Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

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Science  12 Feb 2016:
Vol. 351, Issue 6274, pp. 711-714
DOI: 10.1126/science.aad2791

T cells target peptide combos

One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease.

Science, this issue p. 711


T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Type 1 diabetes (T1D) is an autoimmune disease mediated by T cells responding to self-antigens in the pancreatic β cell. Studies in the widely used nonobese diabetic (NOD) mouse model of T1D have shown that CD4 T cell responses to several β cell proteins, especially (pro)insulin, likely contribute to diabetes. It is, however, unclear how pathogenic T cells escape thymic deletion and how (pro)insulin becomes a target of the autoimmune T cell response. To address these questions, we have used our panel of NOD-derived, diabetogenic CD4 T cell clones (including the well-known BDC-2.5 clone) (1), in conjunction with proteomic analysis of β cell extracts, to identify T cell target antigens. Recently, we reported two new autoantigens for CD4 T cells in autoimmune diabetes: chromogranin A (ChgA) (2) and islet amyloid polypeptide (IAPP) (3). Like insulin, ChgA and IAPP are β cell prohormonal secretory granule proteins. WE14, a naturally occurring peptide cleavage product of ChgA, was found to be antigenic in both the NOD mouse (2) and in T1D patients (4). However, because this peptide only stimulates T cells weakly, we hypothesized that the natural ligand for pathogenic CD4 T cells may be a modified form of ChgA.

Posttranslational modification is a well-established property of antigens in many autoimmune diseases (5). A notable exception is T1D in which the investigation of modified peptides as antigenic epitopes has only just begun (69). Using mass spectrometric analysis, we verified the presence of the peptide WE14 in chromatographic fractions of mouse β cell extracts, but the peptide distribution over individual fractions did not follow the antigen distribution of the natural ligand recognized by WE14-responsive T cell clones, including BDC-2.5 (fig. S1A, top). Conversely, the propeptide region (C-peptide) from mouse proinsulin (fig. S1A, bottom) did follow the antigen distribution profile. Furthermore, a broad panel of shorter C-peptide fragments could also be identified in peak antigenic fractions (fig. S1B). The matching antigen–C-peptide distributions suggested that a C-peptide fragment is a component of the natural T cell ligand. The proposed WE14/I-Ag7 binding register (2), in which the peptide WE14 fills only half of the major histocompatibility complex (MHC) II binding groove (positions 5 to 9), leaves MHC II positions 1 to 4 unoccupied. The C-peptide fragments could fill these unoccupied positions, which would lead to increased peptide-MHC binding affinity and provide additional residues for improved TCR recognition. We hypothesized that C-terminal carbonyl groups of C-peptide fragments form covalent bonds with N-terminal amino groups of naturally occurring peptides, such as WE14, which results in the formation of hybrid insulin peptides (HIPs).

To investigate the possibility that BDC-2.5 and additional diabetogenic T cell clones from our panel are activated by HIPs, we used a chemical cross-linking strategy (fig. S2) to synthesize a HIP library for screening of our murine CD4 T cell clones (Fig. 1A). The peptides in the library consist of insulin sequences (left peptides) on the N-terminal side, covalently linked to peptide sequences from other secretory granule proteins (right peptides) on the C-terminal side. As left peptides, we selected C-terminal sequences derived from proinsulin C-peptide and insulin B-chain fragments that were identified through mass spectrometry in high abundance in antigenic β cell fractions (tables S1 and S2). As right peptides, we used N-terminal sequences from the ChgA peptide WE14 and the three natural cleavage products (10) of IAPP (amylin, IAPP1, and IAPP2). As shown in Fig. 1A, the hybrid peptide LQTLAL-WSRMD, containing the C-peptide sequence LQTLAL on the left and the WE14 sequence WSRMD on the right, activates all three WE14-reactive clones (BDC-2.5, BDC-10.1, and BDC-9.46), each of which expresses a distinct T cell receptor (TCR). To further test our hypothesis, we screened the peptide library with a second subset of T cell clones (BDC-6.9 and BDC-9.3) sharing the same TCR. We identified a single HIP sequence (LQTLAL-NAARD) recognized by both BDC-6.9 and BDC-9.3 (Fig. 1A). This peptide contains, on the left, the same C-peptide sequence LQTLAL present in the HIP recognized by the WE14-reactive T cell clones and, on the right, the IAPP propeptide 2 (IAPP2) sequence NAARD. We concluded that the endogenous ligands for the two sets of pathogenic T cell clones are HIPs containing the C-peptide sequence LQTLAL on the left side and the natural cleavage products, WE14 or IAPP2, on the right side.

