Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that ß-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles.

Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin.

The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPß/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPß/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic ß-cells with deficient expression of either TRAPß/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPß/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPß/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5 minutes. The proinsulin and insulin levels in TRAPß/SSR2 or TRAPδ/SSR4 null mutant ß-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in ß-cells with diminished expression of TRAPß/SSR2, strongly suggesting that TRAPß/SSR2 is needed to support TRAPα/SSR1 function.

A pilot study of preproinsulin peptides reactivity in Chinese patients with type 1 diabetes.

BACKGROUND: The aim of our study is to investigate whether preproinsulin (PPI) could trigger a proinflammatory CD4+ T cell response in Chinese patients with type 1 diabetes (T1D). METHODS: Peripheral blood mononuclear cells were stimulated by a pool of 13 PPI peptides. Additional five PPI peptides previously proved to be antigenic in other cohorts of patients with T1D were also used. PPI reactive T cell responses were measured by interferon (IFN)-γ ELISPOT assay. RESULTS: Fifty-one Chinese patients with T1D were enrolled in this study and 72.34% of them were positive for at least one islet autoantibody. The stimulation index (SI) value of IFN-γ response to PPI peptide pool or peptides with dominant epitopes was below 3 in patients when SI≥3 was used as the positive cut-off value. Two peptides (B9-23 and C19-A3) restricted to DQ8 or DR4 molecule failed to induce positive IFN-γ response in patients with high-risk HLA-DQ8 or HLA-DR4/DR9 alleles. RNA-seq analysis of PPI specific CD4+ T cell lines further showed that most of the IFN-γ associated genes remained unchanged. CONCLUSIONS: This is the first report of CD4+ T cell epitope mapping of PPI in Chinese T1D. The lack of positive IFN-γ response to PPI peptides indicates that PPI might not be the principal antigenic candidate for autoreactive CD4+ T cells in Chinese T1D. Therefore, the efficacy of PPI-based immunotherapies in attenuating proinflammatory CD4+ T cell response requires further investigation.

Pathophysiological characteristics of preproinsulin-specific CD8+ T cells in subjects with juvenile-onset and adult-onset type 1 diabetes: A 1-year follow-up study.

AIMS/HYPOTHESIS: Among the beta-cell associated antigens, preproinsulin (PPI) has been shown to play a key role in the pathogenesis of type 1 diabetes (T1D). PPI-specific autoreactive CD8+ T cells emerge early during beta-cell destruction and persist in peripheral circulation during diabetes progression. However, the influence of insulin therapy on phenotype of autoreactive CD8+ T cells in T1D including, juvenile-onset T1D (JOT1D), and adult-onset T1D (AOT1D) is not yet known. METHODS: We followed the time course of PPI-specific CD8+ T cells in JOT1D and AOT1D subjects that achieved glycemic control after 1 year of insulin therapy, using major histocompatibility complex-I (MHC-I) dextramers by flow cytometry. RESULTS AND DISCUSSION: At follow-up, PPI-specific CD8+ T cells could be detected consistently in peripheral blood of all T1D subjects. Proportion of PPI-specific effector memory (TEM ) subsets decreased, while central memory T (TCM ) cells remained unchanged in both groups. Expression of granzyme-B and perforin in PPI-specific CD8+ T cells also remained unchanged. Further, on analysis of B-chain and signal peptide (SP) specific CD8+ T cell responses separately, we again observed decrease in TEM subset in both the groups, while increase in naive (TN ) subset was observed in B-chain specific CD8+ T cells only. CONCLUSION: Our study shows that PPI-specific CD8+ T cells can be detected in both JOT1D and AOT1D subjects over a period of time with reliable consistency in frequency but variable pathophysiological characteristics. Insulin therapy seems to reduce the PPI-specific TEM subsets; however, the PPI-specific TCM cells continue to persist as attractive targets for immunotherapy.

Inefficient translocation of preproinsulin contributes to pancreatic ß cell failure and late-onset diabetes.

