An Insulin-Chromogranin A Hybrid Peptide Activates DR11-Restricted T Cells in Human Type 1 Diabetes

In type 1 diabetes (T1D), insulin-producing β-cells in the pancreas are destroyed (1). Variation in the HLA class II locus represents the highest genetic risk factor, with individuals positive for HLA-DRB1*03/DQB1*0201 (DR3/DQ2) or HLA-DRB1*04/DQB1*0302 (DR4/DQ8) having the highest risk (2). Protection against disease is seen in individuals with DRB1*1501/DQB1*0602 (3,4), but other haplotypes, including DRB1*1104/DQB1*0301, DRB1*0701/DQB1*0303, and DRB1*1401/DQB1*0503 are considered protective (2). Disease incidence is increasing, and cases in individuals without high-risk HLA haplotypes are becoming more frequent, suggesting that environmental factors trigger disease in individuals with less risk (5).

Self-tolerance can be circumvented through posttranslational modifications (PTMs) that alter HLA-binding or T-cell receptor contact residues within self-peptides. Several classes of PTMs with roles in forming neoantigens have been identified (6). Hybrid insulin peptides (HIPs) are an important class of PTMs specifically linked to T1D (7). HIPs consist of proinsulin peptides covalently linked to other secretory granule peptides, generating sequences not encoded in the genome. T cells isolated from nonobese diabetic (NOD) mice and T-cell lines isolated from human organ donors with diabetes respond to HIPs (8–11). In addition, the formation of HIPs within murine and human islets has been shown by mass spectrometry (8,12,13). A recent study probed T-cell reactivity in patients with new-onset disease and documented T-cell responses toward HIPs in 30% of patients and isolating several HIP-responsive T-cell clones (14).

The aim of the present study was to isolate a T-cell clone specific for a C-peptide–WE14 HIP (namely, HIP9, the human equivalent of the murine 2.5HIP) and investigate responses to this epitope in human disease. We show that a newly isolated HIP9-specific clone is restricted by HLA-DRB1*11, a nonrisk allele. With the corresponding tetramer, we observed increased frequencies of HIP9 tetramer-labeled T cells in individuals with diabetes compared with control participants. These results provide direct evidence that this insulin–chromogranin A (CgA) HIP is relevant in human diabetes and show for the first time, to our knowledge, that HIP epitopes can be recognized in the context of nonrisk HLA haplotypes.

Peripheral blood was obtained from White individuals with T1D and from healthy control participants. All procedures were approved by the ethics committee of UZ Leuven and the institutional review boards at the Benaroya Research Institute and the University of Colorado. Study participants’ attributes are summarized in Table 1 and Supplementary Table 1. Peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Cederlane Laboratories) and frozen in 10% DMSO containing AIM V Medium (Gibco).

Recombinant HIP9 peptide (SLQPLAL-WSKMDQL) was synthesized by CHI Scientific at a purity of >95%. Recombinant PPI 9-28 (PLLALLALWGPDPAAAFVNQ) was synthesized by Genscript at a purity of >85%. Recombinant DRB1*11:01 monomers were purified from insect cell cultures as previously described (15), loaded with peptide (0.2 mg/mL) in the presence of n-dodecyl-β-maltoside (0.2 mg/mL) and Pefabloc (1 mmol/L; Sigma) for 72 h at 37°C. Peptide-loaded monomers were conjugated into tetramers using R-phycoerythrin (Southern Biotech) at a molar ratio of 8:1.

T-cell clone isolation was performed as described elsewhere (14). Briefly, carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled PBMCs were cultured with HIP9 peptide in AIM V medium (Gibco) containing human AB serum (Gemini BioProduct). CFSE^dim CD25^hi T cells were single-cell sorted and expanded in 96-well plates in the presence of recombinant human interleukin 2 (20 units/mL; Hoffman-LaRoche), interleukin 4 (5 ng/mL; R&D Systems), anti-CD3 (30 ng/mL; OKT3, eBioscience), irradiated PBMCs (1 × 10⁶/mL), and irradiated PRIESS cells (1 × 10⁶/mL; a gift from Maki Nakayama [University of Colorado Denver]).

For interferon-γ (IFN-γ) ELISA, supernatants were collected 48 h after culture and analyzed using a kit from Invitrogen (catalog 88-73160-88) according to the manufacturer’s protocol.

