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

Introduction: In 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. Methods: Purified 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. Results: The 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. Discussion: MHC-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.

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.

Activated but functionally impaired memory Tregs are expanded in slow progressors to type 1 diabetes.

AIMS/HYPOTHESIS: Slow progressors to type 1 diabetes are individuals positive for multiple pancreatic islet autoantibodies who have remained diabetes-free for at least 10 years; regulation of the autoimmune response is understudied in this group. Here, we profile CD4+ regulatory T cells (Tregs) in a small but well-characterised cohort of extreme slow progressors with a median age 43 (range 31-72 years), followed up for 18-32 years. METHODS: Peripheral blood samples were obtained from slow progressors (n = 8), age- and sex-matched to healthy donors. One participant in this study was identified with a raised HbA1c at the time of assessment and subsequently diagnosed with diabetes; this donor was individually evaluated in the analysis of the data. Peripheral blood mononuclear cells (PBMCs) were isolated, and to assess frequency, phenotype and function of Tregs in donors, multi-parameter flow cytometry and T cell suppression assays were performed. Unsupervised clustering analysis, using FlowSOM and CITRUS (cluster identification, characterization, and regression), was used to evaluate Treg phenotypes. RESULTS: Unsupervised clustering on memory CD4+ T cells from slow progressors showed an increased frequency of activated memory CD4+ Tregs, associated with increased expression of glucocorticoid-induced TNFR-related protein (GITR), compared with matched healthy donors. One participant with a raised HbA1c at the time of assessment had a different Treg profile compared with both slow progressors and matched controls. Functional assays demonstrated that Treg-mediated suppression of CD4+ effector T cells from slow progressors was significantly impaired, compared with healthy donors. However, effector CD4+ T cells from slow progressors were more responsive to Treg suppression compared with healthy donors, demonstrated by increased suppression of CD25 and CD134 expression on effector CD4+ T cells. CONCLUSIONS/INTERPRETATIONS: We conclude that activated memory CD4+ Tregs from slow progressors are expanded and enriched for GITR expression, highlighting the need for further study of Treg heterogeneity in individuals at risk of developing type 1 diabetes.

Regulatory B Cells: Role in Type 1 Diabetes.

Regulatory B cells (Bregs) have an anti-inflammatory role and can suppress autoimmunity, by employing both cytokine secretion and cell-contact mediated mechanisms. Numerous Breg subsets have been described and have overlapping phenotypes in terms of their immune expression markers or cytokine production. A hallmark feature of Bregs is the secretion of IL-10, although IL-35 and TGFß-producing B cells have also been identified. To date, few reports have identified an impaired frequency or function of Bregs in individuals with type 1 diabetes; thus our understanding of the role played by these Breg subsets in the pathogenesis of this condition is limited. In this review we will focus on how regulatory B cells are altered in the development of type 1 diabetes, highlighting both frequency and function and discuss both human and animal studies.

Natural Protection From Type 1 Diabetes in NOD Mice Is Characterized by a Unique Pancreatic Islet Phenotype.

The 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 characterizing this cohort are very limited. Here, using both immunofluorescence and multiparameter 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 characterized 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 toward 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 toward 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.

Regulatory B Cells in Type 1 Diabetes.

Type 1 diabetes is an organ-specific autoimmune disease characterized by immune-mediated beta cell destruction in pancreatic islets, which results in deficient insulin production. B cells have a dual role in type 1 diabetes pathogenesis. A pathogenic role for B cells has been widely described and is supported by the observation of a delay in the loss of C-peptide following B-cell depletion by Rituximab, in the first year after diagnosis. However, it is now clear that B cells, under certain conditions, can delay and prevent the onset of type 1 diabetes as demonstrated in mouse models. In this chapter, we describe the methods required to study the phenotype and function of regulatory B cells in the context of diabetes.

Assessing Immune Responses in the Nonobese Diabetic Mouse Model of Type 1 Diabetes.