Among the defects in the early events of insulin biosynthesis, proinsulin misfolding and endoplasmic reticulum (ER) stress have drawn increasing attention as causes of ß cell failure. However, no studies have yet addressed potential defects at the cytosolic entry point of preproinsulin into the secretory pathway. Here, we provide the first evidence that inefficient translocation of preproinsulin (caused by loss of a positive charge in the n region of its signal sequence) contributes to ß cell failure and diabetes. Specifically, we find that, after targeting to the ER membrane, preproinsulin signal peptide (SP) mutants associated with autosomal dominant late-onset diabetes fail to be fully translocated across the ER membrane. The newly synthesized, untranslocated preproinsulin remains strongly associated with the ER membrane, exposing its proinsulin moiety to the cytosol. Rather than accumulating in the ER and inducing ER stress, untranslocated preproinsulin accumulates in a juxtanuclear compartment distinct from the Golgi complex, induces the expression of heat shock protein 70 (HSP70), and promotes ß cell death. Restoring an N-terminal positive charge to the mutant preproinsulin SP significantly improves the translocation defect. These findings not only reveal a novel molecular pathogenesis of ß cell failure and diabetes but also provide the first evidence of the physiological and pathological significance of the SP n region positive charge of secretory proteins.

Preproinsulin specific CD8+ T cells in subjects with latent autoimmune diabetes show lower frequency and different pathophysiological characteristics than those with type 1 diabetes.

Latent autoimmune diabetes in adults (LADA) resembles type 1 diabetes (T1D) in disease presentation except that its onset is slow. We compared pathophysiological characteristics of CD8+ T cells recognizing preproinsulin (PPI) derived epitopes in both disease groups using MHC-I dextramers (DMRs) in peripheral blood and after in-vitro stimulation with PPI. Subjects with T1D harbored higher frequency of DMR+ CD8+ T cells with relatively higher frequency of effector T cell subsets. Following stimulation with PPI, an increase in DMR+ CD8+ T cells, particularly the central-memory subset was observed in T1D group, whereas no significant change in DMR+ CD8+ T cell subsets was observed in LADA group. Intracellular expression of Granzyme-B and Perforin in DMR+ CD8+ T cells was comparable in both the groups. In conclusion, lower frequency and inferior proliferative potential on account of a relatively restrained central-memory subset of PPI specific CD8+ T cells are associated with slow rate of disease progression in LADA.

Plasma insulin levels are regulated by release, rather than transcription or translation, in barfin flounder, Verasper moseri.

We evaluated whether transcription or translation of the preproinsulin gene or insulin release into plasma is the primary regulator of plasma insulin level in barfin flounder. Three experimental groups were used: one tested 2h after feeding (Fed), one tested after fasting for 5 days (Fasted), and one tested 2 h after feeding following 5 days of fasting (Refed). No significant differences in insulin transcription, insulin concentrations in the principal islets (PI), or plasma total insulin-like growth factor-I (IGF-I) levels were observed between the three groups. In contrast, plasma insulin level in the Fasted group was significantly lower (P<0.002) than that in the other groups. These results suggest that insulin release is the primary regulator of plasma insulin level and is more sensitive to short-term changes in nutritional conditions than IGF-I level. Furthermore, we estimated the capacity for insulin release. Based on various individual measures, the average insulin stored in the PI was 82.8 µg/kg body weight (BW), and the maximum plasma content of insulin was estimated to be <1.7 µg/kg BW. The half-life of plasma insulin in diabetogenic chemically (alloxan) treated flounder injected with insulin was estimated to be 2.79 h, which is much longer than that in mammals, assuming a two-compartment model for the ß phase. These results suggest that the capacity for insulin release in fish is ensured by at least two systems, such as the ability to store excess insulin in Brockman bodies, and enhanced efficiency of insulin storage by elongating its half-life.

Pancreatic ß cell derived extracellular vesicles containing surface preproinsulin are involved in glucose stimulated insulin secretion.

