Hybrid insulin peptides (HIPs) formed through covalent cross-linking of proinsulin fragments to secretory granule peptides are detectable within murine and human islets. The 2.5HIP (C-peptide–chromogranin A [CgA] HIP), recognized by the diabetogenic BDC-2.5 clone, is a major autoantigen in the nonobese diabetic mouse. However, the relevance of this epitope in human disease is currently unclear. A recent study probed T-cell reactivity toward HIPs in patients with type 1 diabetes, documenting responses in one-third of the patients and isolating several HIP-reactive T-cell clones. In this study, we isolated a novel T-cell clone and showed that it responds vigorously to the human equivalent of the 2.5HIP (designated HIP9). Although the responding patient carried the risk-associated DRB1*04:01/DQ8 haplotype, the response was restricted by DRB1*11:03 (DR11). HLA class II tetramer staining revealed higher frequencies of HIP9-reactive T cells in individuals with diabetes than in control participants. Furthermore, in DR11+ participants carrying the DRB4 allele, HIP9-reactive T-cell frequencies were higher than observed frequencies for the immunodominant proinsulin 9-28 epitope. Finally, there was a negative correlation between HIP9-reactive T-cell frequency and age at diagnosis. These results provide direct evidence that this C-peptide–CgA HIP is relevant in human type 1 diabetes and suggest a mechanism by which nonrisk HLA haplotypes may contribute to the development of β-cell autoimmunity.

Article Highlights
  • A hybrid insulin peptide formed from C-peptide–WE14 is a potent ligand for the diabetogenic BDC-2.5 clone, but its relevance in human disease is unclear.

  • A human T-cell clone specific for the C-peptide–WE14 HIP is restricted by HLA-DRB1*11, a nonrisk allele.

  • Using an HLA class II tetramer, we observed increased frequencies of HIP-reactive cells T cells in individuals with diabetes compared with control participants and an inverse correlation between frequency and age of onset.

  • These results suggest a pathogenic role for the C-peptide–WE14 HIP and provide a plausible mechanism for the expansion of disease-driving T-cell responses in individuals with nonrisk HLA haplotypes.

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 (811). 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.

Human Participants and Peripheral Blood Mononuclear Cell Isolation

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).

Table 1

Characteristics of peripheral blood donors

Donor no.*SexAge (years)HLA-DRB1HLA-DQB1T1D duration
(years)
AutoantibodiesBMI
(kg/m2)
3977§ 0401/1103 0301/0302 INS, GAD, IA2, ZnT8 UNK 
T1D 01 28 07/11 UNK GAD, IA2 24.2 
T1D 02 22 0701/1101 UNK IA2 22.1 
T1D 03 26 0404/11 UNK — 27.2 
T1D 04 25 0102/1101 0301/0501 GAD, IA2 24.5 
T1D 05 22 0401/11 0301/0302 GAD, IA2 27.2 
T1D 06 34 07/11 02/0301 INS, GAD 30.5 
T1D 07 38 0101/1101 UNK 10 INS 23.4 
T1D 08 44 0701/1104 0202/0301 10 UNK 27.4 
T1D 09 47 1101/1201 0301/0301 13 INS, GAD 23.2 
T1D 10 29 0404/1104 0301/0302 14 INS, GAD, IA2 26.5 
T1D 11 32 0801/1104 0301/0402 23 IA2 24.6 
CTR 01 21 0401/11 UNK NA NA UNK 
CTR 02 44 1101/1301 0301/0601 NA NA UNK 
CTR 03 51 1101/1501 0301/0602 NA NA UNK 
CTR 04 25 11/15 UNK NA NA UNK 
CTR 05 27 11/13 UNK NA NA UNK 
CTR 06 22 07/11 UNK NA NA UNK 
CTR 07 30 0401/1101 0301/0302 NA NA UNK 
CTR 08 35 0701/11 02/0301 NA NA UNK 
CTR 09 30 0701/11 0202/0301 NA NA UNK 
CTR 10 47 0401/1101 0301/0301 NA NA UNK 
CTR 11 33 0401/1101 0301/0302 NA NA UNK 
Donor no.*SexAge (years)HLA-DRB1HLA-DQB1T1D duration
(years)
AutoantibodiesBMI
(kg/m2)
3977§ 0401/1103 0301/0302 INS, GAD, IA2, ZnT8 UNK 
T1D 01 28 07/11 UNK GAD, IA2 24.2 
T1D 02 22 0701/1101 UNK IA2 22.1 
T1D 03 26 0404/11 UNK — 27.2 
T1D 04 25 0102/1101 0301/0501 GAD, IA2 24.5 
T1D 05 22 0401/11 0301/0302 GAD, IA2 27.2 
T1D 06 34 07/11 02/0301 INS, GAD 30.5 
T1D 07 38 0101/1101 UNK 10 INS 23.4 
T1D 08 44 0701/1104 0202/0301 10 UNK 27.4 
T1D 09 47 1101/1201 0301/0301 13 INS, GAD 23.2 
T1D 10 29 0404/1104 0301/0302 14 INS, GAD, IA2 26.5 
T1D 11 32 0801/1104 0301/0402 23 IA2 24.6 
CTR 01 21 0401/11 UNK NA NA UNK 
CTR 02 44 1101/1301 0301/0601 NA NA UNK 
CTR 03 51 1101/1501 0301/0602 NA NA UNK 
CTR 04 25 11/15 UNK NA NA UNK 
CTR 05 27 11/13 UNK NA NA UNK 
CTR 06 22 07/11 UNK NA NA UNK 
CTR 07 30 0401/1101 0301/0302 NA NA UNK 
CTR 08 35 0701/11 02/0301 NA NA UNK 
CTR 09 30 0701/11 0202/0301 NA NA UNK 
CTR 10 47 0401/1101 0301/0301 NA NA UNK 
CTR 11 33 0401/1101 0301/0302 NA NA UNK 

