Anne Eugster

Effects of High-Dose Oral Insulin on Immune Responses in Children at High Risk for Type 1 Diabetes: The Pre-POINT Randomized Clinical Trial

IMPORTANCE Exposing the oral mucosa to antigen may stimulate immune tolerance. It is unknown whether treatment with oral insulin can induce a tolerogenic immune response in children genetically susceptible to type 1 diabetes. OBJECTIVE To assess the immune responses and adverse events associated with orally administered insulin in autoantibody-negative, genetically at-risk children. DESIGN, SETTING, AND PARTICIPANTS The Pre-POINT study, a double-blind, placebo-controlled, dose-escalation, phase 1/2 clinical pilot study performed between 2009 and 2013 in Germany, Austria, the United States, and the United Kingdom and enrolling 25 islet autoantibody-negative children aged 2 to 7 years with a family history of type 1 diabetes and susceptible human leukocyte antigen class II genotypes. Follow-up was completed in August 2013. INTERVENTIONS Children were randomized to receive oral insulin (n = 15) or placebo (n = 10) once daily for 3 to 18 months. Nine children received insulin with dose escalations from 2.5 to 7.5 mg (n = 3), 2.5 to 22.5 mg (n = 3), or 7.5 to 67.5 mg (n = 3) after 6 months; 6 children only received doses of 22.5 mg (n = 3) or 67.5 mg (n = 3). MAIN OUTCOMES AND MEASURES An immune response to insulin, measured as serum IgG and saliva IgA binding to insulin, and CD4+ T-cell proliferative responses to insulin. RESULTS Increases in IgG binding to insulin, saliva IgA binding to insulin, or CD4+ T-cell proliferative responses to insulin were observed in 2 of 10 (20% [95% CI, 0.1%-45%]) placebo-treated children and in 1 of 6 (16.7% [95% CI, 0.1%-46%]) children treated with 2.5 mg of insulin, 1 of 6 (16.7%[ 95% CI, 0.1%-46%]) treated with 7.5 mg, 2 of 6 (33.3% [95% CI, 0.1%-71%]) treated with 22.5 mg, and 5 of 6 (83.3% [ 95% CI, 53%-99.9%]) treated with 67.5 mg (P = .02). Insulin-responsive T cells displayed regulatory T-cell features after oral insulin treatment. No hypoglycemia, IgE responses to insulin, autoantibodies to glutamic acid decarboxylase or insulinoma-associated antigen 2, or diabetes were observed. Adverse events were reported in 12 insulin-treated children (67 events) and 10 placebo-treated children (35 events). CONCLUSIONS AND RELEVANCE In this pilot study of children at high risk for type 1 diabetes, daily oral administration of 67.5 mg of insulin, compared with placebo, resulted in an immune response without hypoglycemia. These findings support the need for a phase 3 trial to determine whether oral insulin can prevent islet autoimmunity and diabetes in such children. TRIAL REGISTRATION isrctn.org Identifier: ISRCTN76104595.

Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity

Summary Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery.

High Diversity in the TCR Repertoire of GAD65 Autoantigen-Specific Human CD4+ T Cells

Autoreactive CD4+ T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4+ T cells, and TCR α-chain next-generation sequencing to bulk memory CD4+ T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4+ T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer+ cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer+ CD4+ T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001–1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies.

Measuring t cell receptor and t cell gene expression diversity in antigen-responsive human cd4+ t cells

T cells have diversity in TCR, epitope recognition, and cytokine production, and can be used for immune monitoring. Furthermore, clonal expansion of TCR families in disease may provide opportunities for TCR-directed therapies. We developed methodology for sequencing expressed genes of TCR alpha and beta chains from single cells and applied this to vaccine (tetanus-toxoid)-responsive CD4(+) T cells. TCR alpha and beta chains were both successfully sequenced in 1309 (43%) of 3038 CD4(+) T cells yielding 677 different receptors. TRAV and TRBV gene usage differed between tetanus-toxoid-responsive and non-responsive cells (p=0.004 and 0.0002), and there was extensive TCR diversity in tetanus-toxoid-responsive cells within individuals. Identical TCRs could be recovered in different samples from the same subject: TCRs identified after booster vaccination were frequent in pre-booster memory T cells (31% of pre-booster TCR), and also identified in pre-booster vaccination naïve cells (6.5%). No TCR was shared between subjects, but tetanus toxoid-responsive cells sharing one of their TCR chains were observed within and between subjects. Coupling single-cell gene expression profiling to TCR sequencing revealed examples of distinct cytokine profiles in cells bearing identical TCR. Novel molecular methodology demonstrates extensive diversity of Ag-responsive CD4(+) T cells within and between individuals.

A divergent population of autoantigen-responsive CD4+ T cells in infants prior to β cell autoimmunity