AIMS: Extracellular vesicles (EVs) are involved in intercellular communication and are a topic of increasing interest due to their therapeutic potential. The aim of this study was to determine whether human islet-derived EVs contain insulin, and if so, what role do they play in glucose stimulated insulin secretion. MAIN METHODS: We isolated EVs from human islets culture and plasma to probe for insulin. Plasma from hyperglycemic glucose clamp experiments were also used to isolate and measure EV insulin content in response to a secretory stimulus. We performed immunogold electron microscopy for insulin presence in EVs. Co-culture experiments of isolated EVs with fresh islets were performed to examine the effect of EV cargo on insulin receptor signaling. KEY FINDINGS: EVs isolated from culture medium contained insulin. Glucose treatment of islets increased the level of EV insulin. Hyperglycemic glucose clamp experiments in humans also lead to increased levels of insulin in plasma-derived EVs. Immunogold electron microscopy and proteinase K-digestion experiments demonstrated that insulin in EVs predominantly associated with the exterior surface of EVs while western blot analyses uncovered the presence of only preproinsulin in EVs. Membrane-bound preproinsulin in EVs was capable of activating insulin signaling pathway in an insulin receptor-dependent manner. The physiological relevance of this finding was observed in priming of human naïve islets by EVs during glucose stimulated insulin secretion. SIGNIFICANCE: Our data suggest that (1) human islets secret insulin via an alternate pathway (EV-mediated) other than conventional granule-mediated insulin secretion, and (2) EV membrane bound preproinsulin is biologically active.

Recombinant production of hormonally active human insulin from pre-proinsulin by Tetrahymena thermophila.

Alternative cell factories, such as the unicellular ciliate eukaryotic Tetrahymena thermophila, may be required for the production of protein therapeutics that are challenging to produce in conventional expression systems. T. thermophila (Tt) can secrete proteins with the post-translational modifications necessary for their function in humans. In this study, we tested if T. thermophila could process the human pre-proinsulin to produce hormonally active human insulin (hINS) with correct modifications. Flask and bioreactor culture of T. thermophila were used to produce the recombinant Tt-hINS either with or without an affinity tag from a codon-adapted pre-proinsulin sequence. Our results indicate that T. thermophila can produce a 6 kDa Tt-hINS monomer with the appropriate disulfide bonds after removal of the human insulin signal sequence or endogenous phospholipase A signal sequence, and the C-peptide of the human insulin. Additionally, Tt-hINS can form 12 kDa dimeric, 24 kDa tetrameric, and 36 kDa hexameric complexes. Tt-hINS-sfGFP fusion protein was localized to the vesicles within the cytoplasm and was secreted extracellularly. Assessing the affinity-purified Tt-hINS activity using the in vivo T. thermophila extracellular glucose drop assay, we observed that Tt-hINS induced a significant reduction (approximately 21 %) in extracellular glucose levels, indicative of its functional insulin activity. Our results demonstrate that T. thermophila is a promising candidate for the pharmaceutical and biotechnology industries as a host organism for the production of human protein drugs.

The role of endoplasmic reticulum aminopeptidases in type 1 diabetes mellitus.

Type-I diabetes mellitus (T1DM) is generally considered as a chronic, T-cell mediated autoimmune disease. This notwithstanding, both the endogenous characteristics of ß-cells, and their response to environmental factors and exogenous inflammatory stimuli are key events in disease progression and exacerbation. As such, T1DM is now recognized as a multifactorial condition, with its onset being influenced by both genetic predisposition and environmental factors, among which, viral infections represent major triggers. In this frame, endoplasmic reticulum aminopeptidase 1 (ERAP1) and 2 (ERAP2) hold center stage. ERAPs represent the main hydrolytic enzymes specialized in trimming of N-terminal antigen peptides to be bound by MHC class I molecules and presented to CD8+ T cells. Thus, abnormalities in ERAPs expression alter the peptide-MHC-I repertoire both quantitatively and qualitatively, fostering both autoimmune and infectious diseases. Although only a few studies succeeded in determining direct associations between ERAPs variants and T1DM susceptibility/outbreak, alterations of ERAPs do impinge on a plethora of biological events which might indeed contribute to the disease development/exacerbation. Beyond abnormal self-antigen peptide trimming, these include preproinsulin processing, nitric oxide (NO) production, ER stress, cytokine responsiveness, and immune cell recruitment/activity. The present review brings together direct and indirect evidence focused on the immunobiological role of ERAPs in T1DM onset and progression, covering both genetic and environmental aspects.

Peripheral blood mononuclear cells reactivity in recent-onset type I diabetes patients is directed against the leader peptide of preproinsulin, GAD65271-285 and GAD65431-450.

