Regulatory T cell phenotype and function 4 years after GAD–alum treatment in children with type 1 diabetes

Glutamic acid decarboxylase (GAD)₆₅ formulated with aluminium hydroxide (GAD‐alum) was effective in preserving insulin secretion in a Phase II clinical trial in children and adolescents with recent‐onset type 1 diabetes. In addition, GAD‐alum treated patients increased CD4⁺CD25^hi forkhead box protein 3⁺ (FoxP3⁺) cell numbers in response to in‐vitro GAD₆₅ stimulation. We have carried out a 4‐year follow‐up study of 59 of the original 70 patients to investigate long‐term effects on the frequency and function of regulatory T cells after GAD‐alum treatment. Peripheral blood mononuclear cells were stimulated in vitro with GAD₆₅ for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells (CD4⁺CD25^hiCD127^lo) and effector T cells (CD4⁺CD25^–CD127⁺) were further sorted, expanded and used in suppression assays to assess regulatory T cell function after GAD‐alum treatment. GAD‐alum‐treated patients displayed higher frequencies of in‐vitro GAD₆₅‐induced CD4⁺CD25⁺CD127⁺ as well as CD4⁺CD25^hiCD127^lo and CD4⁺FoxP3⁺ cells compared to placebo. Moreover, GAD₆₅ stimulation induced a population of CD4^hi cells consisting mainly of CD25⁺CD127⁺, which was specific of GAD‐alum‐treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD‐alum‐ and placebo‐treated individuals. Regulatory T cell frequency did not correlate with C‐peptide secretion throughout the study. In conclusion, GAD‐alum treatment induced both GAD₆₅‐reactive CD25⁺CD127⁺ and CD25^hiCD127^lo cells, but no difference in regulatory T cell function 4 years after GAD‐alum treatment.     

Immunoglobulin subclass profiles of anti-idiotypic antibodies to GAD65Ab differ between type 1 diabetes patients and healthy individuals

Previously we reported the presence of anti-idiotypic antibodies (anti-Id)-specific to autoantibodies against GAD65 (GAD65Ab) in healthy individuals while the activity of anti-Id directed to GAD65Ab in type 1 diabetes (T1D) patients was significantly lower. These anti-Id recognize the antigen-binding site of GAD65Ab, thus preventing their binding to GAD65. Here, we characterized the IgG subclass profile of these anti-Id (GAD65Ab specific) and of the associated GAD65Ab themselves. The IgG subclass response of anti-Id in healthy individuals (n = 16) was IgG3-dominated, while in T1D patients (n = 8) IgG1 was the major IgG subclass. The GAD65Ab bound by anti-Id in both healthy individuals (n = 38) and GAD65Ab-negative T1D patients (n = 35) showed a predominant rank order of IgG1 > IgG2 > IgG4 > IgG3. However, the frequency of GAD65Ab of the IgG4 subclass was significantly higher in T1D patients (P < 0.05). We conclude that the IgG subclass profile of anti-Id (GAD65Ab specific) in healthy individuals differs from that in T1D patients. These differences may provide insights into the development of these antibodies.     

Immune responses to glutamic acid decarboxylase and insulin in patients with gestational diabetes

Pregnancy is a natural state of immunoprotection and tolerance. We studied subjects with gestational diabetes (GDM) to evaluate the influence of pregnancy on the humoral immune response to the autoantigen GAD and to injected insulin. Antibodies against glutamic acid decarboxylase (GADA) subclasses and epitope reactivity were determined in 34 GADA-positive pregnant patients with GDM, in 20 GADA-positive relatives of people with TID and in 25 GADA-positive patients with newly diagnosed TID. Partum levels of insulin antibodies (IA), IgG1- and IgG4-IA were measured in 131 women with GDM treated with human insulin from the time of diabetes diagnosis (including 22 with GADA) and were compared to 19 patients with TID after 3 months of insulin treatment. GADA titre and subclasses were similar among all groups. GADA in GDM patients bound fewer epitopes than GADA in relatives of patients with TID (all epitopes being present in 23%versus 65%, P < 0.01). In particular, antibodies to the minor GADA epitopes GAD6596-249, GAD651-100 and GAD67 were less frequent in patients with GDM compared to relatives (P < 0.01). Antibodies to insulin (IA) were found in 17% of patients with GDM. They were more frequent in GDM patients with GADA compared to GADA-negative patients (41%versus 12%, P < 0.005). IgG1 was the dominant insulin antibody subclass response in both patients with GDM and TID but levels of IgG1-IA and IgG4-IA were significantly lower in patients with GDM compared to patients with TID (P < 0.004). Antibody responses in women with gestational diabetes appear to be dampened and restricted, but without change in subclass usage.     

GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution,GAD65 enzyme activity and humoral response

We have previously shown that two injections of 20 μg GAD-alum to recent onset type 1 diabetic children induced GADA levels in parallel to preservation of insulin secretion. Here we investigated if boosted GADA induced changes in IgG1, 2, 3 and 4 subclass distributions or affected GAD₆₅ enzyme activity. We further studied the specific effect of GAD-alum through analyses of IA-2A, tetanus toxoid and total IgE antibodies. Serum from children receiving GAD-alum or placebo was collected pre-treatment and after 3, 9, 15 and 21 months. At 3 months a reduced percentage of IgG1 and increased IgG3/IgG4 were detected in GAD-alum treated. Further, IA-2A, IgE and tetanus toxoid antibodies, as well as GAD₆₅ enzyme activity, were unaffected confirming the specific effect of treatment. In the GAD-alum group, higher pre-treatment GADA were associated to more pronounced C-peptide preservation. The induced IgG3/IgG4 and reduced IgG1 suggest a Th2 deviation of the immune response.     

CD4+ T cells recognize diverse epitopes within GAD65: implications for repertoire development and diabetes monitoring

Type 1 diabetes is associated with T‐cell responses to β‐cell antigens such as GAD65. Single T‐cell epitopes have been investigated for immune monitoring with some success, but multiple epitopes may be required to fully characterize responses in all subjects. We used a systematic approach to examine the diversity of the GAD65‐specific T‐cell repertoire in subjects with DRB1*04:01 haplotypes. Using class II tetramers, we observed responses to 15 GAD65 epitopes, including five novel epitopes. The majority were confirmed to be processed and presented. Upon stimulation with peptides, GAD‐specific responses were equally broad in subjects with diabetes and healthy controls in the presence or absence of CD25⁺ T cells, suggesting that a susceptible HLA is sufficient to generate a potentially autoreactive repertoire. Without depleting CD25⁺ cells, GAD_113–132 and GAD_265–284 responses were significantly stronger in subjects with diabetes. Although nearly every individual responded to at least one GAD65 epitope, most were seen in less than half of the subjects tested, suggesting that multiple epitopes are recommended for immune monitoring.     

The glutamic acid decarboxylase 65 immunoglobulin G subclass profile differs between adult-onset type 1 diabetes and latent autoimmune diabetes in adults (LADA) up to 3 years after clinical onset

Autoantibodies against glutamic acid decarboxylase 65 (GADA) are found frequently in patients with autoimmune diabetes. Immunoglobulin (Ig)G₁ is the most frequent subclass among the GADA IgG subclasses. IgG₄ is a more common subclass in latent autoimmune diabetes in adults (LADA) at clinical onset compared to type 1 diabetes. The aim of this work was to study the different GADA-IgG subclass profiles during a 3-year follow-up in these groups of autoimmune diabetes. Adult-onset subjects, classified as either type 1 (n = 40) or LADA (n = 43), were included in the study. New samples were collected every year from these patients. In addition to conventional GADA analyses, GADA-IgG subclasses were also analysed with a radioimmunoprecipitation assay using biotin-conjugated antibodies (directed against human IgG subclasses and IgM) and streptavidin Sepharose. During 3 years'' follow-up, all the IgG subclass levels decreased in type 1 diabetes – IgG₁: P < 0·001; IgG₂: P < 0·001; IgG₃: P < 0·001; IgG₄: P < 0·05 (Friedman''s’ test) – while levels remained stable for all four subclasses in LADA. GADA IgM, however, decreased in both groups (P < 0·001). Patients with LADA have higher GADA IgG₃ and IgG₄ at clinical onset and seem to maintain the levels and profile of their IgG subclasses up to 3 years after clinical onset, while all the GADA IgG subclass levels decrease in type 1 diabetic patients. This indicates a persistent different immune response in LADA compared to type 1 diabetes and further indicates the difference in pathogenesis.     