A divergent autoantigen-responsive CD4+ T cell population is present in infancy, before autoimmunity in type 1 diabetes. Baby β cells in danger Susceptibility to type 1 diabetes involves genetic risk factors and potential environmental triggers, making it harder to predict which individuals will actually develop disease. Longitudinal samples collected starting shortly after birth from children at risk for type 1 diabetes were examined by Heninger et al. to scrutinize early immune responses. Some, but not all, of these children eventually developed autoimmunity; CD4+ T cells from those that progressed had a distinct phenotype after presentation of autoantigen, suggesting that they had already been somewhat activated, even before autoantibodies were detectable. These exciting findings indicate that cues that dictate type 1 diabetes development may be happening even earlier than expected, in infancy. Autoimmune diabetes is marked by sensitization to β cell self-antigens in childhood. We longitudinally followed at-risk children from infancy and performed single-cell gene expression in β cell antigen–responsive CD4+ T cells through pre- and established autoimmune phases. A striking divergence in the gene signature of β cell antigen–responsive naïve CD4+ T cells from children who developed β cell autoimmunity was found in infancy, well before the appearance of β cell antigen–specific memory T cells or autoantibodies. The signature resembled a pre–T helper 1 (TH1)/TH17/T follicular helper cell response with expression of CCR6, IL21, TBX21, TNF, RORC, EGR2, TGFB1, and ICOS, in the absence of FOXP3, IL17, and other cytokines. The cells transitioned to an IFNG-TH1 memory phenotype with the emergence of autoantibodies. We suggest that the divergent naïve T cell response is a consequence of genetic or environmental priming during unfavorable perinatal exposures and that the signature will guide future efforts to detect and prevent β cell autoimmunity.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

Allogeneic stem cell transplantation is potentially curative, but associated with post‐transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR‐α repertoire and its clinical relevance in patients following stem cell transplantation. Using next‐generation sequencing we examined the TCR‐α repertoire of CD8+ T cells and CMV‐specific CD8+ T cells in four patients. Additionally, we performed single‐cell TCR‐αβ sequencing of CMV‐specific CD8+ T cells. The TCR‐α composition of human leucocyte antigen (HLA)‐A*0201 CMVpp65– and CMVIE‐specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8+ T cells and half of the total CD8+ T cell repertoire was dominated by few CMV‐reactive clonotypes. Some TCR‐α clonotypes were shared between patients. Gene expression of the circulating CMV‐specific CD8+ T cells was consistent with chronically activated effector memory T cells. The CD8+ T cell response to CMV reactivation resulted in an expansion of a few TCR‐α clonotypes to dominate the CD8+ repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti‐viral T cell response in this setting.

Abundant CMV reactive clonotypes in the CD8+ T cell receptor alpha repertoire following allogeneic transplantation

Allogeneic stem cell transplantation is potentially curative, but associated with post‐transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR‐α repertoire and its clinical relevance in patients following stem cell transplantation. Using next‐generation sequencing we examined the TCR‐α repertoire of CD8+ T cells and CMV‐specific CD8+ T cells in four patients. Additionally, we performed single‐cell TCR‐αβ sequencing of CMV‐specific CD8+ T cells. The TCR‐α composition of human leucocyte antigen (HLA)‐A*0201 CMVpp65– and CMVIE‐specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8+ T cells and half of the total CD8+ T cell repertoire was dominated by few CMV‐reactive clonotypes. Some TCR‐α clonotypes were shared between patients. Gene expression of the circulating CMV‐specific CD8+ T cells was consistent with chronically activated effector memory T cells. The CD8+ T cell response to CMV reactivation resulted in an expansion of a few TCR‐α clonotypes to dominate the CD8+ repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti‐viral T cell response in this setting.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors

Islet-reactive CD8+ T cells are common in the periphery, but home to the pancreas preferentially in the context of type 1 diabetes. At home in the pancreas Type 1 diabetes (T1D) is associated with enrichment of autoreactive CD8+ T cells that target destruction of pancreatic islets. Culina et al. studied islet-reactive CD8+ T cells reactive to the zinc transporter 8186–194 (ZnT8186–194) and other islet epitopes in healthy individuals and T1D patients, which showed similar functionality and similar frequencies and naïve phenotypes in the peripheral circulation across both groups. In contrast, ZnT8186–194-reactive CD8+ T cells were enriched in the pancreas of T1D patients relative to healthy controls and showed cross-reactivity to an epitope from the commensal Bacteroides stercoris. These results indicate that incomplete central tolerance may allow the survival of these islet-reactive CD8+ T cells in the periphery, and that proinflammatory conditions in the islets can contribute to T1D progression. The human leukocyte antigen–A2 (HLA-A2)–restricted zinc transporter 8186–194 (ZnT8186–194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186–194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186–194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186–194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186–194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186–194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Incomplete immune response to coxsackie B viruses associates with early autoimmunity against insulin

Viral infections are associated with autoimmunity in type 1 diabetes. Here, we asked whether this association could be explained by variations in host immune response to a putative type 1 etiological factor, namely coxsackie B viruses (CVB). Heterogeneous antibody responses were observed against CVB capsid proteins. Heterogeneity was largely defined by different binding to VP1 or VP2. Antibody responses that were anti-VP2 competent but anti-VP1 deficient were unable to neutralize CVB, and were characteristic of children who developed early insulin-targeting autoimmunity, suggesting an impaired ability to clear CVB in early childhood. In contrast, children who developed a GAD-targeting autoimmunity had robust VP1 and VP2 antibody responses to CVB. We further found that 20% of memory CD4+ T cells responding to the GAD65247-266 peptide share identical T cell receptors to T cells responding to the CVB4 p2C30-51 peptide, thereby providing direct evidence for the potential of molecular mimicry as a mechanism for GAD autoimmunity. Here, we highlight functional immune response differences between children who develop insulin-targeting and GAD-targeting autoimmunity, and suggest that children who lose B cell tolerance to insulin within the first years of life have a paradoxical impaired ability to mount humoral immune responses to coxsackie viruses.

Different developmental histories of beta-cells generate functional and proliferative heterogeneity during islet growth

The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states.Βeta-cells have recently been shown to be heterogeneous with regard to morphology and function. Here, the authors show that β-cells in zebrafish switch from proliferative to functional states with increasing time since β-cell birth, leading to functional and proliferative heterogeneity.