Fig. 1 Identification of HIP sequences that are recognized by pathogenic T cell clones.

(A) A library of 32 HIPs was synthesized. Left peptides are C-terminal amino acid sequences of various mouse insulin C-peptide and B-chain fragments. Right peptides reflect the N-terminal amino acid sequences of mouse WE14 (WSRMD), IAPP1 (TPVRS), amylin (KCNTA), and IAPP2 (NAARD). T cell clones BDC-2.5, BDC-9.46, BDC-10.1, BDC-9.3, and BDC-6.9 were used to screen the peptide library. Black squares indicate positive T cell responses measured through production of interferon-γ (IFN-γ) to individual HIPs. Data shown are representative of three independently replicated experiments. (B) To confirm antigenicity of HIPs, pure peptides (>95%) were obtained commercially for assay with the T cell clones. Response of WE14-reactive clone BDC-2.5 to 2.5HIP (solid squares) compared with unmodified WE14 (open diamonds), Ins2 C-peptide (open circles), or the Ins2 C-peptide fragment ending with the amino acid sequence DLQTLAL (solid circles). (C and D) Response of IAPP-reactive clones, BDC-6.9 (C) and BDC-9.3 (D), to the 6.9HIP (solid squares) compared with IAPP2 alone (open diamonds), Ins2 C-peptide (open circles), or the Ins2 C-peptide fragment (solid circles). Data are representative of at least three separate experiments.

To confirm the antigenicity of the peptides identified from the library, we obtained commercially synthesized preparations of 2.5 and 6.9HIPs (HIPs antigenic for BDC-2.5 and BDC-6.9) spanning the full-length insulin II (Ins2) C-peptide fragment ending in LQTLAL on the N-terminal ends and the entire WE14 or IAPP2 sequences on the C-terminal ends (see peptide section in Materials and Methods). WE14-reactive T cell clones such as BDC-2.5 recognize the 2.5HIP at low nanomolar concentrations, whereas unmodified WE14 is poorly antigenic for these clones (2), which mandates high peptide concentrations (>10 μM) for T cell activation (Fig. 1B); hence, the HIP was >10,000 times as potent as a stimulator for BDC-2.5 than WE14. The T cell clones BDC-6.9 and BDC-9.3 recognize the 6.9HIP, also at low nanomolar concentrations (Fig. 1, C and D). BDC-6.9 and BDC-9.3 do not react to the unmodified IAPP2 peptide, and none of the clones respond to the full-length Ins2 C-peptide or the C-peptide fragment ending with the amino acid sequence LQTLAL. T cell reactivity with WE14 or IAPP2 was not altered if these peptides were coincubated (without cross-linking) with the C-peptide fragment ending in LQTLAL, which indicated that the covalent attachment of the peptides is required to obtain full T cell activation.

To validate the in vivo presence of HIPs in β cells, we fractionated murine β cell extracts by reversed-phase high-performance liquid chromatography (RP-HPLC) and performed mass spectrometric (MS) analyses on antigenic fractions. The T cell clone BDC-2.5 responded to two chromatographic regions, which indicated that at least two distinct ligands (left and right peak) exist for this T cell clone (Fig. 2A). After proteolytic digestion and tandem MS (MS/MS) analysis of the left antigen peak (Fig. 2A), we identified the peptide DLQTLAL-WSRM (Fig. 2B and fig. S3). This spectrum provides physical evidence verifying the presence of the 2.5HIP in murine β cells. We have not identified a HIP in the antigen peak on the right, possibly because of limited instrumental sensitivity, poor peptide ionization, or the presence of yet another unknown HIP sequence. Purification of the endogenous ligand recognized by BDC-6.9 and BDC-9.3 (fig. S4A), followed by MS analysis, led to the identification of the corresponding 6.9HIP sequence DLQTLAL-NAAR (fig. S4, B and C). Both the 2.5HIP and the 6.9HIP contain the C-peptide fragment ending with the amino acid sequence DLQTLAL, a naturally occurring cleavage product of insulin C-peptide formed within the secretory granules of β cells (11). This fragment may thus provide a preferred ligation site for the formation of HIPs, but additional insulin ligation sites may also exist.