Introduction: T cell reactivity against pancreatic autoantigens is considered one of the main contributors to the destruction of insulin-producing cells in type 1 diabetes (T1D). Over the years, peptide epitopes derived from these autoantigens have been described in NOD mice and in both HLA class II transgenic mice and humans. However, which ones are involved in the early onset or in the progressive phases of the disease is still unclear. Methods: In this work we have investigated, in early-onset T1D pediatric patients and HLA-matched controls from Sardinia, the potential of preproinsulin (PPI) and glutamate decarboxylase 65 (GAD65)-derived peptides to induce spontaneous T cell proliferation responses of peripheral blood mononuclear cells (PBMCs). Results: Significant T cell responses against PPI1-18, PPI7-19 and PPI31-49, the first two belonging to the leader sequence of PPI, and GAD65271-285 and GAD65431-450, were found in HLA-DR4, -DQ8 and -DR3, -DQ2 T1D children. Conclusions: These data show that cryptic epitopes from the leader sequence of the PPI and GAD65271-285 and GAD65431-450 peptides might be among the critical antigenic epitopes eliciting the primary autoreactive responses in the early phases of the disease. These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy.

Designing Personalized Antigen-Specific Immunotherapies for Autoimmune Diseases-The Case for Using Ignored Target Cell Antigen Determinants.

We have proposed that antigen-specific immunotherapies (ASIs) for autoimmune diseases could be enhanced by administering target cell antigen epitopes (determinants) that are immunogenic but ignored by autoreactive T cells because these determinants may have large pools of naïve cognate T cells available for priming towards regulatory responses. Here, we identified an immunogenic preproinsulin determinant (PPIL4-20) that was ignored by autoimmune responses in type 1 diabetes (T1D)-prone NOD mice. The size of the PPIL4-20-specific splenic naive T cell pool gradually increased from 2-12 weeks in age and remained stable thereafter, while that of the major target determinant insulin B-chain9-23 decreased greatly after 12 weeks in age, presumably due to recruitment into the autoimmune response. In 15-16 week old mice, insulin B-chain9-23/alum immunization induced modest-low level of splenic T cell IL-10 and IL-4 responses, little or no spreading of these responses, and boosted IFNγ responses to itself and other autoantigens. In contrast, PPIL4-20/alum treatment induced robust IL-10 and IL-4 responses, which spread to other autoantigens and increased the frequency of splenic IL-10-secreting Treg and Tr-1-like cells, without boosting IFNγ responses to ß-cell autoantigens. In newly diabetic NOD mice, PPIL4-20, but not insulin B-chain9-23 administered intraperitoneally (with alum) or intradermally (as soluble antigen) supplemented with oral GABA induced long-term disease remission. We discuss the potential of personalized ASIs that are based on an individual's naïve autoantigen-reactive T cell pools and the use of HLA-appropriate ignored autoantigen determinants to safely enhance the efficacy of ASIs.

A Novel Nonsense INS Mutation Causes Inefficient Preproinsulin Translocation Into the Endoplasmic Reticulum.

Preproinsulin (PPI) translocation across the membrane of the endoplasmic reticulum (ER) is the first and critical step of insulin biosynthesis. Inefficient PPI translocation caused by signal peptide (SP) mutations can lead to ß-cell failure and diabetes. However, the effect of proinsulin domain on the efficiency of PPI translocation remains unknown. With whole exome sequencing, we identified a novel INS nonsense mutation resulting in an early termination at the 46th residue of PPI (PPI-R46X) in two unrelated patients with early-onset diabetes. We examined biological behaviors of the mutant and compared them to that of an established neonatal diabetes causing mutant PPI-C96Y. Although both mutants were retained in the cells, unlike C96Y, R46X did not induce ER stress or form abnormal disulfide-linked proinsulin complexes. More importantly, R46X did not interact with co-expressed wild-type (WT) proinsulin in the ER, and did not impair proinsulin-WT folding, trafficking, and insulin production. Metabolic labeling experiments established that, despite with an intact SP, R46X failed to be efficiently translocated into the ER, suggesting that proinsulin domain downstream of SP plays an important unrecognized role in PPI translocation across the ER membrane. The study not only expends the list of INS mutations associated with diabetes, but also provides genetic and biological evidence underlying the regulation mechanism of PPI translocation.

