Abstract/Flash Poster Session A

 
We recently established that hybrid insulin peptides (HIPs), formed spontaneously in islet beta-cells by fusion of insulin C-peptide fragments to peptides of Chromogranin A (ChgA) or Islet Amyloid Polypeptide (IAPP), are ligands for diabetogenic CD4 T cell clones. The goal of this study was to investigate whether HIP-reactive T cells were indicative of ongoing autoimmunity in human T1D patients. To investigate their relevance to human disease, we used IFN-gamma ELISPOT to determine whether HIP-reactive T cells with an inflammatory phenotype were present in PBMCs of new onset T1D patients. We observed that PBMCs from new onset T1D patients responded to 8 of 16 HIPs tested and that nearly half of the patients tested responded to one or more HIPs. We used a CFSE based assay to further characterize T cells reactive to HIP peptides. CD4+CFSEdimCD25+ T cells were sorted single into single wells using FACS and cloned. T cell clonality was confirmed using TCR deep sequencing and T cell reactivity to specific HIPs was confirmed by ELISA. A total of 6 T cell clones were isolated and show a variety of TCR usage. One T cell clone was isolated from the same patient on two different blood draws, indicating a high frequency of this T cell clone in the peripheral blood. While all the T cell clones isolated secreted IFN-gamma in response to HIPs, they also produced other inflammatory cytokines such as TNF-alpha and GM-CSF and reacted to HIPs in the low nanomolar range. Our findings provide new evidence that HIP-reactive T cells are critical players in the pathogenesis of T1D and indicate that HIP-reactive T cells present in the peripheral blood can potentially serve as a biomarker of disease in a subset of patients.
 

Multiple studies suggest that the strength of the T cell receptor (TCR) signal leading to regulatory T cell (Treg) development and function is unique and distinct from effector T cells (Teffs). However, functional implications of a unique Treg TCR repertoire in autoimmune diabetes are largely unknown. We have recently shown that the strength of TCR signaling in islet infiltrating Tregs modulates their functional profile and regulatory capacity. We hypothesize that distinct TCR repertoires of islet infiltrating Tregs have intrinsic functional differences, and this leads to differential signaling between Teffs and Tregs. Consistent with this hypothesis, islet infiltrating insulin specific Tregs exhibit increased TCR signaling, based on Nur77-GFP reporter of TCR activation.To address this hypothesis, we are characterizing TCR repertoire, function, and signaling characteristics of islet infiltrating Treg TCRs.We were able to obtain the profile of Treg and Teff TCR repertoires specific for a single epitope by isolating and sequencing insulin tetramer binding Teffs and Tregs from islets of NOD mice expressing a fixed alpha chain of an insulin specific TCR. In this epitope-focused approach, sequencing data showed relatively high similarity (Morisita-Horn index: 0.27) between Treg and Teff TCR repertoires, where a substantial portion (46.4%) of Tregs expressed Teff TCRs. Current work is focused on comparing the in vitro and in vivo functional differences between Treg and Teff derived TCRs. Our preliminary data suggests that Treg TCRs can span a wide range of reactivity to insulin, but are more tolerant of changes at the N- and C-terminus of peptide epitope. Collectively, our data suggest that Treg and Teffs specific for a single epitope can share TCRs; however, Treg development and function is associated with increased TCR signaling and increased potential for antigenic promiscuity. 