Reversal of type 1 diabetes by a new MHC II‐peptide chimera: “Single‐epitope‐mediated suppression” to stabilize a polyclonal autoimmune T‐cell process

Polyclonality of self‐reactive CD4⁺ T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II‐peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T‐cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3 : 383–391]. Since most of the glutamic acid decarboxylase 65 (GAD65)‐specific CD4⁺ T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T‐regulatory cell population (GAD65_217–230‐specific CD4⁺ T cells) using a soluble I‐A/GAD65_217–230/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T‐cell process. Activated GAD65_217–230‐specific T cells and a fraction of the diabetogenic (B_9–23‐specific) T cells were polarized toward the IL‐10‐secreting T‐regulatory type 1‐like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post‐therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self‐reactive T‐cell process by a single epitope‐mediated bystander suppression. Dimeric MHC class II‐peptide chimeras‐like approach may provide rational grounds for the development of more efficient antigen‐specific therapies in type 1 diabetes.     

Isolation and Characterization of Human Monoclonal Autoantibodies to Glutamic Acid Decarboxylase

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M_r 65,000 (GAD₆₅), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD₆₅ autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD₆₅ were produced using standard techniques. F(ab')₂s from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to ¹²⁵I-labelled GAD₆₅ (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M_r chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD₆₅ of 2.2 &#50 10⁹, 5.8 &#50 10⁹, 1.3 &#50 10¹⁰ mol/l^{&#109 1}; affinities of the mouse monoclonals (n = 5) ranged from 1.1 &#50 10⁸ to 5.4 &#50 10¹⁰ mol/l^{&#109 1}. The binding of each of the human monoclonals was inhibited by GAD6 F(ab')₂ and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')₂s suggesting that the epitopes for these antibodies were overlapping. Studies with GAD₆₅/GAD₆₇ chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to ¹²⁵I-labelled GAD₆₅. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD₆₅ in the donor's serum and in the sera of patients with type-1 diabetes.     

Comparative study of GAD65-specific CD4+ T cells in healthy and type 1 diabetic subjects

Glutamic acid decarboxylase 65 (GAD65) is a putative autoantigen associated with the pathogenesis of type 1 diabetes (T1D). The prevalence of autoreactive CD4+ T cells towards the immunodominant GAD65_555–567 epitope in DR4 healthy and T1D subjects was investigated with class II tetramers. A slightly higher percentage of diabetic subjects had GAD65_555–567 tetramer-positive T cells upon GAD65_555–567 peptide stimulation on the total CD4+ T-cell populations compared to healthy subjects. In contrast, three quarters of subjects in both groups had tetramer-positive T cells resulting from stimulation of the CD4+CD25+ regulatory T-cell depleted CD4+ T cells. The frequencies and TCR Vβ gene usages of GAD65_555–567 T cells were also similar in both groups. Experiments demonstrated that GAD65_555–567-reactive T cells in healthy and diabetic subjects had different CD45RA phenotypes. For the healthy group, GAD65_555–567-reactive T cells were generally found in the CD45RA+ naïve T-cell pool while GAD65_555–567-reactive T cells from T1D subjects were present in both CD45RA+ naïve and CD45RA− memory T-cell pools. These findings suggested that there is no difference in thymic selection of DR4 restricted GAD-reactive T cells amongst healthy and T1D individuals but GAD65_555–567-reactive T cells have been preferentially activated in diabetic patients.     

Characterization of hydroxyl radical modified GAD65: A potential autoantigen in type 1 diabetes

Glutamic acid decarboxylase-65 (GAD₆₅) is an immunological marker of type 1 autoimmune diabetes. High titre of autoantibodies against GAD₆₅ (GAD₆₅Abs) have also been detected in some other autoimmune diseases. In search of a potential immunological marker of type 1 diabetes, in vitro GAD₆₅ was modified by hydroxyl radical followed by the study of structural and conformational perturbed protein by different spectroscopic techniques (UV, fluorescence and CD) and thermal denaturation profile. Binding studies of circulating autoantibodies from diabetic groups (type 1 and type 2) with native and reactive oxygen species (ROS) modified GAD₆₅, exhibited high recognition of type 1 diabetic serum autoantibodies with modified antigen (p < 0.001) over unmodified GAD₆₅. Binding specificity of isolated IgG of patients (n = 17) from each diabetic group and control group (n = 10) was checked by inhibition enzyme linked immunosorbent assay (ELISA) and quantitative precipitin titration assay. Relative affinity of ROS-GAD₆₅Abs for modified and native GAD₆₅ was in the order of 1.56 × 10^{? 6} and 2.72 × 10^{? 7} M, as calculated by Langmuir plot. In coherence, ROS oxidation of GAD₆₅ causes conformational perturbation, generating highly immunogenic unique neoepitopes that may be one of the factors in antigen-driven induction of type 1 diabetes autoantibodies that can serve as a potential marker in early diagnosis/prognosis of the disease.     

Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: protean additions to the autoimmune central neuropathies

Stiff Person Syndrome (SPS) is a rare autoimmune neurological disease attributable to autoantibodies to glutamic acid decarboxylase (anti-GAD) more usually associated with the islet beta cell destruction of autoimmune type 1 diabetes (T1D). SPS is characterized by interference in neurons with the synthesis/activity of the inhibitory neurotransmitter gamma amino butyric acid (GABA) resulting in the prototypic progressive spasmodic muscular rigidity of SPS, or diverse neurological syndromes, cerebellar ataxia, intractable epilepsy, myoclonus and several others. Remarkably, a single autoantibody, anti-GAD, can be common to widely different disease expressions, i.e. T1D and SPS. One explanation for these data is the differences in epitope engagement between the anti-GAD reactivity in SPS and T1D: in both diseases, anti-GAD antibody reactivity is predominantly to a conformational epitope region in the PLP- and C-terminal domains of the 65 kDa isoform but, additionally in SPS, there is reactivity to conformational epitope(s) on GAD67, and short linear epitopes in the C-terminal region and at the N-terminus of GAD65. Another explanation for disease expressions in SPS includes ready access of anti-GAD to antigen sites due to immune responsiveness within the CNS itself according to intrathecal anti-GAD-specific B cells and autoantibody. Closer study of the mysterious stiff-person syndrome should enhance the understanding of this disease itself, and autoimmunity in general.     

Autoantibody epitopes to the smaller isoform of glutamate decarboxylase do not differ in Swedish and Japanese type 1 diabetes patients and may be associated with high-risk human leucocyte antigen class II alleles

Type 1 diabetes (T1D) is an autoimmune disease with a strong human leucocyte antigen (HLA) class II association. Depending on geographic locations, the disease-associated HLA class II alleles vary. We evaluated the beta cell-specific autoimmunity reflected in autoantibodies directed to the smaller isoform of glutamate decarboxylase (GAD65) in Japanese and Swedish T1D patients. GAD65Ab epitope specificities were assessed using GAD65-specific recombinant Fab. GAD65Ab epitope specificities did not differ between Swedish and Japanese patients. Only recognition of the MICA-4-defined middle epitope was significantly stronger in the Japanese T1D patient group compared to the Swedish T1D patients (P = 0.001). Binding to the b96.11-defined middle epitope was substantial in both groups and showed significant associations with high-risk HLA class II haplotypes. In the Japanese T1D group the association was with haplotype DRB1*0802-DQB1*0302 (P = 0.0008), while in the Swedish T1D patients binding to the b96.11-defined epitope as associated with the presence of high-risk HLA genotypes DR3-DQB1*0201 and/or DR4-DQB1*0302 (P = 0.02). A significant association between reduction in binding in the presence of recombinant Fab (rFab) DPD and high-risk allele DQB1*0201 was found (P = 0.008) in the Swedish T1D patients only. We hypothesize that epitope-specific autoantibodies effect the peptide presentation on HLA class II molecules by modulating antigen uptake and processing. Molecular modelling of the high-risk HLA class II molecules will be necessary to test whether these different molecules present similar peptide-binding specificities.     

Antibody cross-reactivity induced by the homologous regions in glutamic acid decarboxylase (GAD65) and 2C protein of coxsackievirus B4