Proinsulin-Reactive CD4 T Cells in the Islets of Type 1 Diabetes Organ Donors.

Proinsulin is an abundant protein that is selectively expressed by pancreatic beta cells and has been a focus for development of antigen-specific immunotherapies for type 1 diabetes (T1D). In this study, we sought to comprehensively evaluate reactivity to preproinsulin by CD4 T cells originally isolated from pancreatic islets of organ donors having T1D. We analyzed 187 T cell receptor (TCR) clonotypes expressed by CD4 T cells obtained from six T1D donors and determined their response to 99 truncated preproinsulin peptide pools, in the presence of autologous B cells. We identified 14 TCR clonotypes from four out of the six donors that responded to preproinsulin peptides. Epitopes were found across all of proinsulin (insulin B-chain, C-peptide, and A-chain) including four hot spot regions containing peptides commonly targeted by TCR clonotypes derived from multiple T1D donors. Of importance, these hot spots overlap with peptide regions to which CD4 T cell responses have previously been detected in the peripheral blood of T1D patients. The 14 TCR clonotypes recognized proinsulin peptides presented by various HLA class II molecules, but there was a trend for dominant restriction with HLA-DQ, especially T1D risk alleles DQ8, DQ2, and DQ8-trans. The characteristics of the tri-molecular complex including proinsulin peptide, HLA-DQ molecule, and TCR derived from CD4 T cells in islets, provides an essential basis for developing antigen-specific biomarkers as well as immunotherapies.

A Humanized Mouse Strain That Develops Spontaneously Immune-Mediated Diabetes.

To circumvent the limitations of available preclinical models for the study of type 1 diabetes (T1D), we developed a new humanized model, the YES-RIP-hB7.1 mouse. This mouse is deficient of murine major histocompatibility complex class I and class II, the murine insulin genes, and expresses as transgenes the HLA-A*02:01 allele, the diabetes high-susceptibility HLA-DQ8A and B alleles, the human insulin gene, and the human co-stimulatory molecule B7.1 in insulin-secreting cells. It develops spontaneous T1D along with CD4+ and CD8+ T-cell responses to human preproinsulin epitopes. Most of the responses identified in these mice were validated in T1D patients. This model is amenable to characterization of hPPI-specific epitopes involved in T1D and to the identification of factors that may trigger autoimmune response to insulin-secreting cells in human T1D. It will allow evaluating peptide-based immunotherapy that may directly apply to T1D in human and complete preclinical model availability to address the issue of clinical heterogeneity of human disease.

Oral Fc-Coupled Preproinsulin Achieves Systemic and Thymic Delivery Through the Neonatal Fc Receptor and Partially Delays Autoimmune Diabetes.

Tolerogenic vaccinations using beta-cell antigens are attractive for type 1 diabetes prevention, but clinical trials have been disappointing. This is probably due to the late timing of intervention, when multiple auto-antibodies are already present. We therefore devised a strategy to introduce the initiating antigen preproinsulin (PPI) during neonatal life, when autoimmunity is still silent and central tolerance mechanisms, which remain therapeutically unexploited, are more active. This strategy employs an oral administration of PPI-Fc, i.e. PPI fused with an IgG Fc to bind the intestinal neonatal Fc receptor (FcRn) that physiologically delivers maternal antibodies to the offspring during breastfeeding. Neonatal oral PPI-Fc vaccination did not prevent diabetes development in PPI T-cell receptor-transgenic G9C8.NOD mice. However, PPI-Fc was efficiently transferred through the intestinal epithelium in an Fc- and FcRn-dependent manner, was taken up by antigen presenting cells, and reached the spleen and thymus. Although not statistically significant, neonatal oral PPI-Fc vaccination delayed diabetes onset in polyclonal Ins2-/-.NOD mice that spontaneously develop accelerated diabetes. Thus, this strategy shows promise in terms of systemic and thymic antigen delivery via the intestinal FcRn pathway, but the current PPI-Fc formulation/regimen requires further improvements to achieve diabetes prevention.

Translocon-Associated Protein Complex (TRAP) is Crucial for Co-Translational Translocation of Pre-Proinsulin.