Type 1 diabetes is an autoimmune disease resulting in the loss of insulin production and, consequently, hyperglycemia. The nonobese diabetic (NOD) mouse develops spontaneous diabetes with considerable similarity to the disease in humans. Immunological studies using the NOD mouse model allow for the investigation of the natural history of the disease and leukocyte and lymphocyte pathogenic and regulatory functions, as well as testing potential therapies for intervention. The analyses of the cellular events leading up to diabetes may utilize different in vitro cellular assays, immunohistochemistry, and in vivo adoptive transfer, to study mechanisms of the disease and the effects of therapeutic intervention. In this chapter, we describe some common techniques for phenotyping and mechanistic analyses of function, particularly of CD8+ T cells.

Dendritic cells license regulatory B cells to produce IL-10 and mediate suppression of antigen-specific CD8 T cells.

Regulatory B cells (Bregs) suppress and reduce autoimmune pathology. However, given the variety of Breg subsets, the role of Bregs in the pathogenesis of type 1 diabetes is still unclear. Here, we dissect this fundamental mechanism. We show that natural protection from type 1 diabetes in nonobese diabetic (NOD) mice is associated with increased numbers of IL-10-producing B cells, while development of type 1 diabetes in NOD mice occurs in animals with compromised IL-10 production by B cells. However, B cells from diabetic mice regain IL-10 function if activated by the innate immune receptor TLR4 and can suppress insulin-specific CD8 T cells in a dendritic cell (DC)-dependent, IL-10-mediated fashion. Suppression of CD8 T cells is reliant on B-cell contact with DCs. This cell contact results in deactivation of DCs, inducing a tolerogenic state, which in turn can regulate pathogenic CD8 T cells. Our findings emphasize the importance of DC-Breg interactions during the development of type 1 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.

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.

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.

Immune and Pancreatic ß Cell Interactions in Type 1 Diabetes.

The autoimmune destruction of the pancreatic islet ß cells is due to a targeted lymphocyte attack. Different T cell subsets communicate with each other and with the insulin-producing ß 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 ß 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.

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.

A novel pathogenic RBP-3 peptide reveals epitope spreading in persistent experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) in the C57BL/6J mouse is a model of non-infectious posterior segment intraocular inflammation that parallels clinical features of the human disease. The purpose of this study was to analyse the immune response to the four murine subunits of retinol binding protein-3 (RBP-3) to identify pathogenic epitopes to investigate the presence of intramolecular epitope spreading during the persistent inflammation phase observed in this model of EAU. Recombinant murine subunits of the RBP-3 protein were purified and used to immunize C57BL/6J mice to induce EAU. An overlapping peptide library was used to screen RBP-3 subunit 3 for immunogenicity and pathogenicity. Disease phenotype and characterization of pathogenic subunits and peptides was undertaken by topical endoscopic fundal imaging, immunohistochemistry, proliferation assays and flow cytometry. RBP-3 subunits 1, 2 and 3 induced EAU in the C57BL/6J mice, with subunit 3 eliciting the most destructive clinical disease. Within subunit 3 we identified a novel uveitogenic epitope, 629-643. The disease induced by this peptide was comparable to that produced by the uveitogenic 1-20 peptide. Following immunization, peptide-specific responses by CD4(+) and CD8(+) T-cell subsets were detected, and cells from both populations were present in the retinal inflammatory infiltrate. Intramolecular epitope spreading between 629-643 and 1-20 was detected in mice with clinical signs of disease. The 629-643 RBP-3 peptide is a major uveitogenic peptide for the induction of EAU in C57BL/6J mice and the persistent clinical disease induced with one peptide leads to epitope spreading.

Tissue-resident exhausted effector memory CD8+ T cells accumulate in the retina during chronic experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis is a model for noninfectious posterior segment intraocular inflammation in humans. Although this disease is CD4(+) T cell dependent, in the persistent phase of disease CD8(+) T cells accumulate. We show that these are effector memory CD8(+) T cells that differ from their splenic counterparts with respect to surface expression of CD69, CD103, and Ly6C. These retinal effector memory CD8(+) T cells have limited cytotoxic effector function, are impaired in their ability to proliferate in response to Ag-specific stimulation, and upregulate programmed death 1 receptor. Treatment with fingolimod (FTY720) during the late phase of disease revealed that retinal CD8(+) T cells were tissue resident. Despite signs of exhaustion, these cells were functional, as their depletion resulted in an expansion of retinal CD4(+) T cells and CD11b(+) macrophages. These results demonstrate that, during chronic autoimmune inflammation, exhausted CD8(+) T cells become established in the local tissue. They are phenotypically distinct from peripheral CD8(+) T cells and provide local signals within the tissue by expression of inhibitory receptors such as programmed death 1 that limit persistent inflammation.