GAD₆₅ contains an amino acid sequence which is highly homologous with a sequence in the 2C protein of coxsackievirus B4 (CBV4-2C). In the present study the possibility that this region could contain an epitope capable of inducing immunological cross-reactivity between CBV4-2C and GAD₆₅ was evaluated. Six out of seven rabbit sera, which were raised against seven different synthetic peptides carrying various modifications of the homology sequence, showed cross-reactivity between 2C, GAD₆₅ and GAD₆₇ derived peptides in ELISA. There was substantial cross-reactivity between 2C and GAD₆₅ peptides, but not between 2C and GAD₆₇ peptides. The most cross-reactive peptides were those corresponding to the 2C sequences FIEWLKVKILPEVKEK and KILPEVKEKHEFLSRL. When the binding of the four 2C peptide-specific sera to the GAD₆₅ protein was analysed in immunoprecipitation, two sera were found to be cross-reactive (anti-FIEWLKVKILPEVKEK and anti-WLKVKILPEVKEKHEF). One of these (anti-WLKVKILPEVKEKHEF) reacted also with coxsackie B virus (CBV)-infected cells. Antibodies against this epitope were induced during enterovirus (including CBV) infections in initially healthy children who later progressed to clinical insulin-dependent diabetes mellitus (IDDM). Antibody responses were frequent also in constantly GAD₆₅ antibody-negative non-diabetic children, and antibody levels did not differ between newly diagnosed IDDM patients and matched control subjects. Blocking experiments confirmed that the observed reactivity of both rabbit and human antibodies was immunologically specific. The results suggest that the epitope is antigenically highly similar in 2C and GAD₆₅, and that peptide immunization induces antibodies which cross-react with these molecules. However, the significance of this phenomenon in the pathogenesis of IDDM remains to be confirmed, as the peptide antibody levels were similar in patients with recent-onset IDDM and in control subjects.     

‘25‐Hydroxyvitamin D,Autoantigenic and Total Antibody Concentrations: Results from a Danish Case–control Study of Newly Diagnosed Patients with Childhood Type 1 Diabetes and their Healthy Siblings’

B cells have recently entered the stage as an important accessory player in type 1 diabetes (T1D) etiopathogenesis. Experimental studies suggest regulatory functions of vitamin D on B cells. However, only a few human studies, with considerable methodological limitations, have been conducted within this field. Our objective was to investigate whether higher 25‐hydroxyvitamin D (25(OH )D) concentrations were inversely associated with β‐cell autoantigens glutamic acid decarboxylase (isoform 65) (GADA ) and insulinoma‐associated antigen‐2A (IA ‐2A). Further, we also wanted to examine the relationship between 25(OH )D and total antibody concentrations. We randomly selected 500 patients with newly diagnosed T1D and 500 siblings for 25(OH )D, antibody and genetic analysis from the population‐based Danish Registry of Childhood and Adolescent Diabetes. The relative change (RC ) in the mean concentration of GADA , IA ‐2A and antibody isotypes by a 10 nmol/l increase in 25(OH )D concentration was modelled by a robust log‐normal regression model. We found no association between 25(OH )D and GADA [adjusted RC per 10 nmol/l increase: 1.00; 95% confidence interval (CI ): 0.98–1.02] and IA ‐2A [adjusted RC per 10 nmol/l increase: 0.92; CI : 0.76–1.12]. Further, 25(OH )D was not associated with the total concentration of antibody isotypes [immunoglobulin (Ig)A, IgE, IgG and IgM]. All null findings were unaltered after adjustment for genetic variation in the vitamin D pathway. Physiological concentrations of 25(OH )D are unlikely to have a clinically important effect on antibody concentrations in a paediatric population of newly diagnosed patients with T1D and their healthy siblings.

     

Thermally Sensitive N‐Type Thermoelectric Aniline Oligomer‐Block‐Polyethylene Glycol‐Block‐Aniline Oligomer ABA Triblock Copolymers

High‐performance n‐type thermoelectric polymers are vital in the development of polymer thermoelectric generators (PTEGs). However, the progress is very slow due to their low stability and low thermoelectric properties. Here, aniline oligomer‐block‐polyethylene glycol‐block‐aniline oligomer (ANI)_n‐b‐PEO‐b‐(ANI)_n) (n = 4, 8, 16) are prepared and studied. The results indicate that the (ANI)_n‐b‐PEO‐b‐(ANI)_n show unique temperature sensitivity and switch from p‐type to n‐type when the temperature is above 300 K. Differential scanning calorimetry curves show that the T_g of the (ANI)_n‐b‐PEO‐b‐(ANI)_n is lower than 300 K, indicating that PEO segments of the (ANI)_n‐b‐PEO‐b‐(ANI)_n easily move above 300 K and have high flexibility. Temperature‐dependent Raman spectra confirm that there are strong interactions between PEO segments and aniline oligomers during the raising temperature, and PEO chains might provide plenty of negative charge at high temperature, which can convert the (ANI)_n‐b‐PEO‐b‐(ANI)_n into n‐type thermoelectric materials. To adjust the number of aniline unit, (ANI)₈‐b‐PEO‐b‐(ANI)₈ shows excellent n‐type characteristics with Seebeck coefficient as large as ?1171 µV K^?1 at 381 K. The strong interaction between PEO and aniline oligomers plays a dominant role in the n‐type thermoelectric performance of the triblock copolymers, which have potential application in the field of PTEGs and temperature sensors.     