Background. Tolerogenic vaccinations using beta-cell antigens (Ags) targeted by autoimmune T-cells are attractive for T1D prevention due to their selectivity and safety, but have not hold their promise in clinical trials. This is probably due to the late time of intervention, once the autoimmune reaction is already on going. We therefore devised a strategy to introduce the triggering Ag preproinsulin (PPI) during neonatal life, when autoimmunity is still silent and central tolerance mechanisms, which remain therapeutically unexploited, are more active. This strategy takes advantage of an Fc-fused PPI (PPI-Fc), which can be transported across the intestinal epithelium through the neonatal Fc receptor (FcRn), the pathway that physiologically delivers maternal antibodies to the offspring.
Methods. PPI-Fc was orally administered to 1-day-old G9C8 NOD mice, which are transgenic for a TCR recognizing the PPI-B15-23 epitope. PPI-Fc transfer through the gut epithelium was documented by in vivo imaging, confocal microscopy and ELISA. Its cellular uptake by lamina propria Ag-presenting cells and subsequent effects on pathogenic and regulatory T-cells were followed along with diabetes incidence at the adult age.
Results. One single PPI-Fc oral dose prevented diabetes in G9C8 NOD mice. PPI-Fc was efficiently transferred through the gut epithelium via the FcRn pathway. It was taken up by macrophages and migratory dendritic cells in the lamina propria and reached the thymus. This bio-distribution was associated with a decrease in PPI-reactive CD8+ T-cells and an increase in thymic-derived CD4+ Tregs 4 weeks after treatment. Oral PPI-Fc vaccination proved superior to Fc-devoid PPI at all these steps and may represent a novel strategy for T1D prevention in auto-antibody-positive and genetically at-risk relatives.

Background
The CRISPR-Cas9 system has been developed into a phenotype screening strategy. Using lentiviral delivery of a large collection of gRNAs, thousands of genes can be targeted for Cas9-mediated disruption in mammalian cells. Growing evidence supports ER stress as a target to prevent the initiation of the autoimmune reaction, propagation of inflammation, and b cell death in type 1 diabetes.
 
Methods
We used a genome-wide library of more than 60,000 gRNAs to perform a loss-of-function screen in NIT-1 beta cells. We implanted mutant NIT-1 cells into NOD.scid mice in which we also transplanted splenocyte from diabetic NOD mice. We recovered surviving NIT-1 beta cells two months after implantation and extracted genomic DNA from explanted cells to measure gRNA enrichment by next-generation sequencing.
 
Results
We identified 13 gRNAs covering 12 genes in beta cells that survived autoimmunity in transplanted mice. Several of these gene mutations conferred resistance to ER stress-induced cell death, and one mutation abrogated both CD4+ and CD8+ T cell activation by NIT-1 cell stimulation in vitro. Significantly, two of the genes identified in this genome-wide screen have been associated with human diabetes by GWAS.
We have identified a tractable number of candidate genes whose disruption extended the survival of beta cells in mice with autoimmune diabetes. Two of these candidate genes are associated with human diabetes, suggesting that our screening strategy discovered relevant targets for the protection of beta cells in type 1 diabetes.

Background: The reeducation of the immune system is crucial to tackle autoimmunity in type 1 diabetes (T1D). Since self–tolerance is induced by apoptotic cell clearance (a process known as efferocytosis), a nanotherapy consisting of liposomes rich in phosphatidylserine (PS) encapsulating beta–cell autoantigens was designed to mimic apoptotic beta–cells. This strategy halted autoimmunity and prevented experimental T1D in the non–obese diabetic (NOD) model through the generation of tolerogenic dendritic cells (DCs). To move forward, this study aimed to validate the effect of PS–liposomes in human DCs. Methods: PS–liposomes loaded with human insulin peptides as autoantigens were generated with optimum size and composition for DCs phagocytosis. Human DCs were derived in vitro from peripheral blood monocytes from adult and paediatric patients with T1D. Phagocytosis kinetics of PS–liposomes as well as the changes induced in DCs’ phenotype, function and transcriptome were determined. Results: Human DCs displayed optimal phagocytosis kinetics of PS–liposomes, and their rapid engulfment was dependent on the presence of PS. Remarkably, DCs from paediatric patients at T1D onset captured liposomes more efficiently than DCs from paediatric patients 1–3 years after diagnosis. DCs’ viability was unaffected by PS–liposomes engulfment, although the phenotypic profile was modulated, as evidenced by a decreased expression of efferocytosis receptors and preserved low levels of antigen-presenting, activation and costimulatory molecules. After PS–liposomes uptake, DCs showed anti–inflammatory cytokine secretion and impaired ability to stimulate autologous T lymphocyte proliferation. Furthermore, transcriptomic analysis revealed differential expression of immunoregulatory genes in DCs after PS–liposomes engulfment, indicating tolerance induction. Conclusion: Phagocytosis of apoptotic–mimicking PS–liposomes induced similar effects in DCs from adult and paediatric patients with T1D, thus validating the tolerogenic effect prompted in NOD mice. As PS–liposomes call for apoptotic cell clearance, a physiological and memory–inducing approach, these results reinforce the potential of this biomimicry–based antigen–specific immunotherapy to restablish tolerance to self.

