H.-Michael Dosch

Normalization of obesity-associated insulin resistance through immunotherapy

Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4+ T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor Vα repertoires, suggesting antigen-specific expansion. CD4+ T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic interferon-γ (IFN-γ)-secreting T helper type 1 (TH1) cells, overwhelming static numbers of TH2 (CD4+GATA-binding protein-3 (GATA-3)+) and regulatory forkhead box P3 (Foxp3)+ T cells. CD4+ (but not CD8+) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through TH2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD3-specific antibody or its F(ab′)2 fragment, reduces the predominance of TH1 cells over Foxp3+ cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4+ T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.

TRPV1+ Sensory Neurons Control β Cell Stress and Islet Inflammation in Autoimmune Diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.

Obesity predisposes to Th17 bias

Obesity is associated with numerous inflammatory conditions including atherosclerosis, autoimmune disease and cancer. Although the precise mechanisms are unknown, obesity‐associated rises in TNF‐α, IL‐6 and TGF‐β are believed to contribute. Here we demonstrate that obesity selectively promotes an expansion of the Th17 T‐cell sublineage, a subset with prominent pro‐inflammatory roles. T‐cells from diet‐induced obese mice expand Th17 cell pools and produce progressively more IL‐17 than lean littermates in an IL‐6‐dependent process. The increased Th17 bias was associated with more pronounced autoimmune disease as confirmed in two disease models, EAE and trinitrobenzene sulfonic acid colitis. In both, diet‐induced obese mice developed more severe early disease and histopathology with increased IL‐17+ T‐cell pools in target tissues. The well‐described association of obesity with inflammatory and autoimmune disease is mechanistically linked to a Th17 bias.

Autoimmune islet destruction in spontaneous type 1 diabetes is not β-cell exclusive

Pancreatic islets of Langerhans are enveloped by peri-islet Schwann cells (pSC), which express glial fibrillary acidic protein (GFAP) and S100β. pSC-autoreactive T- and B-cell responses arise in 3- to 4-week-old diabetes-prone non-obese diabetic (NOD) mice, followed by progressive pSC destruction before detectable β-cell death. Humans with probable prediabetes generate similar autoreactivities, and autoantibodies in islet-cell autoantibody (lCA) –positive sera co-localize to pSC. Moreover, GFAP-specific NOD T-cell lines transferred pathogenic peri-insulitis to NOD/severe combined immunodeficient (NOD/SCID) mice, and immunotherapy with GFAP or S100β prevented diabetes. pSC survived in rat insulin promoter Iymphocytic choriomeningitis virus (rip–LCMV) glycoprotein/CD8+ T-cell receptorgp double-transgenic mice with virus-induced diabetes, suggesting that pSC death is not an obligate consequence of local inflammation and β-cell destruction. However, pSC were deleted in spontaneously diabetic NOD mice carrying the CD8+/8.3 T-cell receptor transgene, a T cell receptor commonly expressed in earliest islet infiltrates. Autoimmune targeting of pancreatic nervous system tissue elements seems to be an integral, early part of natural type 1 diabetes.

Effects of Exenatide Alone and in Combination With Daclizumab on β-Cell Function in Long-Standing Type 1 Diabetes

OBJECTIVE In patients with long-standing type 1 diabetes, we investigated whether improved β-cell function can be achieved by combining intensive insulin therapy with agents that may 1) promote β-cell growth and/or limit β-cell apoptosis and 2) weaken the anti–β-cell autoimmunity. RESEARCH DESIGN AND METHODS For this study, 20 individuals (mean age 39.5 ± 11.1 years) with long-standing type 1 diabetes (21.3 ± 10.7 years) were enrolled in this prospective open-label crossover trial. After achieving optimal blood glucose control, 16 subjects were randomized to exenatide with or without daclizumab. Endogenous insulin production was determined by repeatedly measuring serum C-peptide. RESULTS In 85% of individuals with long-standing type 1 diabetes who were screened for participation in this trial, C-peptide levels ≥0.05 ng/ml (0.02 nmol/l) were found. Residual β-cells responded to physiological (mixed-meal) and pharmacological (arginine) stimuli. During exenatide treatment, patients lost 4.1 ± 2.9 kg body wt and insulin requirements declined significantly (total daily dose on exenatide 0.48 ± 0.11 vs. 0.55 ± 0.13 units · kg−1 · day−1 without exenatide; P = 0.0062). No signs of further activation of the underlying autoimmune disease were observed. Exenatide delayed gastric emptying, suppressed endogenous incretin levels, but did not increase C-peptide secretion. CONCLUSIONS In long-standing type 1 diabetes, which remains an active autoimmune disease even decades after its onset, surviving β-cells secrete insulin in a physiologically regulated manner. However, the combination of intensified insulin therapy, exenatide, and daclizumab did not induce improved function of these remaining β-cells.

Analysis of T-Cell Assays to Measure Autoimmune Responses in Subjects With Type 1 Diabetes Results of a Blinded Controlled Study

Type 1 diabetes is a chronic autoimmune disease mediated by autoreactive T-cells. Several experimental therapies targeting T-cells are in clinical trials. To understand how these therapies affect T-cell responses in vivo, assays that directly measure human T-cell function are needed. In a blinded, multicenter, case-controlled study conducted by the Immune Tolerance Network, we tested responses in an immunoblot and T-cell proliferative assay to distinguish type 1 diabetic patients from healthy control subjects. Peripheral blood cells from 39 healthy control subjects selected for DR4 and 23 subjects with recently diagnosed type 1 diabetes were studied. Autoantibody responses were measured in serum samples. Positive responses in both assays were more common in peripheral blood mononuclear cells from new-onset type 1 diabetic patients compared with control subjects. The proliferative, immunoblot, and autoantibody assays had sensitivities of 58, 91, and 78% with specificities of 94, 83, and 85%, respectively. When cellular assays were combined with autoantibody measurements, the sensitivity of the measurements was 75% with 100% specificity. We conclude that cellular assays performed on peripheral blood have a high degree of accuracy in discriminating responses in subjects with type 1 diabetes from healthy control subjects. They may be useful for assessment of cellular autoimmune responses involved in type 1 diabetes.

