Historical and new insights into pathogenesis of type 1 diabetes (2).

In this second and final part of the collection of articles for the Immunology of Diabetes Society review series on insights into pathogenesis of type 1 diabetes, we present two articles. The first of these covers a debate that took place in the Immunology of Diabetes Society meeting in London 2018, in which five investigators presented a case for specific immune cells/targets to be the 'Achilles Heel of type 1 diabetes'. The second article presents further insights into the generation of post-translationally modified peptides. It focuses upon mechanisms and processes that lead to new potentially autoantigenic targets for CD8+ T cells, and complements the review of new hybrid peptide targets for CD4+ T cells in the first part of our series.

Identifying the 'Achilles heel' of type 1 diabetes.

When Thetis dipped her son Achilles into the River Styx to make him immortal, she held him by the heel, which was not submerged, and thus created a weak spot that proved deadly for Achilles. Millennia later, Achilles heel is part of today's lexicon meaning an area of weakness or a vulnerable spot that causes failure. Also implied is that an Achilles heel is often missed, forgotten or under-appreciated until it is under attack, and then failure is fatal. Paris killed Achilles with an arrow 'guided by the Gods'. Understanding the pathogenesis of type 1 diabetes (T1D) in order to direct therapy for prevention and treatment is a major goal of research into T1D. At the International Congress of the Immunology of Diabetes Society, 2018, five leading experts were asked to present the case for a particular cell/element that could represent 'the Achilles heel of T1D'. These included neutrophils, B cells, CD8+ T cells, regulatory CD4+ T cells, and enteroviruses, all of which have been proposed to play an important role in the pathogenesis of type 1 diabetes. Did a single entity emerge as 'the' Achilles heel of T1D? The arguments are summarized here, to make this case.

Historical and new insights into pathogenesis of type 1 diabetes.

In recent years, there have been exciting new insights into pathogenesis of type 1 diabetes in a number of areas of immunology. In this edition, a collection of four review articles are presented, which encompass new findings presented at the Immunology of Diabetes Society meeting in London 2018. The articles are focused particularly in 4 related areas of investigation, which include autoantibodies in type 1 diabetes, new autoantigenic targets for CD4 T cells, trafficking of immune cells to the pancreas and islet-immune interactions in the pancreas.

Persistent C-peptide is associated with reduced hypoglycaemia but not HbA1c in adults with longstanding Type 1 diabetes: evidence for lack of intensive treatment in UK clinical practice?

AIMS: Most people with Type 1 diabetes have low levels of persistent endogenous insulin production. The Diabetes Control and Complications Trial showed that close to diagnosis preserved endogenous insulin was associated with lower HbA1c , hypoglycaemia and complication rates, when intensively treated. We aimed to assess the clinical impact of persistent C-peptide on rate of hypoglycaemia and HbA1c in those with long duration (> 5 years) Type 1 diabetes. METHODS: We conducted a cross-sectional case-control study of 221 people (median age 24 years) with Type 1 diabetes. We confirmed ongoing endogenous insulin secretion by measuring C-peptide after a mixed-meal tolerance test. We compared self-reported hypoglycaemia (n = 160), HbA1c , insulin dose and microvascular complications (n = 140) in those with preserved and low C-peptide. RESULTS: Stimulated median (IQR) C-peptide was 114 (43, 273) pmol/l and < 3 (< 3, < 3) pmol/l in those with preserved and low C-peptide respectively. Participants with preserved C-peptide had lower reported monthly rates of hypoglycaemia, with 21% fewer symptomatic episodes, 5.9 vs. 7.5 [incidence rate ratio (IRR) 0.79, P = 0.001], and 65% fewer asymptomatic episodes, 1.0 vs. 2.9 (IRR 0.35, P < 0.001). Those with preserved C-peptide had a lower insulin dose (0.68 vs. 0.81 units/kg, P = 0.01) but similar HbA1c (preserved 69 vs. low 67 mmol/mol, P = 0.06). CONCLUSIONS: Adults with Type 1 diabetes and preserved endogenous insulin production receiving usual care in the UK have lower daily insulin doses and fewer self-reported hypoglycaemic episodes, but no difference in HbA1c . This is consistent with non-intensive treatment in previous studies, and suggests a need to consider therapy intensification to gain full benefit of preserved endogenous insulin.

Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D. Here we describe some of the assays used currently to detect autoreactive T cells in human blood and review critically their strengths and weaknesses. The challenges and future prospects for the T cell assays are discussed.

Allograft-specific cytokine profiles associate with clinical outcome after islet cell transplantation.