 
Background. Type 1 diabetes (T1D) is an autoimmune disease in which the immune system destroys insulin-producing pancreatic beta-cells. Although pro-insulin is probably the primary autoantigen, other non-beta-cell specific proteins behave as autoantigens. Data indicate that such proteins can become beta-cell specific target antigens when posttranslationally modified, as this could explain their escape from thymic tolerization. We have previously demonstrated that GRP78 is citrullinated upon inflammatory stress and is recognized as an autoantigen in non-obese diabetic (NOD) mice. Moreover, first evidence of autoreactivity against citrullinated GRP78 epitope 292-305 (292-305X) was shown in CD4+ T-cell outgrowth from an islet of a T1D patient. The aim of this project was to investigate whether this GRP78 peptide 292-305X can induce tolerance in NOD mice.
Methods. 3-week old pre-diabetic female NOD mice (n=17) were injected s.c. with 50µg of 292-305X (emulsified 1:1 in IFA) and followed-up for 26 weeks. As controls, mice were injected with the native peptide (292-305R in IFA, n=12) or IFA alone (n=17).
Results. Diabetes incidence was decreased in the group injected with 292-305X (18%) when compared with the IFA group (53%, p p<0.05) and the group vaccinated with the native peptide (67%, p<0.01). At 26 weeks of age, mice vaccinated with 292-305X showed a trend of reduced insulitis as compared to controls. The percentages of CD4+ and CD8+ effector memory cells (CD62L- CD44+) in the spleen, pancreatic lymph nodes and pancreas did not significantly differ between the groups. However, a significant increase in percentage of regulatory T cells (CD4+ CD25+ Foxp3+) was observed in pancreatic lymph nodes of protected mice vaccinated with 292-305X (p<0.05), as compared to the IFA group. These results indicate that peptide 292-305X can induce tolerance in pre-diabetic NOD mice by increasing regulatory T-cell frequencies. These findings may open new pathways for the development of therapeutic approaches for T1D.

Neonatal period is a critical window when central tolerance is established in an Autoimmune Regulator (Aire). However, not all self-antigens are expressed downstream of Aire and tolerance to some tissue restricted antigens might rely in part on peripheral dendritic cells (DCs). The relative contribution of thymically and peripherally derived antigens in the generation of neonatal β cell antigen specific CD4+ regulatory T cells (Tregs) is not well understood. We have recently shown ectopic expression of Insulin B9-23 and the insulin mimetope R22E within antigen presenting cells early in thymocyte development can protect mice from developing Type 1 Diabetes (T1D) through central tolerance mechanisms. However, it remains to be determined whether peripherally derived neo-antigens such as post-translationally modified (PTM) peptides, may also be transported to the thymus to impact thymocyte development. Pathogenic T cells that are specific for neo-antigens uniquely expressed in the pancreas may escape thymic selection. Therefore, we examined the role of a known PTM peptide, the hybrid insulin/chromogranin A (2.5HIP) peptide, on neonatal development of β cell specific T cells and Tregs. We found that adoptively transferred DCs pulsed with ChgA peptides into neonatal and adult BDC2.5 TCR Tg and WT NOD mice readily migrate to the thymus and impact the development of ChgA specific T cells. Additionally, we found targeting thymic Langerin+ dendritic cells with anti-Langerin linked to ChgA peptides also altered T cell development. In both experimental approaches, we found a robust increase in the ratio and number of ChgA specific Tregs in the presence of 2.5HIP PTM while also observing an increase in negative selection. Co-administration of exogenous antibody/IL-2 complexes greatly enhanced the frequency and number of thymic Foxp3+ Tregs. Together our data suggests neonatal exposure to PTM peptides can efficiently drive thymic development of β cell specific Tregs and may alter T1D pathogenesis.

