Immune and pancreatic β cell interactions in type 1 diabetes

The autoimmune destruction of the pancreatic islet β cells is due to a targeted lymphocyte attack. Different T cell subsets communicate with each other and with the insulin-producing β cells in this process, with evidence not only of damage to the tissue cells but also of lymphocyte regulation. Here we explore the various components of the immune response as well as the cellular interactions that are involved in causing or reducing immune damage to the β cells. We consider these in the light of the possibility that understanding them may help us identify therapeutic targets to reduce the damage and destruction leading to type 1 diabetes. Trends In type 1 diabetes (T1D), β cells are highly sensitive to selective damage and recruit immune cells by chemokine production. These immune cells directly damage β cells and induce enzymes and cytokines that cause free radical- and cytokine-induced apoptosis. Damaged islets express innate immune receptors, engagement of which may amplify β cell destruction contributing to their own destruction. Interestingly, damaged and functional islets coexist. Immune regulatory cells and regulatory mechanisms induced by islet cells counterbalance inflammation. Communication between immune cells and resident islet β cells during inflammation is dependent on the pancreatic microenvironment. Therapeutically targeting the direct and indirect mediators of β cell damage to prevent further destruction combined with boosting β cell numbers and function are important joint targets in developing therapies for T1D

Natural protection from Type 1 Diabetes in Non obese diabetic (Nod) mice is characterised by a unique pancreatic islet phenotype

The non-obese diabetic (NOD) mouse develops spontaneous type 1 diabetes, with some features of disease that are very similar to the human disease. However, a proportion of NOD mice are naturally-protected from developing diabetes, and currently studies characterising this cohort are very limited. Here, using both immunofluorescence and multi-parameter flow cytometry we focus on the pancreatic islet morphology and immune infiltrate observed in naturally-protected NOD mice. We show that naturally-protected NOD mice are characterised by an increased frequency of insulin-containing, smaller sized, pancreatic islets. Although mice remain diabetes free, florid immune infiltrate remains. However, this immune infiltrate is skewed towards a regulatory phenotype in both T and B-cell compartments. Pancreatic islets have an increased frequency of IL-10 producing B cells and associated cell surface markers. Resident memory CD69+CD8+ T cells show a significant shift towards reduced CD103 expression, while CD4+ T cells have increased FoxP3+CTLA4+ expression. These data indicate that naturally-protected NOD mice have a unique islet signature and provide new insight into regulatory mechanisms within pancreatic islets

B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model

Aims/hypothesis: Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Methods: Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. Results: There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4+ and CD8+ T cells. These CD8+ T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Conclusions/interpretation: Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes

Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

Aims/hypothesis Autoreactive B cells escape immune tolerance and contribute to the pathogenesis of type 1 diabetes. While global B cell depletion is a successful therapy for autoimmune disease, the fate of autoreactive cells during this treatment in autoimmune diabetes is unknown. We aimed to identify and track anti-insulin B cells in pancreatic islets and understand their repopulation after anti-CD20 treatment. Methods We generated a double transgenic system, the VH125.hCD20/NOD mouse. The VH125 transgenic mouse, expressing an increased frequency of anti-insulin B cells, was crossed with a human CD20 (hCD20) transgenic mouse, to facilitate B cell depletion using anti-CD20. B cells were analysed using multiparameter and ImageStream flow cytometry. Results We demonstrated that anti-insulin B cells were recruited to the pancreas during disease progression in VH125.hCD20/NOD mice. We identified two distinct populations of anti-insulin B cells in pancreatic islets, based on CD19 expression, with both populations enriched in the CD138int fraction. Anti-insulin B cells were not identified in the plasma-cell CD138hi fraction, which also expressed the transcription factor Blimp-1. After anti-CD20 treatment, anti-insulin B cells repopulated the pancreatic islets earlier than non-specific B cells. Importantly, we observed that a CD138intinsulin+CD19− population was particularly enriched after B cell depletion, possibly contributing to the persistence of disease still observed in some mice after anti-CD20 treatment. Conclusions/interpretation Our observations may indicate why the loss of C-peptide is only temporarily delayed following anti-CD20 treatment in human type 1 diabetes

Peripheral proinsulin expression controls low-avidity proinsulin-reactive CD8 T Cells in type 1 diabetes

Low-avidity autoreactive CD8 T cells (CTLs) escape from thymic negative selection, and peripheral tolerance mechanisms are essential for their regulation. We report the role of proinsulin (PI) expression on the development and activation of insulin-specific CTLs in the NOD mouse model of type 1 diabetes. We studied insulin B-chain–specific CTL from different T-cell receptor transgenic mice (G9Cα−/−) expressing normal PI1 and PI2 or altered PI expression levels. In the absence of PI2 (Ins2−/−), CTL in pancreatic lymph nodes (PLNs) were more activated, and male G9Cα−/− mice developed T1D. Furthermore, when the insulin-specific CTLs developed in transgenic mice lacking their specific PI epitope, the CTLs demonstrated increased cytotoxicity and proliferation in vitro and in vivo in the PLNs after adoptive transfer into NOD recipients. Dendritic cell–stimulated proliferation of insulin-specific T cells was reduced in the presence of lymph node stromal cells (LNSCs) from NOD mice but not from mice lacking the PI epitope. Our study shows that LNSCs regulate CTL activation and suggests that exposure to PI in the periphery is very important in maintenance of tolerance of autoreactive T cells. This is relevant for human type 1 diabetes and has implications for the use of antigen-specific therapy in tolerance induction