Validity and Reproducibility of Measurement of Islet Autoreactivity by T-Cell Assays in Subjects With Early Type 1 Diabetes

OBJECTIVE Type 1 diabetes results from an immunemediated destruction of β-cells, likely to be mediated by T lymphocytes, but the sensitivity, specificity, and other measures of validity of existing assays for islet autoreactive T-cells are not well established. Such assays are vital for monitoring responses to interventions that may modulate disease progression. RESEARCH DESIGN AND METHODS We studied the ability of cellular assays to discriminate responses in patients with type 1 diabetes and normal control subjects in a randomized blinded study in the U.S. and U.K. We evaluated the reproducibility of these measurements overall and to individual analytes from repeat collections. RESULTS Responses in the cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays could differentiate patients from control subjects with odds ratios of 21.7, 3.44, and 3.36, respectively, with sensitivity and specificity as high as 74 and 88%. The class II tetramer and U.S. ELISPOT assays performed less well. Despite the significant association of the responses with type 1 diabetes, the reproducibility of the measured responses, both overall and individual analytes, was relatively low. Positive samples from normal control subjects (i.e., false positives) were generally isolated to single assays. CONCLUSIONS The cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays can distinguish responses from patients with type 1 diabetes and healthy control subjects. The limited reproducibility of the measurements overall and of responses to individual analytes may reflect the difficulty in detection of low frequency of antigen-specific T-cells or variability in their appearance in peripheral blood.

Increased T Cell Proliferative Responses to Islet Antigens Identify Clinical Responders to Anti-CD20 Monoclonal Antibody (Rituximab) Therapy in Type 1 Diabetes

Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of β-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of β-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20– and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated β-cell loss. The way in which these responses affect the disease course remains unknown.

ICA69null Nonobese Diabetic Mice Develop Diabetes, but Resist Disease Acceleration by Cyclophosphamide

ICA69 (islet cell Ag 69 kDa) is a diabetes-associated autoantigen with high expression levels in β cells and brain. Its function is unknown, but knockout of its Caenorhabditis elegans homologue, ric-19, compromised neurotransmission. We disrupted the murine gene, ica-1, in 129-strain mice. These animals aged normally, but speed-congenic ICA69null nonobese diabetic (NOD) mice developed mid-life lethality, reminiscent of NOD-specific, late lethal seizures in glutamic acid decarboxylase 65-deficient mice. In contrast to wild-type and heterozygous animals, ICA69null NOD congenics fail to generate, even after immunization, cross-reactive T cells that recognize the dominant Tep69 epitope in ICA69, and its environmental mimicry Ag, the ABBOS epitope in BSA. This antigenic mimicry is thus driven by the endogenous self Ag, and not initiated by the environmental mimic. Insulitis, spontaneous, and adoptively transferred diabetes develop normally in ICA69null NOD congenics. Like glutamic acid decarboxylase 65, ICA69 is not an obligate autoantigen in diabetes. Unexpectedly, ICA69null NOD mice were resistant to cyclophosphamide (CY)-accelerated diabetes. Transplantation experiments with hemopoietic and islet tissue linked CY resistance to ICA69 deficiency in islets. CY-accelerated diabetes involves not only ablation of lymphoid cells, but ICA69-dependent drug toxicity in β cells that boosts autoreactivity in the regenerating lymphoid system.

Peptide Dose, MHC Affinity, and Target Self-Antigen Expression Are Critical for Effective Immunotherapy of Nonobese Diabetic Mouse Prediabetes

Cross-reactive T cells that recognize both Tep69 (dominant nonobese diabetic (NOD) T cell epitope in ICA69 (islet cell autoantigen of 69 kDa)) and ABBOS (dominant NOD T cell epitope in BSA) are routinely generated during human and NOD mouse prediabetes. Here we analyzed how systemic administration of these mimicry peptides affects progressive autoimmunity in adoptively transferred and cyclophosphamide-accelerated NOD mouse diabetes. These models were chosen to approximate mid to late stage prediabetes, the typical status of probands in human intervention trials. Unexpectedly, high dose (100 μg) i.v. ABBOS prevented, while Tep69 exacerbated, disease in both study models. Peptide effects required cognate recognition of endogenous self-Ag, because both treatments were ineffective in ICA69null NOD congenic mice adoptively transferred with wild-type, diabetic splenocytes. The affinity of ABBOS for NOD I-Ag7 was orders of magnitude higher than that of Tep69. This explained 1) the expansion of the mimicry T cell pool following i.v. Tep69, 2) the long-term unresponsiveness of these cells after i.v. ABBOS, and 3) precipitation of the disease after low dose i.v. ABBOS. Disease precipitation and prevention in mid to late stage prediabetes are thus governed by affinity profiles and doses of therapeutic peptides. ABBOS or ABBOS analogues with even higher MHC affinity may be candidates for experimental intervention strategies in human prediabetes, but the dose translation from NOD mice to humans requires caution.