Background
Inflammatory cytokines, particularly the interferons, have been implicated in the pathogenesis of type 1 diabetes (T1D). Interferon-stimulated genes, including those that encode MHC proteins, are pathological hallmarks of the pancreatic islet during the development of T1D in both humans and the non-obese diabetic (NOD) mouse model. Further, many studies have shown that interferons promote the destructive interaction between antigen-specific CD8+ T cells and the beta cell. Surprisingly, deficiency of individual interferons or their receptors in NOD mice does not protect against the development of T1D, possibly due to signaling redundancy amongst the three receptors for type I, II and III interferons.
Methods
We used CRISPR/Cas9 mutagenesis to generate NOD mice that lack in all three interferon receptors (Ifnar1, Ifngr1 and Ifnlr1).
Results
Remarkably, while development of insulitis in the triple interferon receptor knockout NOD mice was delayed, mice developed diabetes with the same frequency as wild-type NOD mice. This result indicated that the lack of protection observed in triple knockout NOD mice was not due to functional redundancy between the three receptors. The frequency of islet specific autoreactive T cells was increased in these mice. Furthermore, they did not express PD-L1 on beta cells, and were resistant to diabetes acceleration by anti-PD-L1 antibody treatment, suggesting that normal interferon regulatory pathways were inhibited in these mice.
Conclusions
Collectively, these results indicate that in addition to the proinflammatory role of interferons they also are important for ‘adaptive tolerance’ that limits the action of pathogenic T cells. Hence, we observed a ‘neutral’ effect on progression of diabetes in NOD mice lacking interferon receptors. Loss of interferon-dependent adaptive tolerance appears to explain the lack of effect of interferon neutralisation on diabetes frequency.
 

Background Recently, we reported a novel β cell population with compromised β cell signatures that are able to survive and proliferate as a result of autoimmunity during the disease progression in NOD mice. The intrinsic vulnerability of β cells to ER stress and oxidative stress-induced dysfunction was noted in previous reports, in this study we are interrogating the possible role of the novel β cells in feeding the intra-islet inflammation through IL-6 amplifier, which leads to chemokines production, immune cells accumulation and β cells destruction.
Methods RNA-seq was performed to profile the difference between the normal and novel β cells. NOD mice was followed and analyzed for Tet methylcytosine dioxygenase 2 (Tet2) induction in β cells during the disease progression. Mouse and human islets were analyzed in vitro culture for IL-6 induction in β cells. Tet2 deficient mice were used to study the Tet2-IL6 axis in β cells.
Results Our RNA-seq data from NOD mice show that the novel β cells produce significantly more chemokines than the normal β cells. Tet2 can be induced in both human and mice islets culture with cytokines as well as in NOD mice during disease progression, in which Tet2 is consistently higher in normal β than the novel β cells. Cytokines present in the inflamed islets can synergistically induce IL-6 production in β cells, the level of which is even higher when Tet2 itself or Tet2 activity is absent in either mice or human islets.
In summary, we postulated that Tet2 can target IL-6 and tame the inflammation in β cells. The lack of Tet2-IL6 regulation in the novel β cells might account for high IL-6 production that feed-forward through the inflammation amplifier that accumulates immune cells and destruct real β cells.