Fig. 2 Identification of HIPs in antigenic β cell fractions.

(A) Size-exclusion chromatography fractions highly enriched for antigen were further fractionated by RP-HPLC (black line). IFN-γ production by T cells in response to individual fractions is shown for BDC-2.5 (gray line). (B) After proteolytic digestion with the endoproteinase AspN (which releases the core amino acid sequence of the hybrid peptide), the targeted MS/MS analysis of antigen-containing fractions reveals the spectrum of the HIP that contains the C-peptide amino acid sequence DLQTLAL and the WE14 sequence WSRM.

Identifying the autoreactive T cells involved in the pathogenic process is a major objective for T1D research, and achieving this goal may enable us to monitor the disease process and devise antigen-specific methods to prevent and/or reverse disease. MHC class II tetramer reagents can be used to specifically identify pathogenic T cells (12). To determine whether HIP-reactive T cells could be identified in diabetic NOD mice, we designed a peptide:MHC class II (I-Ag7) tetramer containing the HIP sequence antigenic for BDC-2.5; specificity of the 2.5HIP tetramer was demonstrated through staining of the three 2.5HIP-reactive CD4 T cell clones (fig. S5A). To examine the presence of HIP-reactive T cells in NOD mice, cell suspensions from spleens, pancreatic lymph nodes (pLNs), and pancreata of diabetic NOD mice were stained with the 2.5HIP tetramer (Fig. 3 and fig. S5B). Two additional tetramers, pS3 and 2.5mi, containing peptide mimotope sequences recognized by BDC-2.5 (2, 13), were used as positive controls. We found that 2.5HIP tetramer–positive cells were present in both the spleen and pancreas of NOD mice. In both tissues, the 2.5HIP tetramer stained a higher percentage of CD4 T cells compared with the 2.5mi and the pS3 tetramers, a difference that was statistically significant in the pancreas and that indicated that the 2.5HIP tetramer is more efficient in the detection of 2.5HIP-reactive CD4 T cells. Our data show that most of the 2.5HIP tetramer–positive cells in the spleen were CD44 high, which indicated that a significant proportion of these cells were antigen-experienced and further substantiated their involvement in the pathogenic process.

Fig. 3 Tetramer analysis of pancreas and spleen of diabetic NOD mice.

Single-cell suspensions were prepared from pancreas and spleen of diabetic NOD female mice (n = 8) and stained with tetramers, antibodies, and a dead cell marker (7AAD). Gates were set on a lymphocyte gate and CD45+, CD4+, Lin cells. (A) Tetramer staining in the pancreas and the spleen of a representative mouse. (B) Summary of CD4+ tetramer–positive cells present in the pancreas. (C) Summary of CD4+CD44hi tetramer–positive cells present in the spleen. Each symbol represents a different mouse. Averages are indicated as a black horizontal bar. Data are from four independent experiments and were analyzed by two-tailed unpaired t test. Statistical significance * was defined at a *P < 0.05.

To determine whether human CD4 T cells of individuals with T1D recognize HIPs, we screened a selection of HIPs (Fig. 4A) using CD4 T cells isolated from the residual islets of two patients. Corresponding human sequences of naturally occurring peptides found in mouse β cell extracts formed the right portion of the HIPs. The central C-peptide amino acid sequences ELGG or ELGGG, which form the left half of HIPs 1 to 8, satisfy parameters for binding to DQ8 positions 1 and 4 (14). Risk of developing T1D is strongly associated with human leukocyte antigen (HLA) alleles, and HLA-DQ8 confers the highest risk of developing T1D of any single HLA allele (15). For the left sides of HIPs 9 to 16, we used the human version (SLQPLAL) of the murine peptide sequence present in the 2.5 and 6.9HIPs. From one organ donor, 17 CD4 T cell lines were isolated from residual islets, expanded, and tested for reactivity against eight of the HIPs (Fig. 4A, peptides 1 to 8) with Priess B cells (DR4+/+,DQ8+/+) as presenters. The CD4 line MG1 responded to the HIP formed by fusion of proinsulin C-peptide to the pancreatic peptide, neuropeptide Y (GQVELGGG-SSPETLI) (Fig. 4B). Using CD4 T cell clones isolated from a second organ donor (16), we screened all 16 HIPs (Fig. 4A). Two clones responded to the same HIP that was formed by the fusion of proinsulin C-peptide to IAPP2 (GQVELGGG-NAVEVLK) (Fig. 4, C and D). Both clones responded weakly to proinsulin C-peptide (proinsulin 37–54), but the HIP was 1000 to 10,000 times as potent. Both clones recognized HIP peptides presented by HLA-DQ8 (DQA1*03:01; DQB1*03:02) (fig. S6). Although pathogenicity of human T cells cannot be directly demonstrated, the presence of HIP-specific CD4 T cells within the pancreatic islets of individuals with T1D supports the contention that these cells play a pathogenic role in human T1D.