Many unanswered questions remain in understanding the biosynthesis of the peptide hormone insulin. Here we elucidate new aspects in the mechanism of co-translational translocation initiation of pre-proinsulin in the endoplasmic reticulum. We utilize a translational arrest peptide derived from the x-box-binding protein (Xbp1) to induce ribosomal stalling and generate translocation intermediates. We find that the insulin signal sequence is rather weakly gating and requires the assistance of auxiliary translocon components to initiate translocation. Probing the translational intermediates with chemical crosslinking, we identified an early interaction with the translocon-associated protein (TRAP) complex. The TRAPß subunit interacts with pre-proinsulin before the peptide enters the Sec61 translocon channel in a signal sequence-dependent manner. We describe the substrate sequence determinants that are recognized by TRAP on the cytosolic site of the membrane to facilitate substrate-specific opening of the Sec61 translocon channel. Our findings support the hypothesis that the TRAP-dependence is in part determined by the content of glycine and proline residues mainly within the signal sequence.

Preproinsulin Designer Antigens Excluded from Endoplasmic Reticulum Suppressed Diabetes Development in NOD Mice by DNA Vaccination.

DNA vaccines against autoimmune type 1 diabetes (T1D) contain a nonpredictable risk to induce autoreactive T cell responses rather than a protective immunity. Little is known if (and how) antigen expression and processing requirements favor the induction of autoreactive or protective immune responses by DNA immunization. Here, we analyzed whether structural properties of preproinsulin (ppins) variants and/or subcellular targeting of ppins designer antigens influence the priming of effector CD8+ T cell responses by DNA immunization. Primarily, we used H-2b RIP-B7.1 tg mice, expressing the co-stimulator molecule B7.1 in beta cells, to identify antigens that induce or fail to induce autoreactive ppins-specific (Kb/A12-21 and/or Kb/B22-29) CD8+ T cell responses. Female NOD mice, expressing the diabetes-susceptible H-2g7 haplotype, were used to test ppins variants for their potential to suppress spontaneous diabetes development. We showed that ppins antigens excluded from expression in the endoplasmic reticulum (ER) did not induce CD8+ T cells or autoimmune diabetes in RIP-B7.1 tg mice, but efficiently suppressed spontaneous diabetes development in NOD mice as well as ppins-induced CD8+ T cell-mediated autoimmune diabetes in PD-L1 -/- mice. The induction of a ppins-specific therapeutic immunity in mice has practical implications for the design of immune therapies against T1D in individuals expressing different major histocompatibility complex (MHC) I and II molecules.

Conventional and Neo-antigenic Peptides Presented by ß Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors.

Although CD8+ T-cell-mediated autoimmune ß cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by ß cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known ß cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by ß cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.

Factors affecting Salmonella-based combination immunotherapy for prevention of type 1 diabetes in non-obese diabetic mice.

We previously reported the development of an oral vaccine for diabetes based on live attenuated Salmonella-expressing preproinsulin (PPI) as the autoantigen. When combined with host cell-expressed TGFß, the vaccine prevented the onset of diabetes in non-obese diabetic (NOD) mice. Herein, we investigated factors that could affect vaccine efficacy including vaccination number, optimization of the autoantigen codon sequence, Salmonella SPI2-TTSS promoter/effector combinations, concurrent short-course low-dose anti-CD3. We also evaluated autoantigen GAD65 and cytokine IL10 treatment upon vaccine efficacy. T-cells we employed to elucidate the mechanism of the vaccine action. Our results showed that GAD65+TGFß or PPI+TGFß+IL10 prevented the onset of diabetes in the NOD mice and maintained glucose tolerance. However, increasing the number of vaccine doses, codon-optimization of the autoantigen(s) or use of other Salmonella promoter/effector combinations had no in vivo effect. Interestingly, two doses of vaccine (PPI+TGFß+IL10) combined with a sub-therapeutic dose of anti-CD3 prevented diabetes and decreased hyperglycemia in mice. The combined therapy also increased splenic Tregs and local Tregs in pancreatic lymph nodes (PLN) and increased regulatory (IL10 and IL2) but reduced inflammatory (IFNγ and TNFα) cytokines. Together, these results indicate that the combination of low vaccine dose number, less vaccine autoantigen expression and short-course low-dose anti-CD3 can increase regulatory mechanisms and suppress autoimmunity.