To confirm HIP specificity and determine HLA restriction of the HIP9 clone, 10⁴ T cells were plated in triplicate in the presence of 10⁵ irradiated autologous Epstein-Barr virus–transformed B cells or irradiated PBMCs with partially matched HLA class II as antigen-presenting cells (APCs) and pulsed with HIP9 peptide (10 μg/mL) in the presence or absence of anti–DR L243 and/or anti–DQ SPV-L3 antibodies (10 μg/mL). After incubation for 72 h at 37°C, cells were pulsed with ³H-thymidine (1 μCi/well), and incorporation was measured 18 h later with a scintillation counter. To further determine HLA specificity, triplicate wells were coated with a peptide-loaded HLA class II monomer overnight before incubation with the T-cell clone and pulsation with ³H-thymidine. Data are represented as stimulation index values, calculated by normalizing the proliferation of each clone on the basis of ³H-thymidine incorporation of control, nonpulsed APC-stimulated wells.

T cells (n = 1 × 10⁵ to 2 × 10⁵) were added to triplicate wells coated overnight with a peptide-loaded HLA class II monomer in the presence of anti–CD40 antibody (2 µg/mL) in a 37°C incubator. Cells were then stained with CD4-FITC, CD69-APC, and CD154-phycoerythrin (PE) for 20 min at 37°C, run on a BD LSR II Cytometer, and data analyzed using FlowJo (Treestar Inc.).

PBMCs (n = 3 × 10⁶) were stimulated with HIP9 in 48-well plates for 14 days. Starting on day 7, human interleukin 2 (10 units/mL) was added every 2 days. Expanded cells were stained with HIP9 tetramer for 1 h at 37°C and with CD3-BV510, CD4-APC, and CD25-FITC for 20 min at 4°C. Cells were run on a BD FACSSymphony and data analyzed using FlowJo.

Cryopreserved PBMCs (n = 20 × 10⁶ to 30 × 10⁶) were treated with dasatinib (50 nmol/L) for 10 min at 37°C and stained with 20 μg/mL PE-labeled tetramer for 90 min at room temperature. Cells were washed and labeled with anti-PE magnetic beads for 20 min at 4°C. A small fraction (2%) of the cells (pre-enriched fraction) was reserved to determine the total cell number. The remaining cells were enriched using Miltenyi Biotec MS magnetic columns. Both fractions were stained for 20 min at 4°C with CD4-BUV395, CD14-FITC, CD19-FITC, CD45RA-AF700, CXCR3-BV786, CCR7-BV650, CCR6-BV510, and CCR4-BUV605. After washing, cells were labeled with Sytox Green (ThermoFisher). Cells were run on a BD FACSSymphony and data analyzed using FlowJo.

Data were analyzed using GraphPad Prism 9 (GraphPad, La Jolla, CA). Statistical tests included an unpaired t test with Welch correction and simple linear regression. Reported error intervals all indicate SD. All significant differences are reported, with P < 0.05 considered statistically significant.

The data sets generated and analyzed during this study are available from the corresponding author upon reasonable request.

PBMCs from a 6-year-old girl (donor no. 3977) within 2 weeks of diagnosis were stimulated with HIP9 peptide, no antigen, or Pediarix (Fig. 1A). CFSE^dim CD25^hi cells were single-cell sorted and expanded to isolate a HIP9-specific T-cell clone (Fig. 1B). IFN-γ production in response to HIP9 (SLQPLAL-WSKMDQL), but not the left (GSLQPLALEGSLQKRG) or right (KEWEDSKRWSKMDQLA) peptides, confirmed that clone B4 was specific for HIP9 (Fig. 1C).

To determine the HLA restriction of the HIP9 clone, proliferation assays were performed in the presence or absence of anti–DR and/or anti–DQ blocking antibodies. Decreased proliferation with anti–DR blocking indicated the response is HLA-DR restricted (Fig. 2A). Genotyping of donor 3977 indicated DRB1*0401/DQB1*0302 and DRB1*1103/DQB1*0301 haplotypes (Supplementary Table 1). The presence of DRB1*0401 and DRB1*1103 indicates the presence of secondary DRB3 (always found with DRB1*11) and DRB4 (always found with DRB1*04) loci. Therefore, the HIP9 response could be DRB1*0401, DRB1*1103, DRB3, or DRB4 restricted. To define the HLA restriction, we performed assays using partially mismatched PBMCs as APCs. Responsiveness to DRB1*0701/DRB1*1103/DRB3/DRB4 PBMCs proved that HIP9 was recognized in the context of DRB1*1103, DRB3 or DRB4, but not DRB1*0401 (Fig. 2B). Failure to respond to DRB1*0401/DRB4 and DRB1*0901/DRB4 PBMCs confirmed the clone was not DRB4 restricted (Fig. 2B). Failure to respond to DRB1*0301/DRB3 PBMCs suggested the clone might not be DRB3 restricted, although distinct subtypes of DRB3 exist (Fig. 2B). Therefore, the clone most likely recognizes HIP9 in the context of DRB1*1103. In support of this conclusion, based on standard HLA-binding prediction methods, the HIP9 sequence is likely to bind to DRB1*1103 and other DR11 subtypes and much less likely to bind to DRB1*0401 or DRB4 (Supplementary Table 2).