Islet cell transplantation can cure type 1 diabetes, but allograft rejection and recurrent autoimmunity may contribute to decreasing insulin independence over time. In this study we report the association of allograft-specific proliferative and cytokine profiles with clinical outcome. Peripheral blood mononuclear cells were obtained of 20 islet recipients. Cytokine values in mixed lymphocyte cultures (MLC) were determined using stimulator cells with graft-specific HLA class II. Qualitative and quantitative cytokine profiles were determined before and after islet transplantation, blinded from clinical outcome. Cytotoxic T Lymphocyte precursor (CTLp) assays were performed to determine HLA class I alloreactivity. Allograft-specific cytokine profiles were skewed toward a Th2 or regulatory (Treg) phenotype after transplantation in insulin-independent, but not in insulin-requiring recipients. IFNgamma/IL10 ratio and MLC proliferation decreased after transplantation in insulin-independent recipients (p = 0.006 and p = 0.01, respectively). Production of the Treg cytokine IL10 inversely correlated with proliferation in alloreactive MLC (p = 0.008) and CTLp (p = 0.005). Production of IL10 combined with low-MLC reactivity associated significantly with insulin independence. The significant correlation between allograft-specific cytokine profiles and clinical outcome may reflect the induction of immune regulation in successfully transplanted recipients. Islet donor-specific IL10 production correlates with low alloreactivity and superior islet function.

Increased resistance to CD4+CD25hi regulatory T cell-mediated suppression in patients with type 1 diabetes.

Type I diabetes (T1D) is a T cell-mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin-producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4+CD25hi naturally occurring regulatory T cells (T(regs)), defects in which could contribute to loss of self-tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4+CD25hi T(regs) of autologous CD4+CD25- responder cells. Here we demonstrate that this defective regulation is also present in subjects with long-standing T1D (> 3 years duration; P = 0.009). No difference was observed in forkhead box P3 or CD127 expression on CD4+CD25hi T cells in patients with T1D that could account for this loss of suppression. Cross-over co-culture assays demonstrate a relative resistance to CD4+CD25hi T(reg)-mediated suppression within the CD4+CD25- T cells in all patients tested (P = 0.002), while there appears to be heterogeneity in the functional ability of CD4+CD25hi T(regs) from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.

Promiscuous binding of proinsulin peptides to Type 1 diabetes-permissive and -protective HLA class II molecules.

AIMS/HYPOTHESIS: Presentation of peptide epitopes derived from beta-cell autoantigens, such as insulin and its precursor molecules, by MHC class II molecules to autoreactive T-cells is believed to play a role in the development of Type 1 diabetes. However, little is known about the interaction between peptides of (prepro)insulin and MHC class II molecules permissive and protective for Type 1 diabetes. In this study therefore, peptides spanning the human preproinsulin sequence were assessed for their binding characteristics to Type 1 diabetes-protective and -permissive HLA molecules. METHODS: HLA-DR2, -DQ6.2 (Type 1 diabetes-protective) and HLA-DR4, -DQ8 (Type 1 diabetes permissive) molecule binding affinity for overlapping synthetic 20mer peptides spanning human preproinsulin was measured in a direct competition binding assay against a biotinylated indicator peptide. RESULTS: All HLA molecules tested showed similarity in their binding characteristics across the preproinsulin molecule, with regions of the insulin A-chain showing the highest affinity and C-peptide regions the lowest affinity for all HLA molecules tested. Furthermore, an insulin peptide implicated as a major CD4+ T-cell target in disease pathogenesis (B9-23) had high affinity binding to both protective and permissive HLA molecules but did not represent the highest affinity region of (prepro)insulin identified in either case. CONCLUSION/INTERPRETATION: The results suggest that peptide binding affinity alone is unlikely to be the major determinant of disease susceptibility in relation to interactions between (prepro)insulin epitopes and HLA molecules. The identification of epitopes derived from beta-cell autoantigens that bind promiscuously to diabetes-permissive HLA molecules could be important in the design of peptide-based immunotherapeutic strategies for the prevention of Type 1 diabetes.

Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.

The identification, quantification, and characterization of T-cells reactive with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphatase-like molecules IA-2 and phogrin are major research goals in type 1 diabetes. In the Immunology of Diabetes Society First Workshop on Autoreactive T-Cells, the quality of recombinant preparations of these autoantigens was identified as a significant weakness, a finding that may account for much of the inconsistency in published studies of peripheral blood T-cell reactivity to islet autoantigens. Poor antigen quality has also hampered the development of novel technologies for the detection of islet-reactive T-cells. For these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to stimulate a panel of relevant T-cell clones, and inhibitory effects on proliferation to unrelated third-party antigens. Through this process, we have been able to identify preparations of GAD65 and IA-2, generated in insect cells using the baculovirus expression system, that stimulate relevant clones and display low inhibitory effects on third-party antigens. In addition, we characterized a PI preparation generated in bacteria as being free of effects on proliferation to third-party antigens and low in endotoxin content. These preparations are important to promote the development of robust and sensitive assays of islet-reactive T-cells in patients with type 1 diabetes or patients at high risk for developing the disease.