Gene expression profiling in NOD mice reveals that B cells are highly educated by the pancreatic environment during autoimmune diabetes

Aims/hypothesis: B cells play an important role in driving the development of type 1 diabetes; however, it remains unclear how they contribute to local beta cell destruction during disease progression. Here, we use gene expression profiling of B cell subsets identified in inflamed pancreatic tissue to explore their primary functional role during the progression of autoimmune diabetes. Methods: Transcriptional profiling was performed on FACS-sorted B cell subsets isolated from pancreatic islets and the pancreatic lymph nodes of NOD mice. Results: B cells are highly modified by the inflamed pancreatic tissue and can be distinguished by their transcriptional profile from those in the lymph nodes. We identified both a discrete and a core shared gene expression profile in islet CD19+CD138– and CD19+CD138+ B cell subsets, the latter of which is known to have enriched autoreactivity during diabetes development. On localisation to pancreatic islets, compared with CD138– B cells, CD138+ B cells overexpress genes associated with adhesion molecules and growth factors. Their shared signature consists of gene expression changes related to the differentiation of antibody-secreting cells and gene regulatory networks associated with IFN signalling pathways, proinflammatory cytokines and Toll-like receptor (TLR) activation. Finally, abundant TLR7 expression was detected in islet B cells and was enhanced specifically in CD138+ B cells. Conclusions/interpretation: Our study provides a detailed transcriptional analysis of islet B cells. Specific gene signatures and interaction networks have been identified that point towards a functional role for B cells in driving autoimmune diabetes. Graphical abstract

Novel engineered B lymphocytes targeting islet-specific T cells inhibit the development of type 1 diabetes in non-obese diabetic Scid mice

IntroductionIn this study, we report a novel therapeutic approach using B lymphocytes to attract islet-specific T cells in the non-obese diabetic (NOD) mouse model and prevent the development of autoimmune diabetes. Rather than using the antibody receptor of B cells, this approach utilizes their properties as antigen-presenting cells to T cells.MethodsPurified splenic B cells were treated with lipopolysaccharide, which increases regulatory B (Breg) cell function, then electroporated with mRNA encoding either chimeric MHC-I or MHC-II molecules covalently linked to antigenic peptides. Immunoregulatory functions of these engineered B cells (e-B cells) were tested by in vitro assays and in vivo co-transfer experiments with beta-cell-antigen-specific CD8+ or CD4+ T cells in NOD.Scid mice, respectively.ResultsThe e-B cells expressing chimeric MHC-I-peptide inhibited antigen-specific CD8+ T-cell cytotoxicity in vitro. The e-B cells expressing chimeric MHC-II-peptide induced antigen-specific CD4+ T cells to express the regulatory markers, PD-1, ICOS, CTLA-4, Lag3, and Nrp1. Furthermore, e-B cells encoding the chimeric MHC-I and MHC-II peptide constructs protected NOD.Scid mice from autoimmune diabetes induced by transfer of antigen-specific CD8+ and CD4+ T cells.DiscussionMHC–peptide chimeric e-B cells interacted with pathogenic T cells, and protected the host from autoimmune diabetes, in a mouse model. Thus, we have successfully expressed MHC–peptide constructs in B cells that selectively targeted antigen-specific cells, raising the possibility that this strategy could be used to endow different protective cell types to specifically regulate/remove pathogenic cells

Re-programming immunosurveillance in persistent non-infectious ocular inflammation

Ocular function depends on a high level of anatomical integrity. This is threatened by inflammation, which alters the local tissue over short and long time-scales. Uveitis due to autoimmune disease, especially when it involves the retina, leads to persistent changes in how the eye interacts with the immune system. The normal pattern of immune surveillance, which for immune privileged tissues is limited, is re-programmed. Many cell types, that are not usually present in the eye, become detectable. There are changes in the tissue homeostasis and integrity. In both human disease and mouse models, in the most extreme cases, immunopathological findings consistent with development of ectopic lymphoid-like structures and disrupted angiogenesis accompany severely impaired eye function. Understanding how the ocular environment is shaped by persistent inflammation is crucial to developing novel approaches to treatment

Re-programming immunosurveillance in persistent non-infectious ocular inflammation

Ocular function depends on a high level of anatomical integrity. This is threatened by inflammation, which alters the local tissue over short and long time-scales. Uveitis due to autoimmune disease, especially when it involves the retina, leads to persistent changes in how the eye interacts with the immune system. The normal pattern of immune surveillance, which for immune privileged tissues is limited, is re-programmed. Many cell types, that are not usually present in the eye, become detectable. There are changes in the tissue homeostasis and integrity. In both human disease and mouse models, in the most extreme cases, immunopathological findings consistent with development of ectopic lymphoid-like structures and disrupted angiogenesis accompany severely impaired eye function. Understanding how the ocular environment is shaped by persistent inflammation is crucial to developing novel approaches to treatment