Increased prevalence of autoimmunity in Turner syndrome – influence of age

Individuals with Turner syndrome (TS) are prone to develop autoimmune conditions such as coeliac disease (CD), thyroiditis and type 1 diabetes (T1DM). The objective of the present study was to examine TS of various karyotypes for autoantibodies and corresponding diseases. This was investigated in a prospective cross‐sectional study of Danish TS patients (n = 107, median age 36·7 years, range: 6–60 years). A medical history was recorded and a blood sample was analysed for autoantibodies against gliadin, transglutaminase, adrenal cortex, intrinsic factor, anti‐thyroid peroxidase (anti‐TPO) and glutamic‐acid‐decarboxylase 65 (GAD‐65). Autoantibodies were present in 58% (n = 61) of all patients, whereof 18% (11) had autoantibodies targeting more than one organ. Patients with autoantibodies were significantly older than those without (P = 0·001). Anti‐TPO was present in 45% (48) of patients, of whom 33% (16) were hypothyroid. Overall, 18% (19) presented with CD autoantibodies, of whom 26% (five) had CD. Anti‐TPO and CD autoantibodies co‐existed in 9% (10). Immunoglobulin A deficiency was found in 3% (three) of patients, who all had CD autoantibodies without disease. Among four patients with anti‐GAD‐65 none had T1DM, but two were classified as having T2DM. One patient had adrenocortical autoantibodies but not adrenal failure. Autoantibodies against intrinsic factor were absent. Anti‐GAD‐65 was increased in isochromosomal karyotypes (3/23 versus 1/84, P = 0·008) with no other association found between autoantibodies and karyotype. In conclusion, TS girls and women face a high prevalence of autoimmunity and associated disease with a preponderance towards hypothyroidism and CD. Thus, health care providers dealing with this patient group should be observant and test liberally for these conditions even before clinical symptoms emerge.     

Humoral autoimmune responses to glutamic acid decarboxylase have similar target epitopes and subclass that show titer-dependent disease association

Glutamic acid decarboxylase (GAD) is an autoantigen in stiff man syndrome (SMS) and type 1 diabetes (T1DM). Different GAD autoantibody characteristics in these disorders have suggested distinct underlying mechanisms of autoimmunity. Here, it is shown that increased prevalence of autoantibodies to GAD65 amino terminal and GAD67 epitopes and autoantibodies of IgG2, IgG3, or IgG4 subclass in patients with SMS (P < 0.001 vs. T1DM) are secondary to the markedly higher autoantibody titers in SMS patients (P < 0.0001) and that autoantibody epitopes and subclasses were similar when patients were matched for autoantibody titer. Exposure to autoantigen in the disorders is likely to involve similar humoral antigenic determinants, but different B cell regulation.     

Immunoglobulin (Ig)G1 and IgG4 anti‐cyclic citrullinated peptide (CCP) associate with shared epitope,whereas IgG2 anti‐CCP associates with smoking in patients with recent‐onset rheumatoid arthritis (the Swedish TIRA project)

