Sefina Arif

Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health

According to the quality of response they mediate, autoreactive T cells recognizing islet beta cell peptides could represent both disease effectors in the development of type 1 diabetes (T1DM) and directors of tolerance in nondiabetic individuals or those undergoing preventative immunotherapy. A combination of the rarity of these cells, inadequate technology, and poorly defined epitopes, however, has hampered examination of this paradigm. We have identified a panel of naturally processed islet epitopes by direct elution from APCs bearing HLA-DR4. Employing these epitopes in a sensitive, novel cytokine enzyme-linked immunosorbent spot assay, we show that the quality of autoreactive T cells in patients with T1DM exhibits extreme polarization toward a proinflammatory Th1 phenotype. Furthermore, we demonstrate that rather than being unresponsive, the majority of nondiabetic, HLA-matched control subjects also manifest a response against islet peptides, but one that shows extreme T regulatory cell (Treg, IL-10-secreting) bias. We conclude that development of T1DM depends on the balance of autoreactive Th1 and Treg cells, which may be open to favorable manipulation by immune intervention.

CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

The final pathway of beta cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill beta cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide-specific CD8+ T cells killed human beta cells in vitro. Critically, at high glucose concentration, beta cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human beta cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing beta cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining beta cells.

Retargeting of Human T Cells to Tumor-Associated MUC1: The Evolution of a Chimeric Antigen Receptor

MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3ζ endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-γ and IL-17), and elicit brisk killing of MUC1+ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.

Peripheral and Islet Interleukin-17 Pathway Activation Characterizes Human Autoimmune Diabetes and Promotes Cytokine-Mediated β-Cell Death

OBJECTIVE CD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to β-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for β-cells. RESEARCH DESIGN AND METHODS Peripheral blood CD4 T-cell responses to β-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat β-cells, and the rat insulinoma cell line INS-1E were examined. RESULTS A total of 27 patients (54%) showed IL-17 reactivity to one or more β-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1β/interferon (IFN)-γ–induced and tumor necrosis factor (TNF)-α/IFN-γ–induced apoptosis in human islets, rat β-cells, and INS-1E cells, in association with significant upregulation of β-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB. CONCLUSIONS Circulating IL-17+ β-cell–specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to β-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target.

Proinsulin peptide immunotherapy in type 1 diabetes: report of a first‐in‐man Phase I safety study

Immunotherapeutic strategies under consideration for type 1 diabetes include modification of the autoimmune response through antigen‐specific routes. Administration of short peptides representing T cell epitopes targeted by patients with the disease represents one approach. This study evaluated safety and mechanistic outcomes during first‐in‐man intradermal administration of a human leucocyte antigen‐DR4 (HLA‐DR4)‐restricted peptide epitope of proinsulin (C19‐A3). This randomized, open‐label study assessed two major theoretical risks of peptide immunotherapy, namely induction of allergic hypersensitivity and exacerbation of the proinflammatory autoimmune response, using clinical assessment and mechanistic assays in vitro. Patients with long‐standing type 1 diabetes and HLA‐DRB1*0401 genotype received 30 µg (n = 18) or 300 µg (n = 18) of peptide in three equal doses at 0, 1 and 2 months or no intervention (n = 12). Proinsulin peptide immunotherapy in the dosing regimen used is well tolerated and free from risk of systemic hypersensitivity and induction/reactivation of proinsulin‐specific, proinflammatory T cells. Peptide‐specific T cells secreting the immune suppressive cytokine interleukin (IL)‐10 were observed at month 3 in four of 18 patients in the low‐dose group (versus one of 12 in the control group; P = not significant). Mean IL‐10 response to peptide in the low‐dose group increased between 0 and 3 months (P = 0·05 after stimulation with 5 µM peptide in vitro) and then declined to baseline levels between 3 and 6 months (P = 0·01 at 10 µM peptide in vitro). These studies pave the way for future investigations in new‐onset patients designed to examine whether proinsulin peptide immunotherapy has beneficial effects on markers of T cell autoimmunity and preservation of β cell mass.

Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes

Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ–positive, multiautoantibody-positive) and partially regulated (interleukin-10–positive, pauci-autoantibody–positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed “hyper-immune CD20Hi” and “pauci-immune CD20Lo.” Of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.

Posttranslational Modification of HLA-DQ Binding Islet Autoantigens in Type 1 Diabetes

Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis–restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-γ, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.

Naturally Arising Human CD4 T-Cells That Recognize Islet Autoantigens and Secrete Interleukin-10 Regulate Proinflammatory T-Cell Responses via Linked Suppression

OBJECTIVE Regulatory T-cells (Tregs) recognizing islet autoantigens are proposed as a key mechanism in the maintenance of self-tolerance and protection from type 1 diabetes. To date, however, detailed information on such cells in humans, and insight into their mechanisms of action, has been lacking. We previously reported that a subset of CD4 T-cells secreting high levels of the immunosuppressive cytokine interleukin-10 (IL-10) is significantly associated with late onset of type 1 diabetes and is constitutively present in a majority of nondiabetic individuals. Here, we test the hypothesis that these T-cells represent a naturally generated population of Tregs capable of suppressing proinflammatory T-cell responses. RESEARCH DESIGN AND METHODS We isolated and cloned islet-specific IL-10–secreting CD4+ T-cells from nondiabetic individuals after brief ex vivo exposure to islet autoantigens using cytokine capture technology and examined their phenotype and regulatory potential. RESULTS Islet-specific IL-10+ CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen-presenting cell (APC). The regulatory T-cells secrete perforin and granzymes, and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells. CONCLUSIONS This hitherto undescribed population of islet autoantigen–specific Tregs displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen β-cell–specific tolerance.

Multi-parametric flow cytometric and genetic investigation of the peripheral B cell compartment in human type 1 diabetes

The appearance of circulating islet‐specific autoantibodies before disease diagnosis is a hallmark of human type 1 diabetes (T1D), and suggests a role for B cells in the pathogenesis of the disease. Alterations in the peripheral B cell compartment have been reported in T1D patients; however, to date, such studies have produced conflicting results and have been limited by sample size. In this study, we have performed a detailed characterization of the B cell compartment in T1D patients (n = 45) and healthy controls (n = 46), and assessed the secretion of the anti‐inflammatory cytokine interleukin (IL)‐10 in purified B cells from the same donors. Overall, we found no evidence for a profound alteration of the B cell compartment or in the production of IL‐10 in peripheral blood of T1D patients. We also investigated age‐related changes in peripheral B cell subsets and confirmed the sharp decrease with age of transitional CD19+CD27−CD24hiCD38hi B cells, a subset that has recently been ascribed a putative regulatory function. Genetic analysis of the B cell compartment revealed evidence for association of the IL2–IL21 T1D locus with IL‐10 production by both memory B cells (P = 6·4 × 10−4) and islet‐specific CD4+ T cells (P = 2·9 × 10−3). In contrast to previous reports, we found no evidence for an alteration of the B cell compartment in healthy individuals homozygous for the non‐synonymous PTPN22 Trp620 T1D risk allele (rs2476601; Arg620Trp). The IL2–IL21 association we have identified, if confirmed, suggests a novel role for B cells in T1D pathogenesis through the production of IL‐10, and reinforces the importance of IL‐10 production by autoreactive CD4+ T cells.

Prevalence of autoantibodies to autonomic nervous tissue structures in Type 1 diabetes mellitus

Aims The pathogenesis of diabetic autonomic neuropathy is multifactorial, but recent studies have suggested a link between the presence of autoantibodies to nervous tissue structures and severe, symptomatic autonomic neuropathy. The present study was designed to examine the true prevalence of these autoantibodies in a large clinic‐based population of Type 1 diabetic patients compared to nondiabetic controls.