Tetramer-based assays were applied to confirm DRB1*11 restriction and investigate shared recognition between DRB1*11 subtypes: DRB1*1101 tetramers were used to stain the HIP9 clone and a tetramer-stimulated proliferation assay was performed. Strong proliferation (stimulation index of 57) indicated shared recognition of the peptide between DRB1*1101 and DRB1*1103 (Fig. 2C). Activation of the clone with HIP9 presented by DRB1*1101 resulted in 26.5% CD4⁺ activated cells (CD69 and CD154 double positive), but no activation was observed in the negative control conditions (0.064% and 0.19%), confirming recognition in the context of DRB1*1101 (Supplementary Fig. 1). Staining of the HIP9 clone with DRB1*1101 tetramer resulted in 95.6% positive staining (Fig. 2D). Therefore, the clone is DR11-restricted and HIP9 can be effectively presented by DRB1*1101 and DRB1*1103.

PBMCs of three DR11⁺ individuals with T1D and three individuals without T1D were stimulated in vitro for 14 days with HIP9, followed by tetramer staining. In vitro stimulation of PBMCs of a 47-year-old individual with 13 years of disease with HIP9 resulted in a distinct tetramer-positive population (Fig. 2E), providing evidence that DRB1*11-restricted HIP9 responses can be detected in peripheral blood.

To assess disease relevance, HIP9 responses were compared in individuals with and without diabetes. PBMCs were isolated from 11 DR11⁺ individuals with diabetes and 11 DR11⁺ control participants. The average age was 31.54 ± 8.07 years for individuals with diabetes and 33.18 ± 9.67 years for control participants.

Some DR11⁺ individuals had heterozygous haplotypes that included the disease-associated DRB4 allele. In these participants, T-cell frequencies for the previously reported PPI 9-28 epitope were also determined (16). Individuals with diabetes had significantly higher HIP9 frequencies than did control participants (respective mean, 10.18 cells per million vs. 3.30 cells per million) (Fig. 3A). Furthermore, HIP9 frequencies were qualitatively similar if not higher than PPI 9-28 frequencies (5.53 cells per million) (Fig. 3A). The percentage of naïve (CD45RA⁺) HIP9-reactive cells was not different between control participants and individuals with diabetes (Supplementary Fig. 2).

We next looked for correlations between HIP9 T-cell frequency and characteristics such as age, autoantibody status, or disease duration. There was no correlation between T-cell frequencies and disease duration, but a significant correlation with age at diagnosis was observed, with individuals diagnosed at a younger age having higher frequencies (Fig. 3B). Autoantibody status at diagnosis was not available for all participants (reducing the number to 9–10 participants), but individuals with IA2 antibodies had an insignificant trend toward increased HIP9 T-cell frequencies (Fig. 3C). To conclude, individuals with T1D had higher frequencies of T cells recognizing HIP9, and frequencies correlated with age at diagnosis.

In this study, we showed that HIP9 is recognized by HLA-DRB1*11–restricted T cells, providing, to our knowledge, the first direct evidence that a CgA HIP is significantly associated with disease in human diabetes. Although DRB1*11 is not a risk haplotype, we observed increased frequencies of HIP9-reactive T cells in individuals with diabetes compared with control participants. This contrasts with the findings of a prior study that observed HIP9 responses above background in only two participant (results for the remaining 33 participants were negative in the enzyme-linked immunosorbent spot assay used). One of those participants was DRB1*0301/DQB1*02:01 homozygous, and the other was DRB1*0701/DQB1*02:02 and DRB1*1302/DQB1*06:04 positive, suggesting that HIP9 is presented by other HLA class II proteins. However, no obvious HLA allele was shared between the two responding participants. In our study, we observed an inverse correlation between the frequency of HIP9-reactive cells and age of onset, suggesting a possible pathogenic role for these T cells.

To date, all documented HIP-reactive T-cell responses have been restricted by risk-associated HLA molecules (17–19). For example, HIP-reactive T cells that recognize secretogranin I, secretogranin V, GRP78/, or insulin/insulin HIPs were restricted by DRB1*0401 (17). A T-cell clone specific for HIP11 (a C-peptide/C-peptide HIP) isolated from a patient with diabetes, was HLA-DQ2 restricted, whereas a T-cell receptor cloned from pancreatic organ donor was shown to recognize HIP11 in the context of HLA-DQ8 and HLA-DQ8trans in a different binding register (18). A pro-islet amyloid polypeptide HIP has a preferred peptide-binding motif for HLA-DQ8, and T-cell clones recognizing this HIP were isolated both from peripheral blood and pancreatic infiltrates (19). We show here (for the first time, to our knowledge) that a T-cell clone restricted by a nonrisk HLA (DRB1*11) recognizes a CgA HIP. This is a fundamentally important observation because this implies that HIPs can be presented to T cells in individuals with diverse HLA haplotypes, including individuals who do not carry risk alleles. This also suggests that T cells restricted by nonrisk HLA may play a role in T1D.

