Cepharanthine Blocks Presentation of Thyroid and Islet Peptides in a Novel Humanized Autoimmune Diabetes and Thyroiditis Mouse Model

Autoimmune polyglandular syndrome type 3 variant (APS3v) refers to an autoimmune condition in which both type 1 diabetes (T1D) and autoimmune thyroiditis (AITD) develop in the same individual. HLA-DR3 confers the strongest susceptibility to APS3v. Previously we reported a unique amino acid signature pocket that predisposes to APS3v. We found that this pocket is flexible and can trigger APS3v by presenting both thyroid (Tg.1571, TPO.758) and islet (GAD.492) peptides to induce autoimmune response. We hypothesized that blocking the specific APS3v-HLA-DR3 pocket from presenting thyroid/islet antigens can block the autoimmune response in APS3v. To test this hypothesis we performed a virtual screen of small molecules blocking APS3v-HLA-DR3, and identified 11 small molecules hits that were predicted to block APS3v-HLA-DR3. Using the baculovirus-produced recombinant APS3v-HLA-DR3 protein we tested the 11 small molecules in an in vitro binding assay. We validated 4 small molecule hits, S9, S5, S53 and S15, that could block the APS3v-HLA-DR3 pocket in vitro. We then developed a novel humanized APS3v mouse model induced by co-immunizing a peptide mix of Tg.1571, TPO.758 and GAD.492. The immunized mice developed strong T-cell and antibody responses to the thyroid/islet peptides, as well as mouse thyroglobulin. In addition, the mice showed significantly lower free T4 levels compared to controls. Using the APS3v mouse model, we showed that one of the 4 small molecules, Cepharanthine (S53), blocked T-cell activation by thyroid/islet peptides ex vivo and in vivo. These findings suggested Cepharanthine may have a therapeutic potential in APS3v patients carrying the specific APS3v-HLA-DR3 pocket.

Index60 Identifies Individuals at Appreciable Risk for Stage 3 Among an Autoantibody-Positive Population With Normal 2-Hour Glucose Levels: Implications for Current Staging Criteria of Type 1 Diabetes

<u>Objective</u><b>:</b> We assessed whether Index60, a composite measure of fasting C-peptide, 60-minute C-peptide, and 60-minute glucose, could improve the metabolic staging of type 1 diabetes for progression to clinical disease (stage 3) among autoantibody positive (Ab+) individuals with normal 2-hour glucose values (<140 mg/dL). <p><u>Research Design and Methods</u>: We analyzed 3058 Type 1 Diabetes TrialNet Pathway to Prevention participants, with 2-hour glucose<140 mg/dL and Index60<1.00 values from baseline OGTTs. Characteristics associated with type 1 diabetes (younger age, greater autoantibody positivity [Ab+], higher HLA DR3-DQ2/DR4-DQ8 prevalence, lower C-peptide) were compared among four mutually exclusive groups: top 2-hour glucose quartile only [HI-2HGLU], top Index60 quartile only [HI-IND60], both top quartiles [HI-BOTH], neither top quartile [LO-BOTH]. Additionally, within the 2-hour glucose distribution of <140 mg/dL, and separately within the Index60<1.00 distribution, comparisons were made between those above or below the medians.</p> <p><u>Results</u>: HI-IND60 and HI-BOTH were younger, with greater frequency of >2 Ab+, and lower C-peptide levels than either HI-2HGLU or LO-BOTH (all p<0.001). The cumulative incidence for stage 3 was greater for HI-IND60 and HI-BOTH than either HI-2HGLU or LO-BOTH (all p<0.001). Those with Index60 values above the median were younger, had higher ≥2Ab+ (p<0.001) and DR3-DQ2/DR4-DQ8 prevalence (p<0.001), and lower AUC C-peptide levels (p<0.001) than those below. Those above the 2-hour glucose median had higher AUC C-peptide levels (p<0.001), but otherwise did not differ from those below. </p> <p><u>Conclusion</u>: Index60 identifies individuals with characteristics of type 1 diabetes at appreciable risk for progression who would otherwise be missed by 2-hour glucose staging criteria. </p>

Index60 Identifies Individuals at Appreciable Risk for Stage 3 Among an Autoantibody-Positive Population With Normal 2-Hour Glucose Levels: Implications for Current Staging Criteria of Type 1 Diabetes

<u>Objective</u><b>:</b> We assessed whether Index60, a composite measure of fasting C-peptide, 60-minute C-peptide, and 60-minute glucose, could improve the metabolic staging of type 1 diabetes for progression to clinical disease (stage 3) among autoantibody positive (Ab+) individuals with normal 2-hour glucose values (<140 mg/dL). <p><u>Research Design and Methods</u>: We analyzed 3058 Type 1 Diabetes TrialNet Pathway to Prevention participants, with 2-hour glucose<140 mg/dL and Index60<1.00 values from baseline OGTTs. Characteristics associated with type 1 diabetes (younger age, greater autoantibody positivity [Ab+], higher HLA DR3-DQ2/DR4-DQ8 prevalence, lower C-peptide) were compared among four mutually exclusive groups: top 2-hour glucose quartile only [HI-2HGLU], top Index60 quartile only [HI-IND60], both top quartiles [HI-BOTH], neither top quartile [LO-BOTH]. Additionally, within the 2-hour glucose distribution of <140 mg/dL, and separately within the Index60<1.00 distribution, comparisons were made between those above or below the medians.</p> <p><u>Results</u>: HI-IND60 and HI-BOTH were younger, with greater frequency of >2 Ab+, and lower C-peptide levels than either HI-2HGLU or LO-BOTH (all p<0.001). The cumulative incidence for stage 3 was greater for HI-IND60 and HI-BOTH than either HI-2HGLU or LO-BOTH (all p<0.001). Those with Index60 values above the median were younger, had higher ≥2Ab+ (p<0.001) and DR3-DQ2/DR4-DQ8 prevalence (p<0.001), and lower AUC C-peptide levels (p<0.001) than those below. Those above the 2-hour glucose median had higher AUC C-peptide levels (p<0.001), but otherwise did not differ from those below. </p> <p><u>Conclusion</u>: Index60 identifies individuals with characteristics of type 1 diabetes at appreciable risk for progression who would otherwise be missed by 2-hour glucose staging criteria. </p>

