1D, and in this study was additionally found to be associated with development of drug blocking antibodies. Promisingly, NOD mice given transient late disease stage BAFFR-Fc mono-therapy were rendered T1D resistant. However, combo treatment abrogated the protective effect of transient BAFFR-Fc mono-therapy. NOD mice receiving transient BAFF blockade were characterized by an enrichment of regulatory B-lymphocytes (Bregs) that inhibit T1D development through IL-10 production, but this population is sensitive to deletion by anti-CD20 treatment. B-lymphocytes from transient BAFFR-Fc treated mice suppressed T-cell proliferation to a greater extent than those from controls. Proportions of B-lymphocytes expressing CD73, an ecto-enzyme operating in a pathway converting pro-inflammatory ATP to immunosuppresive adenosine, were also temporarily increased by transient BAFFR-Fc treatment, but not anti-CD20 therapy. These collective studies indicate transient BAFFR-Fc mediated B-lymphocyte depletion elicits long-term T1D protection by enriching Bregs that are deleted by anti-CD20 co-therapy.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8 T cells recognizing pancreatic β cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-K and/or H2-D class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8 T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8 T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8 T cells. CRISPR/Cas9-mediated genetic attenuation of expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8 T cells. These results indicated that allelic variants of contribute to the efficiency of intrathymic deletion of diabetogenic CD8 T cells. However, although enhancing thymic deletion of pathogenic CD8 T cells, ablating expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.

Improved mouse models for type 1 diabetes (T1D) therapy development are needed. T1D susceptibility is restored to normally resistant NOD.β2m mice transgenically expressing human disease-associated HLA-A*02:01 or HLA-B*39:06 class I molecules in place of their murine counterparts. T1D is dependent on pathogenic CD8 T-cell responses mediated by these human class I variants. NOD.β2m-A2.1 mice were previously used to identify β-cell autoantigens presented by this human class I variant to pathogenic CD8 T cells and for testing therapies to attenuate such effectors. However, NOD.β2m mice also lack nonclassical MHC I family members, including FcRn, required for antigen presentation, and maintenance of serum IgG and albumin, precluding therapies dependent on these molecules. Hence, we used CRISPR/Cas9 to directly ablate the NOD H2-K and H2-D classical class I variants either individually or in tandem (cMHCI). Ablation of the H2-A class II variant in the latter stock created NOD mice totally lacking in classical murine MHC expression (cMHCI/II). NOD-cMHCI mice retained nonclassical MHC I molecule expression and FcRn activity. Transgenic expression of HLA-A2 or -B39 restored pathogenic CD8 T-cell development and T1D susceptibility to NOD-cMHCI mice. These next-generation HLA-humanized NOD models may provide improved platforms for T1D therapy development.