Individuals with HLA-DRB1*1501/DQB1*0602 are protected (DQB1*0602 has an odds ratio of 0.03) but can occasionally develop disease if they are heterozygous for the high-risk DQ8 allele. According to Erlich et al. (4), DRB1*1104 in combination with DQB1*0301 is the fourth most protective haplotype in T1D. Other DR11 subtypes (DRB1*1101, DRB1*1102, and DRB1*1103 in combination with DQB1*0301) were also seen more frequently in control participants (4). It is interesting that we found increased HIP9 frequencies in individuals with diabetes and a significant negative correlation between frequencies and age at disease onset. T1D incidence is increasing, with a higher incidence in individuals with nonrisk HLA haplotypes (20). It has been speculated that unknown epidemiological and environmental factors can promote disease development in individuals with a lower genetic risk. Disease in such individuals would need to be driven by T-cell responses that are restricted by neutral or even protective HLA alleles, such as those we report here.

The age at diagnosis of our DRB1*11 cohort was 22.18 ± 7.70 years, which is older than the typical age of onset (e.g., 13 years in some populations) (21). Because hybrid forms of diabetes that include some features of type 2 diabetes have been described, we scrutinized the BMI of participants in our cohort. The cohort had an average BMI of 25.53, which is not above the populational average. Of the nine participants for whom autoantibody levels against INS, GAD, and IA2 were measured at diagnosis, only one individual was negative for all three autoantibodies. Two individuals were positive for a single autoantibody, five individuals had autoantibodies against either INS and GAD or GAD and IA2, and one individual was positive for three autoantibodies. The presence of autoantibodies in 9 of 10 participants suggests that this cohort has conventional T1D despite having low-risk HLA haplotypes.

In a recent review, authors proposed endotypes in T1D: endotype 1 with an earlier onset (<13 years of age) and primarily T-cell driven, and endotype 2 with an older onset (>13 years of age) and primarily β-cell driven, with β-cell endoplasmic reticulum stress contributing to pathogenesis (22). In our cohort, only one individual was diagnosed at an age <13 years. The absence of high-risk alleles in most individuals in our cohort could have resulted in a shift toward a later disease onset. Individuals with an earlier age at diagnosis within our cohort had higher T-cell frequencies. It would be of interest to investigate a larger cohort of individuals with low-risk HLA genotypes to verify an association between ages of onset and T-cell frequency. However, recruiting such a cohort may be challenging, given the low prevalence of this allele in individuals with T1D.

The murine 2.5HIP is a very potent ligand for the murine BDC-2.5 T-cell clone. T cells recognizing the 2.5HIP are present at high frequencies in the islets of NOD mice as well as in the pancreatic lymph node, where they acquire an antigen-experienced phenotype. Memory T cells reactive with the 2.5HIP were detectable in the peripheral blood of NOD mice at increasing frequencies throughout disease progression (11). NOD mice deficient for CgA are protected from the development of autoimmune diabetes, establishing the importance of CgA-reactive T cells in autoimmune diabetes (23). In this study, we provide the first direct evidence, to our knowledge, that the analogous CgA HIP is relevant in human T1D. The existence of HIP9-reactive T-cell responses that are restricted by nonrisk HLA proteins supports the general conclusion that that T-cell responses restricted by nonrisk HLA can be associated with disease and provides a plausible mechanism for the expansion of disease-driving T-cell responses in individuals with nonrisk HLA haplotypes.

Acknowledgments. The authors thank Hilde Morobé for patient recruitment and collecting blood samples and all the patients and control participants who donated blood samples. We acknowledge Thien-Son Nguyen and the BRI Clinical Core for sample management, the Benaroya Research Institute Flow Cytometry Core Facility for assistance with flow cytometry, and the BRI tetramer core for providing HLA class II proteins.

Funding. This work was supported by the National Institutes of Health National Institute of Allergy and Infectious Diseases (R21 AI133059 and R01 AI146202-01 to R.L.B.); the National Institute of Diabetes and Digestive and Kidney Diseases (grant R01 DK081166 to K.H.); and a JDRF postdoctoral fellowship to A.C. (grant 3-PDF-2023-1328-A-N).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. All authors contributed to the design and conduct of the study; to data analysis and interpretation; writing the manuscript; and approving the final version. E.A.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. This work was previously presented at the 19th Immunology of Diabetes Society Congress, Paris, France, 23–27 May 2023, and the 23rd Annual Meeting of the Federation of Clinical Immunology Societies (FOCIS 2023), Boston, MA, 20–23 June 2023.