Given the possible importance of anti‐citrullinated peptide/protein antibodies (ACPA) for initiation and progression of rheumatoid arthritis (RA), extended knowledge about the different isotypes and subclasses is important. In the present study, we analysed the immunoglobulin (Ig)G subclasses regarding reactivity against cyclic citrullinated peptides (anti‐CCP) among 504 clinically well‐characterized patients with recent‐onset RA in relation to smoking habits, shared epitope (SE) status and IgA and pan‐IgG anti‐CCP antibodies. All patients, regardless of pan‐IgG anti‐CCP status, were analysed for IgG1–4 CCP reactivity. Sixty‐nine per cent were positive in any IgG anti‐CCP subclass, and of these 67% tested positive regarding IgG1, 35% IgG2, 32% IgG3, and 59% IgG4 anti‐CCP. Among ever‐smokers the percentages of IgG2 anti‐CCP (P = 0·01) and IgA anti‐CCP (P = 0·002)‐positive cases were significantly higher compared to never‐smokers. A positive IgG anti‐CCP subclass ‐negative cases. Combining SE and smoking data revealed that IgG1 and IgG4 anti‐CCP were the IgG anti‐CCP isotypes associated with expression of SE, although the lower number of patients positive for IgG2 or IgG3 anti‐CCP could, however, have influenced the results. High levels of IgG2 anti‐CCP were shown to correlate with expression of the ‘non‐SE’ allele human leucocyte antigen (HLA)‐DRB1*15. In conclusion, in this study we describe different risk factor characteristics across the IgG anti‐CCP subclasses, where IgG2 appears similar to IgA anti‐CCP regarding the predominant association with smoking, while IgG1 and IgG4 related more distinctly to the carriage of SE genes.     

The proteasome immunosubunits,PA28 and ER‐aminopeptidase 1 protect melanoma cells from efficient MART‐126‐35‐specific T‐cell recognition

The immunodominant MART‐1_26(27)‐35 epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART‐1/Melan‐A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART‐1_26‐35 epitope generation in tumor cells. Here, we demonstrate that in addition the IFN‐γ‐inducible proteasome subunit β2i/MECL‐1 (multicatalytic endopeptidase complex‐like 1), proteasome activator 28 (PA28), and ER‐resident aminopeptidase 1 (ERAP1) impair MART‐1_26‐35 epitope generation. β2i/MECL‐1 and PA28 negatively affect C‐ and N‐terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART‐1_26‐35 epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART‐1_26‐35 epitope generation even in the absence of IFN‐γ. In summary, our results provide first evidence that activities of different antigen‐processing components contribute to an inefficient MART‐1_26‐35 epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope‐specific immunotherapies.     

The HLA‐DRB, ‐DQB polymorphism and anti‐insulin antibody response in Slovenian patients with type 1 diabetes

A combination of specific HLA class II antigens and the presence of type 1 diabetes (T1D)‐related antibodies has a high positive predictive value for T1D but low sensitivity. The aim of the present study was to determine the frequencies of HLA‐DRB‐DQB deduced haplotypes associated with susceptibility and protection in Slovenian patients with established T1D, to evaluate the relationship between the HLA‐DRB1‐QBP‐DQB1 haplotypes and the presence of insulin autoantibodies (IAA) and glutamic acid decarboxylase antibodies (GADA), and to access the possible impact of polymorphic QBP promoters on this relationship. A cohort of 135 patients with T1D (age 17.5 ± 7.0 years, duration of T1D 9.14 ± 6.3 years) was investigated. HLA‐DRB1 and DQB1 alleles were typed using the polymerase chain reaction (PCR)–reverse line blot method. QBP promoter region alleles were determined using PCR–sequence‐specific oligonucleotide hybridization (SSO) and PCR–sequence‐specific primers (SSP). IAA and GADA antibodies were determined by enzyme‐linked immunosorbent assay (ELISA). The chi‐square test with Yates’ correction was used for statistical analysis. Deduced haplotypes DRB1*0301‐DQB1*0201 (P = 0.0001, OR = 3.4), DRB1*0401‐DQB1*0302 (P = 0.0001, OR = 29.8), and DRB1*0402‐DQB1*0302 (P = 0.008, OR = 4.7) were significantly more common, and DRB1*1501‐DQB1*0602 (P = 0.0001, OR = 0.03) significantly less common in the investigated cohort than in a Slovenian control group. The highest risk and the strongest protective HLA‐DR‐DQ haplotypes found in Slovenian patients with T1D did not differ from those found in other Caucasian populations. While the DRB1*0301‐QBP2.1‐DQB1*0201 haplotype, where QBP2.1 did not help to further distinguish DQB1*0201‐possessing haplotypes in IAA‐positive and IAA‐negative patients, was strongly associated with the presence of IAA, the DRB1*0101‐QBP5.12‐DQB1*0501 haplotype, although not protective compared to the control population, was associated with an absence of IAA in the investigated cohort. It is suggested that there may be a combined influence of the QBP5.12 promoter and the DQB1*0501 functional molecule on reduced IAA production.