Jenny M. Phillips

Highly purified Th17 cells from BDC2.5NOD mice convert into Th1-like cells in NOD/SCID recipient mice

Th17 cells are involved in the pathogenesis of many autoimmune diseases, but it is not clear whether they play a pathogenic role in type 1 diabetes. Here we investigated whether mouse Th17 cells with specificity for an islet antigen can induce diabetes upon transfer into NOD/SCID recipient mice. Induction of diabetes in NOD/SCID mice via adoptive transfer of Th1 cells from BDC2.5 transgenic mice was prevented by treatment of the recipient mice with a neutralizing IFN-γ-specific antibody. This result suggested a major role of Th1 cells in the induction of disease in this model of type 1 diabetes. Nevertheless, transfer of highly purified Th17 cells from BDC2.5 transgenic mice caused diabetes in NOD/SCID recipients with similar rates of onset as in transfer of Th1 cells. However, treatment with neutralizing IL-17-specific antibodies did not prevent disease. Instead, the transferred Th17 cells, completely devoid of IFN-γ at the time of transfer, rapidly converted to secrete IFN-γ in the NOD/SCID recipients. Purified Th17 cells also upregulated Tbet and secreted IFN-γ upon exposure to IL-12 in vitro and in vivo in NOD/SCID recipients. These results indicate substantial plasticity of Th17 commitment toward a Th1-like profile.

Validated germline-competent embryonic stem cell lines from nonobese diabetic mice

Nonobese diabetic (NOD) mice provide an excellent model of type 1 diabetes. The genetic contribution to this disease is complex, with more than 20 loci implicated in diabetes onset. One of the challenges for researchers using the NOD mouse model (and, indeed, other models of spontaneous autoimmune disease) has been the high density of sequence variation within candidate chromosomal segments. Furthermore, the scope for analyzing many putative disease loci via gene targeting has been hampered by the lack of NOD embryonic stem (ES) cells. We describe here the derivation of NOD ES cell lines capable of generating chimeric mice after stable genetic modification. These NOD ES cell lines also show efficient germline transmission, with offspring developing diabetes. The availability of these cells will not only enable the dissection of closely linked loci and the role they have in the onset of type 1 diabetes but also facilitate the generation of new transgenics.

Diabetes in non-obese diabetic mice is not associated with quantitative changes in CD4+ CD25+ Foxp3+ regulatory T cells

The role of regulatory T cells (Tregs) in maintaining self tolerance has been intensively researched and there is a growing consensus that a decline in Treg function is an important step towards the development of autoimmune diseases, including diabetes. Although we show here that CD25+ cells delay diabetes onset in non‐obese diabetic (NOD) mice, we found, in contrast to previous reports, neither an age‐related decline nor a decline following onset of diabetes in the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. Furthermore, we demonstrate that CD4+ CD25+ cells from both the spleen and pancreatic draining lymph nodes of diabetic and non‐diabetic NOD mice are able to suppress the proliferation of CD4+ CD25– cells to a similar extent in vitro. We also found that pretreatment of NOD mice with anti‐CD25 antibody allowed T cells with a known reactivity to islet antigen to proliferate more in the pancreatic draining lymph nodes of NOD mice, regardless of age. In addition, we demonstrated that onset of diabetes in NOD.scid mice is faster when recipients are co‐administered splenocytes from diabetic NOD donors and anti‐CD25. Finally, we found that although diabetic CD4+ CD25+ T cells are not as suppressive in cotransfers with effectors into NOD.scid recipients, this may not indicate a decline in Treg function in diabetic mice because over 10% of CD4+ CD25+ T cells are non‐Foxp3 and the phenotype of the CD25– contaminating population significantly differs in non‐diabetic and diabetic mice. This work questions whether onset of diabetes in NOD mice is associated with a decline in Treg function.

α-Cell Neogenesis in an Animal Model of IDDM

Currently there is debate regarding the capacity of pancreatic islets to regenerate in adult animals. Because pancreatic endocrine cells are thought to arise from duct cells, we examined the pancreatic ductal epithelium of the diabetic NOD mouse for evidence of islet neogenesis. We have evidence of duct proliferation as well as ductal cell differentiation, as suggested by bromodeoxyuridine-labeling and the presence of glucagon-containing cells within these ducts. In addition, the ductal epithelia in diabetic NOD mice expressed the neuroendocrine markers neuropeptide Y and tyrosine hydroxylase. These ducts also expressed the homeobox gene product, insulin promoter factor 1. Ductal cell proliferation and expression of these markers was not observed in transgenic NOD mice (NOD-E), which do not develop clinical or histopathological symptoms of IDDM. This suggests that the observed ductal cell proliferation and differentiation was a direct result of β-cell destruction and insulin insufficiency in these adult diabetic mice, which further suggests that these events are recapitulating islet ontogeny observed during embryogenesis. It is possible that comparable processes occur in the human diabetic pancreas.