—, negative for all three autoantibodies; GAD, glutamic acid decarboxylase 65; IA2, tyrosine phosphatase-related islet antigen 2; INS, insulin; NA, not applicable; UNK, unknown; ZnT8, zinc transporter 8.

*

Donor numbers of DRB4-positive individuals are underlined.

The mean age of patients was 31.54 years; the mean age of control participants was 33.18 years.

The average duration of disease was 9.45 years.

§

ZnT8 was only determined in donor 3977.

GAD and IA2 autoantibody levels are unknown.

Peptides, HLA Class II Protein, and Tetramer Reagents

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

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). CFSEdim CD25hi 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]).

Interferon-γ ELISA

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.

Proliferation Assay

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.

CD154 CD69 Activation Assay

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.).

In Vitro Tetramer Assays

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.

Ex Vivo Tetramer Assays

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.

Statistical Analyses

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.

Data and Resource Availability

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

Isolation of a CD4 T-Cell Clone Specific for HIP9

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). CFSEdim CD25hi 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).

Figure 1

Isolation of a CD4 T-cell clone specific for HIP9. A: CFSE and CD25 staining of CFSE-labeled PBMCs from donor 3977 with T1D cultured with HIP9 peptide, no antigen as negative control, and Pediarix as positive control. B: CFSEdim CD25hi T cells were single-cell sorted and expanded in 96-well plates in the presence of recombinant human interleukin 2 (20 units/mL), interleukin 4 (5 ng/mL), anti-CD3 (30 ng/mL), irradiated PBMCs (1 × 106/mL), and irradiated PRIESS cells (1 × 106/mL). C: IFN-γ production of the HIP9 clone in response to increasing concentrations of HIP9 (SLQPLAL-WSKMDQL) peptide or the left (SLQPLAL) or right (WSKMDQL) control peptides. OD, optical density.

Figure 1

Isolation of a CD4 T-cell clone specific for HIP9. A: CFSE and CD25 staining of CFSE-labeled PBMCs from donor 3977 with T1D cultured with HIP9 peptide, no antigen as negative control, and Pediarix as positive control. B: CFSEdim CD25hi T cells were single-cell sorted and expanded in 96-well plates in the presence of recombinant human interleukin 2 (20 units/mL), interleukin 4 (5 ng/mL), anti-CD3 (30 ng/mL), irradiated PBMCs (1 × 106/mL), and irradiated PRIESS cells (1 × 106/mL). C: IFN-γ production of the HIP9 clone in response to increasing concentrations of HIP9 (SLQPLAL-WSKMDQL) peptide or the left (SLQPLAL) or right (WSKMDQL) control peptides. OD, optical density.

Close modal

The HIP9 T-Cell Clone Is HLA-DRB1*11–Restricted

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).