Fig. 4 Human islet-infiltrating T cells respond to HIPs.

(A) List of HIPs (>95% purity) screened. (B) A CD4 T cell line grown from isolated islets of a 20-year-old male with 7 years of T1D (DR17, DR4, DQ2, and DQ8) secreted IFN-γ when a HIP, formed by the fusion of proinsulin C-peptide to the pancreatic polypeptide, neuropeptide Y (GQVELGGG-SSPETLI), was presented by Priess B cells (DR4+/+, DQ8+/+); all peptides at 20 μg/ml. *P = 0.0395 as compared with the media control (paired Student’s t test). The mean and standard deviation of triplicates is shown. (C and D) CD4 T cell clones A3.10 (C) and A2.11 (D) were isolated from islets of a 19-year-old male with 3 years of T1D and were incubated overnight with the indicated concentration of peptide and HLA-DQ2+, DQ8+ Epstein-Barr virus–transformed B cell line. Responses to peptide were detected by IFN-γ secretion measured by enzyme-linked immunosorbent assay (ELISA). OD, optical density. Solid circles are the HIP (GQVELGGG-NAVEVLK), open circles are proinsulin 37 to 54 (LQVELGGG-PGAGSLQ), and open squares are another HIP incorporating the same region of IAPP2 (SLQPLAL-NAVEVLK). For (C) and (D), one representative of at least two independent experiments is shown. Clone A3.10 and A2.11 (fig. S6) recognize HIPs presented by HLA-DQ8.

HIPs represent not only a novel posttranslational modification in autoimmune disease, but they also constitute a new family of autoantigens in T1D. Hybrid peptides that form through protein splicing have been previously noted in tumor antigen formation, a process that occurs through the proteosomal class I pathway and yields antigens for CD8 T cells (1719). The formation of HIPs in the secretory granules of β cells could be a side reaction of the proteolytic hydrolysis of peptide bonds in the presence of naturally occurring cleavage products (e.g., WE14), and molecular crowding of peptides in secretory granules may favor this reversed proteolytic transpeptidation (20). The identification of HIP-reactive CD4 T cells from the islets of individuals with T1D, together with the demonstration that several distinct pathogenic CD4 T cells from NOD mice target different HIPs in β cells, strongly suggests that HIPs play a central role in the pathogenesis of T1D. We hypothesize that HIPs are key antigens for autoreactive T cells and are responsible for the loss of self-tolerance in human T1D. We would also speculate that hybrid peptides may form in the secretory granules of other endocrine tissues and could be a source of self-antigens in other autoimmune disorders.

Supplementary Materials

Materials and Methods

Figs. S1 to S6

Tables S1 and S2

References (2126)


Acknowledgments: The following funding sources supported this research: NIH grant 1K01DK094941 (T.D.); American Diabetes Association Pathway to Stop Diabetes Grant 1-15-ACE-14 (T.D.); American Diabetes Association grant 1-15-JF-04 (R.L.B.); NIH grant 1R01DK081166 (K.H.); the Australian National Health and Medical Research Council (NHMRC) APP1061961 (S.M.); Juvenile Diabetes Research Foundation, Career Development Award 5-CDA2014210-A-N (S.M.); Operational Infrastructure Support Program from the Victorian State Government of Australia (S.M.); Helmsley Charitable Trust 2015PG-T1D057 (S.C.K.); Juvenile Diabetes Research Foundation grant 2-SRA-2015-68-Q-R (A.C.P., D.M.H.); NIH grant 5U01DK89572 (A.C.P., D.M.H.); NIH grant DK104211 (A.C.P). We thank the NIH tetramer core for providing tetramer reagents. A patent application related to the work on the hybrid peptides has been filed (Colorado University Technology Transfer Office file no. CU3769H-PPA1, Compositions and methods for diagnosing autoimmune diseases; T.D. and K.H. as inventors). The data presented in this manuscript are tabulated in the main paper and in the supplementary materials section.

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