Association of high-affinity autoantibodies with type 1 diabetes high-risk HLA haplotypes

Objective ECL assays are high-affinity autoantibody (Ab) tests that are more specific than Abs detected by traditional radiobinding assays (RBA) for risk screening and prediction of progression to type 1 diabetes. We sought to characterize the association of high-risk HLA haplotypes and genotypes with electrochemiluminescence (ECL) positivity and levels in relatives of individuals with type 1 diabetes. Methods We analyzed 602 participants from the TrialNet Pathway to Prevention Study who were positive for at least one RBA diabetes related Ab (GADA or IAA) and for whom ECL and HLA data were available. ECL and RBA Ab levels were converted to SD units away from mean (Z-scores) for analyses. Results Mean age at initial visit was 19.4+13.7 years; 344 (57.1%) were female and 104 (17.3%) carried the high-risk HLA- DR3/4*0302 genotype. At initial visit 424/602 (70.4%) participants were positive for either ECL-GADA or ECL-IAA, and 178/602 (29.6%) were ECL negative. ECL and RBA-GADA positivity were associated with both HLA-DR3 and DR4 haplotypes (all p&lt;0.05), while ECL and RBA-GADA z-score titers were higher in participants with HLA-DR3 haplotypes only (both p&lt;0.001). ECL-IAA (but not RBA-IAA) positivity was associated with the HLA-DR4 haplotype (p&lt;0.05). Conclusions ECL-GADA positivity is associated with the HLA-DR3 and HLA-DR4 haplotypes and levels are associated with the HLA-DR3 haplotype. ECL-IAA positivity is associated with HLA-DR4 haplotype. These studies further contribute to the understanding of genetic risk and islet autoimmunity endotypes in type 1 diabetes.

Telomere Length is Not a Main factor for the Development of Islet Autoimmunity and Type 1 Diabetes in the TEDDY Study

The Environmental Determinants of Diabetes in the Young (TEDDY) study enrolled 8,676 children, 3-4 months of age, born with HLA-susceptibility genotypes for islet autoimmunity (IA) and type 1 diabetes (T1D). Whole-genome sequencing (WGS) was performed in 1,119 children in a nested case-control study design. Telomere length was estimated from WGS data using five tools: Computel, Telseq, Telomerecat, qMotif and Motif_counter. The estimated median telomere length was 5.10 kb (IQR: 4.52 kb – 5.68 kb) using Computel. The age when the blood sample was drawn had a significant negative correlation with telomere length (P = 0.003). European children, particularly those from Finland (P = 0.041) and from Sweden (P = 0.001), had shorter telomeres than children from the U.S.A. Paternal age (P = 0.019) was positively associated with telomere length. First degree relative status, presence of gestational diabetes in the mother, and maternal age did not have a significant impact on estimated telomere length. HLA-DR4/4 or HLA-DR4/X children had significantly longer telomeres compared to children with HLA-DR3/3 or HLA-DR3/9 haplogenotypes (P = 0.008). Estimated telomere length was not significantly different with respect to any IA (P = 0.377), IAA-first (P = 0.248), GADA-first (P = 0.248) or T1D (P = 0.861). These results suggest that telomere length has no major impact on the risk for IA, the first step to develop T1D. Nevertheless, telomere length was shorter in the T1D high prevalence populations, Finland and Sweden.

A fungal antigenic driver for Löfgren’s syndrome sarcoidosis

Löfgren’s syndrome is an acute form of sarcoidosis that is characterized by the activation of CD4+ T helper cells. In this issue of JEM, Greaves et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210785) identified a peptide derived from an airborne mold species that stimulates T cells of Löfgren’s syndrome patients in an HLA-DR3–restricted manner. An increased serum IgG antibody response to the full-length protein was also observed in those patients, indicating that the fungus Aspergillus nidulans might be the elusive microbial agent that drives acute sarcoidosis.

CD4+ T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope

Löfgren’s syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3–restricted manner. Using ELISPOT analysis, a greater number of IFN-γ– and IL-2–secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

Islet autoantibody level distributions in type 1 diabetes and their association with genetic and clinical characteristics

Positivity for islet autoantibodies is used for diagnosis of type 1 diabetes. However, the importance of the autoantibody level at diagnosis of type 1 diabetes is not clear. Here, we assessed the association of glutamate decarboxylase (GADA), islet antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) autoantibody levels, measured using radiobinding assays, on genetic and clinical characteristics at diagnosis of 1536 participants with diabetes who were positive for these autoantibodies. We show that GADA and IA-2A levels had bimodal distributions, but ZnT8A level did not. The comparison of genetic and clinical characteristics between high and low level categories showed high GADA level was associated with older age at diagnosis, female sex and HLA-DR3-DQ2, whereas high IA-2A level was associated with younger age of diagnosis, ZnT8A positivity and HLA-DR4-DQ8. We replicated our findings in an independent cohort of 427 people with type 1 diabetes where autoantibodies were measured using enzyme-linked immunosorbent assays. In conclusion, Islet autoantibody levels provide additional information over positivity in type 1 diabetes at diagnosis. The bimodality of islet autoantibody levels highlights the novel aspect of heterogeneity of type 1 diabetes which may have implications on prediction, treatment and prognosis.