Figure 2

The HIP9 clone is HLA-DR restricted and recognized in the context of DRB1*1103 and DRB1*1101. A: Proliferation of the HIP9-specific T-cell clone in response to APCs pulsed with HIP9 (10 μg/mL) with or without anti–HLA-DR (aDR) and anti–HLA-DQ (aDQ) blocking antibodies. B: Proliferation of the HIP9-specific T-cell clone in response to partially HLA-matched PBMCs. C: Proliferation of the HIP9-specific T-cell clone in response to DRB1*1101 HLA molecules presenting the HIP9 peptide (10 μg/mL). D: Staining of the HIP clone with HLA-DRB1*1101 tetramer (tmr) loaded with HIP9. The gates were set on the basis of staining with an empty tetramer. E: Tetramer staining of PBMCs stimulated in vitro for 14 days with HIP9 peptide of three individuals with (T1D#2, 5, and 9) and three individuals without (CTR#2, 4, and 5) T1D. A and C: Data are represented as stimulation index values, calculated by normalizing the proliferation of each clone on the basis of 3H-thymidine incorporation of control nonstimulated wells. A stimulation index >3 is considered positive. Data are reported as mean with SEM (technical error bars). Ag, antigen; ctr, control; neg, negative.

Figure 2

The HIP9 clone is HLA-DR restricted and recognized in the context of DRB1*1103 and DRB1*1101. A: Proliferation of the HIP9-specific T-cell clone in response to APCs pulsed with HIP9 (10 μg/mL) with or without anti–HLA-DR (aDR) and anti–HLA-DQ (aDQ) blocking antibodies. B: Proliferation of the HIP9-specific T-cell clone in response to partially HLA-matched PBMCs. C: Proliferation of the HIP9-specific T-cell clone in response to DRB1*1101 HLA molecules presenting the HIP9 peptide (10 μg/mL). D: Staining of the HIP clone with HLA-DRB1*1101 tetramer (tmr) loaded with HIP9. The gates were set on the basis of staining with an empty tetramer. E: Tetramer staining of PBMCs stimulated in vitro for 14 days with HIP9 peptide of three individuals with (T1D#2, 5, and 9) and three individuals without (CTR#2, 4, and 5) T1D. A and C: Data are represented as stimulation index values, calculated by normalizing the proliferation of each clone on the basis of 3H-thymidine incorporation of control nonstimulated wells. A stimulation index >3 is considered positive. Data are reported as mean with SEM (technical error bars). Ag, antigen; ctr, control; neg, negative.

Close modal

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.

Individuals With T1D Have Elevated HIP9-Specific T-Cell Frequencies That Correlate With Clinical Characteristics

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).

Figure 3

Individuals with T1D have increased HIP9 T-cell frequencies, and the frequency correlates with clinical characteristics. A: Total HIP9 and PPI 9-28 CD4+ T-cell frequencies in the enriched fraction in control participants (CTRs) and individuals with T1D. B: Applying linear regression analysis indicated a significant negative correlation between HIP9 frequencies and age at diagnosis (P = 0.012) and no correlation between frequencies and duration of disease. C: Comparison of HIP9 T-cell frequencies in individuals with negative (Neg) versus positive (Pos) insulin antibodies (cutoff, 0.6% binding), negative versus positive GAD autoantibodies (cutoff, 23 units/mL), and negative versus positive IA2 antibodies (cutoff, 1.4 units/mL). Data in A and C are reported as mean with SEM. Unpaired t test with Welch correction (A), simple linear regression (B), and unpaired t test (C) were used. *P < 0.05.

Figure 3

Individuals with T1D have increased HIP9 T-cell frequencies, and the frequency correlates with clinical characteristics. A: Total HIP9 and PPI 9-28 CD4+ T-cell frequencies in the enriched fraction in control participants (CTRs) and individuals with T1D. B: Applying linear regression analysis indicated a significant negative correlation between HIP9 frequencies and age at diagnosis (P = 0.012) and no correlation between frequencies and duration of disease. C: Comparison of HIP9 T-cell frequencies in individuals with negative (Neg) versus positive (Pos) insulin antibodies (cutoff, 0.6% binding), negative versus positive GAD autoantibodies (cutoff, 23 units/mL), and negative versus positive IA2 antibodies (cutoff, 1.4 units/mL). Data in A and C are reported as mean with SEM. Unpaired t test with Welch correction (A), simple linear regression (B), and unpaired t test (C) were used. *P < 0.05.

Close modal

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 (1719). 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.

This article contains supplementary material online at https://doi.org/10.2337/figshare.25103864.

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