Two amino acids in glutamic acid decarboxylase act in concert for maintenance of conformational determinants recognised by Type I diabetic autoantibodies.

AIMS/HYPOTHESIS: Glutamic acid decarboxylase 65 is a major autoantigen in Type I (insulin-dependent) diabetes mellitus, autoimmune polyendocrine syndrome and stiff-man syndrome. These disorders are characterised by the presence of multiple autoantibodies to the autoantigen which can be distinguished in a variety of different ways. We have investigated the role of single amino-acid mutations in glutamic acid decarboxylase 65 in distinguishing the binding of serum antibodies and a variety of patient-derived human IgG monoclonal antibodies directed to different determinants of the autoantigen. METHODS: We identified a mutant of glutamic acid decarboxylase 65 that contained four single amino-acid mutations from the wild-type molecule. The role of these mutations was investigated by site-directed mutagenesis. We investigated the binding of patient-derived serum antibodies to glutamic acid decarboxylase 65 to a number of single and double amino-acid mutants using immunoprecipitation with labelled, recombinant antigen. To overcome the heterogeneity of different anti-glutamic acid decarboxylase 65 antibodies present in a patient's serum, the binding of a panel of eleven patient-derived human monoclonal antibodies recognising different determinants on the autoantigen was also studied. RESULTS: Two replacements in glutamic acid decarboxylase 65 at Asn247Ser and Leu574Pro were identified that preferentially influence the anti-glutamic acid decarboxylase 65 serum antibodies of Type I diabetic patients, without statistically significantly effecting those recognised in other disorders. Single or double amino-acid replacements Asn247Ser and Leu574Pro in the autoantigen showed differential affects on expression of epitopes recognised by the human monoclonals. The double replacement of Asn247Ser and Leu574Pro in glutamic acid decarboxylase 65 resulted in the loss of binding of all eleven human monoclonal antibodies, irrespective of their epitope recognition. In contrast, single replacement of Leu574Pro statistically significantly reduced the binding of some carboxyl terminal-directed antibodies such as MICA 1, MICA 3 and DP-A without influencing the binding of other monoclonals. Replacement of Asn247Ser did not, however, influence the binding of any patients serum or human monoclonal antibodies. CONCLUSION/INTERPRETATION: Two distantly spaced amino acids, Asn247 and Leu574 in glutamic acid decarboxylase 65 were identified that act in concert to greatly influence the conformational structure of the autoantigen and statistically significantly influence the binding of antibodies present in Type I diabetic sera. The single or double amino-acid mutants can be used to distinguish some anti-glutamic acid decarboxylase-65 autoantibodies and could prove useful in distinguishing Type I diabetic from autoimmune polyendocrine syndrome and stiff-man syndrome patients' sera as well as to study changes in antibody patterns during disease progression.

Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients.

Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet beta-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet beta-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet beta-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in beta-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet beta-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.

The novel cuticular collagen Ovcol-1 of Onchocerca volvulus is preferentially recognized by immunoglobulin G3 from putatively immune individuals.

The cDNA sequence encoding an Onchocerca volvulus collagen, Ovcol-1, has been isolated and the corresponding native antigen has been identified. The cDNA encodes an open reading frame of 96 amino acid residues containing an uninterrupted 66-residue Gly-X-Y repeat triple-helical (TH) domain (where X and Y may be any amino acids) flanked by a 26-residue amino non-TH domain and a 4-residue carboxyl non-TH domain. The size (9.7 kDa) and structure of the deduced molecule are unique among previously identified collagen chains. This novel collagen type has been designated "mini-chain collagen." Native Ovcol-1 is aqueous soluble and resolves by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 14.2 kDa under reducing conditions. Immunoelectron microscopy of adult female O. volvulus localized Ovcol-1 to the cuticles of both the adult worm and uterine microfilaria. A group of individuals from an area in Ecuador where O. volvulus is hyperendemic have been classified as putatively immune (PI) to O. volvulus infection. Analysis of the humoral immune responses to Ovcol-1 demonstrated that immunoglobulin G3 (IgG3) of PI individuals preferentially recognized this antigen in comparison to IgG3 of infected individuals.

Resistance to Onchocerca volvulus: differential cellular and humoral responses to a recombinant antigen, OvMBP20/11.