2,2-bis(4-chlorophenyl)-1,1-dichloroethylene stimulates androgen independence in prostate cancer cells through combinatorial activation of mutant androgen receptor and mitogen-activated protein kinase pathways.

Therapy resistance represents a major clinical challenge in disseminated prostate cancer for which only palliative treatment is available. One phenotype of therapy-resistant tumors is the expression of somatic, gain-of-function mutations of the androgen receptor (AR). Such mutant receptors can use noncanonical endogenous ligands (e.g., estrogen) as agonists, thereby promoting recurrent tumor formation. Additionally, selected AR mutants are sensitized to the estrogenic endocrine-disrupting compound (EDC) bisphenol A, present in the environment. Herein, screening of additional EDCs revealed that multiple tumor-derived AR mutants (including T877A, H874Y, L701H, and V715M) are sensitized to activation by the pesticide 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), thus indicating that this agent may impinge on AR signaling in cancer cells. Further investigation showed that DDE induced mutant AR recruitment to the prostate-specific antigen regulatory region, concomitant with an enhancement of target gene expression, and androgen-independent proliferation. By contrast, neither AR activation nor altered cellular proliferation was observed in cells expressing wild-type AR. Activation of signal transduction pathways was also observed based on rapid phosphorylation of mitogen-activated protein kinase (MAPK) and vasodilator-stimulated phosphoprotein, although only MAPK activation was associated with DDE-induced cellular proliferation. Functional analyses showed that both mutant AR and MAPK pathways contribute to the proliferative action of DDE, as evidenced through selective abrogation of each pathway. Together, these data show that exposure to environmentally relevant doses of EDCs can promote androgen-independent cellular proliferation in tumor cells expressing mutant AR and that DDE uses both mutant AR and MAPK pathways to exert its mitogenic activity.

Unique bisphenol A transcriptome in prostate cancer: novel effects on ERbeta expression that correspond to androgen receptor mutation status.

BACKGROUND: Prostatic adenocarcinomas are dependent on androgen receptor (AR) activity for growth and progression, and therapy for disseminated disease depends on ablation of AR activity. Recurrent tumors ultimately arise wherein AR has been re-activated. One mechanism of AR restoration is via somatic mutation, wherein cells containing mutant receptors become susceptible to activation by alternative ligands, including bisphenol A (BPA). In tumors with specific AR mutations, BPA promotes therapeutic bypass, suggesting significant negative impact to the clinical management of prostate cancer. OBJECTIVE: Our goal was to determine the mechanism of BPA action in cancer cells carrying BPA-responsive AR mutants. METHODS: The molecular signature of BPA activity in prostate cancer cells harboring mutant AR was delineated via genetic microarray analysis. Specificity of BPA action was assessed by comparison with the molecular signature elicited by dihydrotestosterone (DHT). RESULTS: BPA and DHT elicited distinct transcriptional signatures in prostate cancer cells expressing the BPA-responsive mutant AR-T877A. BPA dramatically attenuated estrogen receptor beta (ERbeta) expression; this finding was specific to prostate tumor cells in which BPA induces cellular proliferation. CONCLUSIONS: BPA induces a distinct gene expression signature in prostate cancer cells expressing somatic AR mutation, and a major molecular consequence of BPA action is down-regulation of ERbeta. Since ERbeta functions to antagonize AR function and AR-dependent proliferation, these findings reveal a novel mechanism by which BPA likely regulates cellular proliferation. Future investigation directed at dissecting the importance of ERbeta in the proliferative response to BPA will establish the contribution of this event to adverse effects associated with human exposure.