Cutting Edge: Interactions Through the IL-10 Receptor Regulate Autoimmune Diabetes

BDC2.5/nonobese diabetic (NOD) transgenic mice express a TCR from a diabetogenic T cell clone yet do not spontaneously develop diabetes at high incidence. Evidence exists showing that in the absence of endogenous TCR α-chain rearrangements this transgenic mouse spontaneously develops diabetes and that CTLA-4 negatively regulates diabetes onset. This strongly suggests that onset of diabetes in BDC2.5/NOD mice is governed by T cell regulation. We addressed the mechanism of immune regulation in BDC2.5/NOD mice. We find that activated spleen cells from young, but not old, BDC2.5/NOD mice are able to transfer diabetes to NOD-scid recipients. We have used anti-IL-10R to show that the failure of splenocytes from older mice to transfer diabetes is due to dominant regulation. We furthermore found that diabetes developed following anti-IL-10R treatment of 6-wk old BDC2.5/NOD mice indicating that endogenous IL-10 plays a key role in the regulation of diabetes onset in this transgenic mouse.

Patients with chronic pancreatitis have islet progenitor cells in their ducts, but reversal of overt diabetes in NOD mice by Anti-CD3 shows no evidence for islet regeneration

Monoclonal antibodies to T-cell coreceptors have been shown to tolerise autoreactive T-cells and prevent or even reverse autoimmune pathology. In type 1 diabetes, there is a loss of insulin-secreting β-cells, and a cure for type 1 diabetes would require not only tolerance induction but also recovery of the functional β-cell mass. Although we have previously shown that diabetic mice have increased numbers of ductal progenitors in the pancreas, there is no evidence of any increase of insulin-secreting cells in the ducts. In contrast, in the adult human pancreas of patients with chronic pancreatitis, we can demonstrate, in the ducts, increased numbers of insulin-containing cells, as well as cells containing other endocrine and exocrine markers. There are also significantly increased numbers of cells expressing the homeodomain protein, pancreatic duodenal homeobox-1. Anti-CD3 has been shown to reverse overt diabetes in NOD mice; thus, we have used this model to ask whether monoclonal antibody–mediated inhibition of ongoing β-cell destruction enables islet regeneration to occur. We find no evidence that such monoclonal antibody therapy results in either regeneration of insulin-secreting β-cells or of increased proliferation of islet β-cells.

Epigenetic Changes at Il12rb2 and Tbx21 in Relation to Plasticity Behavior of Th17 Cells

Plasticity within Th cell populations may play a role in enabling site-specific immune responses to infections while limiting tissue destruction. Epigenetic processes are fundamental to such plasticity; however, to date, most investigations have focused on in vitro-generated T cells. In this study, we have examined the molecular mechanisms underpinning murine Th17 plasticity in vivo by assessing H3K4 and H3K27 trimethylation marks at Tbx21, Rorc, Il17a, Ifng, and Il12rb2 loci in purified ex vivo-isolated and in vitro-generated Th17 cells. Although both populations had largely comparable epigenetic signatures, including bivalent marks at Tbx21, freshly isolated ex vivo Th17 cells displayed restricted expression from Il12rb2 due to the presence of repressive chromatin modifications. This receptor, however, could be upregulated on isolated ex vivo Th17 cells after in vitro activation or by in vivo immunization and was augmented by the presence of IFN-γ. Such activated cells could then be deviated toward a Th1-like profile. We show that IL-12 stimulation removes H3K27 trimethylation modifications at Tbx21/T-bet leading to enhanced T-bet expression with in vitro Th17 cells. Our study reveals important potential phenotypic differences between ex vivo- and in vitro-generated Th17 cells and provides mechanistic insight into Th17 cell plasticity.

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 μg up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor α was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon γ, IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R+, CD4+ or CD8+ cells were found in the lymph nodes after therapy, but one patient showed activated CD8+ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8+ T cell activation and cytokine release, without CD4+ T cell activation.

Tolerogenic strategies to halt or prevent type 1 diabetes

A variety of therapeutic strategies have been developed to tolerize autoreactive T cells and prevent autoimmune pathology. In terms of type 1 diabetes, prevention strategies can inhibit the priming and expansion of autoreactive T cells; however, a cure for diabetes would require tolerance to be established in the presence of primed effector cells together with replacement of the destroyed β cell mass. Replacement of β cells could be accomplished by transplantation of islets or stem cells or through islet regeneration. We will focus here on tolerogenic strategies that have been used to prevent onset of type 1 diabetes and discuss the potential for a cure.

alpha-Cell neogenesis in an animal model of IDDM.

Currently there is debate regarding the capacity of pancreatic islets to regenerate in adult animals. Because pancreatic endocrine cells are thought to arise from duct cells, we examined the pancreatic ductal epithelium of the diabetic NOD mouse for evidence of islet neogenesis. We have evidence of duct proliferation as well as ductal cell differentiation, as suggested by bromodeoxyuridine-labeling and the presence of glucagon-containing cells within these ducts. In addition, the ductal epithelia in diabetic NOD mice expressed the neuroendocrine markers neuropeptide Y and tyrosine hydroxylase. These ducts also expressed the homeobox gene product, insulin promoter factor 1. Ductal cell proliferation and expression of these markers was not observed in transgenic NOD mice (NOD-E), which do not develop clinical or histopathological symptoms of IDDM. This suggests that the observed ductal cell proliferation and differentiation was a direct result of beta-cell destruction and insulin insufficiency in these adult diabetic mice, which further suggests that these events are recapitulating islet ontogeny observed during embryogenesis. It is possible that comparable processes occur in the human diabetic pancreas.