Persons putatively immune (PI) to Onchocerca volvulus (Ov) infection were identified in Ecuador on the basis of epidemiologic, clinical, and parasitologic findings. Immune responses of PI subjects to a recombinant onchocercal protein, OvMBP20/11, were determined and compared with those of a comparable infected (INF) group from the same Ov-endemic area. PI subjects had significantly less antibody reactivity to this molecule; however, not all INF subjects had an antibody response. IgG1 and IgG4 were the predominant IgG subclasses induced to this molecule, and the amount of IgG1 produced was the only significant difference between the PI and INF groups. In contrast to the antibody responses, proliferative responses to OvMBP20/11 were significantly higher in PI than in INF subjects. Cytokine analysis of peripheral blood mononuclear cell culture supernatants revealed that INF subjects produced significantly more interleukin-10 in response to OvMBP20/11 than did PI subjects. This antigen induced few other cytokines, and there were no differences between study groups.

Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus with homologues in other filarial nematodes and Caenorhabditis elegans.

The full-length cDNA corresponding to an Onchocerca volvulus antigen, OvMBP/11, which had been selected as a serodiagnostic tool was isolated, sequenced, and the native antigen encoded by the cDNA characterised. The cDNA encodes a protein of 20.5 kDa (termed Ov 20) containing a putative signal peptide. Southern blot analysis indicates that there is only a single O. volvulus gene corresponding to Ov 20 but it has significant sequence similarity to genes corresponding to two 20.5-kDa predicted proteins of Caenorhabditis elegans. Homologues of the Ov 20 gene were detected at high stringency by Southern blot in the other Onchocerca species O. gibsoni, and O. gutturosa and at lower stringency in the related filarial nematodes Brugia malayi and Acanthocheilonema viteae. The Ov 20 native antigen has two molecular mass forms, 20 and 22 kDa, in all the life cycle stages studied. These isoforms have different levels of N-linked glycosylation on a peptide backbone of 17.5 kDa. Immunolocalisation and in situ hybridisation studies demonstrated that Ov 20 is transcribed and translated in the body wall of adult females and also in microfilariae, third and fourth stage larvae. Antigen was detected in the supernatant of in vitro cultured adult female nematodes. The B. malayi and A. viteae homologues are antigenically cross-reactive to Ov 20, share the same size peptide backbone but differ in their degree of glycosylation.

Heterogeneity of IgG antibody responses to cloned Onchocerca volvulus antigens in microfiladermia positive individuals from Esmeraldas Province, Ecuador.

The prevalence of IgG antibodies to three recombinant O. volvulus antigens, OvMBP/10, OvMBP/11 and OvMBP/29 was determined in a group of 94 microfilaria positive (mf+) individuals resident in the hyperendemic onchocercal area of Esmeraldas Province, Ecuador. Clone OvMBP/11 was the antigen most frequently recognized by patients sera followed by OvMBP/10 and OvMBP/29. When a cocktail of the three recombinant antigens was used the proportion of positive sera increased to 100%. Antibody responses to the fusion partner maltose binding protein (MBP) were low in comparison with those to the cloned antigens and no correlation of responses between individual antigens was observed. The relative level of antibody response to each of the clones in the cocktail varied between individuals. The distribution of IgG responses to OvMBP/11 was bimodal and those to OvMBP/29 and OvMBP/10 were positively and negatively skewed, respectively. When the three recombinant antigens were used in combination this variation was minimized and the pattern of responses showed a normal distribution as was also seen to crude O. volvulus antigen. The cocktail of recombinants thus offers excellent diagnostic sensitivity in combination with the parasite specificity demonstrated previously.

Onchocerca volvulus: characterization of an immunodominant hypodermal antigen present in adult and larval parasites.

Biochemical and immunological data suggest that a relatively limited number of polypeptide antigens of viable Onchocerca volvulus-infective larvae are available to be recognized by the host's immune system. A partial cDNA clone encoding one such antigen, designated lambda RAL-2, was isolated by screening an expression cDNA library with antisera raised against viable O. volvulus L3. The antigen encoded by this clone was subsequently found to be immunogenic in the majority of individuals exposed to O. volvulus. In the present study, the native antigen corresponding to lambda RAL-2 (Ov17) has been characterized. Immunolocalization and in situ hybridization techniques have been used to localize Ov17 in adult and larval stages of the parasite. In adult females, Ov17 was localized primarily in the hypodermis. Ov17 was accessible to surface labeling reagents in viable adult parasites. Full-length cDNA clones encoding Ov17 suggested that the nascent protein contains a putative leader sequence, which is almost immediately followed by a polyglutamine tract. Analysis of antibody reactivity to recombinant proteins containing and lacking the polyglutamine tract demonstrated that this structure was not a significant B cell epitope in individuals exposed to O. volvulus.