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Recognition of Multiple Hybrid Insulin Peptides by a Single Highly Diabetogenic T-Cell Receptor

The mechanisms underlying the major histocompatibility complex class II (MHCII) type 1 diabetes (T1D) association remain incompletely understood. We have previously shown that thymocytes expressing the highly diabetogenic, I-Ag7-restricted 4.1-T-cell receptor (TCR) are MHCII-promiscuous, and that, in MHCII-heterozygous mice, they sequentially undergo positive and negative selection/Treg deviation by recognizing pro- and anti-diabetogenic MHCII molecules on cortical thymic epithelial cells and medullary hematopoietic antigen-presenting cells (APCs), respectively. Here, we use a novel autoantigen discovery approach to define the antigenic specificity of this TCR in the context of I-Ag7. This was done by screening the ability of random epitope–GS linker–I-Aβg7chain fusion pools to form agonistic peptide–MHCII complexes on the surface of I-Aαd chain-transgenic artificial APCs. Pool deconvolution, I-Ag7-binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif XL(G/A)XEXE(D/E)X that was shared by seven agonistic hybrid insulin peptides (HIPs) resulting from the fusion of several different chromogranin A and/or insulin C fragments, including post-translationally modified variants. These data validate a novel, highly sensitive MHCII-restricted epitope discovery approach for orphan TCRs and suggest thymic selection of autoantigen-promiscuous TCRs as a mechanism for the murine T1D–I-Ag7-association.


INTRODUCTION
Compelling experimental evidence have shown that type 1 diabetes (T1D) onset and progression require the activation and recruitment of autoreactive CD4+ T cells. In turn, these T cells orchestrate the activation of downstream effectors of diabetogenic autoimmunity, such as B cells and beta cell-cytotoxic CD8+ T cells (1). In non-autoimmune-prone individuals, autoreactive T cells are either deleted (thymocyte negative selection) or programmed to become autoreactive regulatory T cells (2). Both pathways require the recognition of cognate autoantigenic epitopes that are either expressed in thymic medullary epithelial cells or are delivered to the thymus by bone marrow-derived antigen-presenting cells (APCs), such as dendritic cells (DCs). As a result, thymocytes expressing TCRs capable of recognizing peptide-major histocompatibility complexes (pMHC) displaying epitopes that are selectively expressed in peripheral tissues, such as post-translationally modified epitopes or hybrid insulin peptides (HIPs), have the highest likelihood to contribute to the initiation and/or progression of autoimmunity when appropriately recruited and activated (3)(4)(5).
It has been established that MHCII polymorphisms, particularly around b-chain position 57 (b57), afford susceptibility/resistance to autoimmune diseases, including T1D, in both mice and humans, through poorly understood mechanisms (2,6,7). Human T1D is primarily associated with HLA-DQ polymorphisms. Whereas alleles encoding DQb chains carrying Ala, Val, or Ser at position 57 afford risk, those carrying Asp at this position afford protection (2,6,7). The nonobese diabetic (NOD) mouse is homozygous for an H-2 haplotype that encodes a unique MHCII molecule, I-A g7 (I-Aa d /I-Ab g7 ) in which the anti-diabetogenic Asp found at b57 in other murine MHCII molecules is replaced by a pro-diabetogenic Ser (2).
We have previously shown that transgenic NOD and NOD.Rag2 -/mice expressing the I-A g7 -restricted 4.1-TCR spontaneously develop an accelerated form of T1D that results in complete destruction of pancreatic beta cells within a few weeks after birth (8). In H-2 heterozygous NOD mice expressing both pro-(I-A g7 ) and anti-diabetogenic MHC class II molecules (H2 g7/b , H2 g7/k , H2 g7/q , and H2 g7/nb1 haplotypes and transgenic I-Ea k , I-Ab d , or I-Ab g7PD molecules), this TCR sequentially undergoes positive and negative selection/Treg cell reprogramming by recognizing the pro-diabetogenic I-A g7 molecule on cortical thymic epithelial cells and antidiabetogenic MHC class II molecules on hematopoietic APCs, respectively, in a b56-67-regulated manner (2,(9)(10)(11)(12)(13). Furthermore, selective expression of the anti-diabetogenic I-A b and I-Ea d molecules on DCs suppressed diabetes development in non-TCR-transgenic NOD mice, by promoting the formation of Treg cells with superior anti-diabetogenic properties than those arising in wild-type NOD mice (12,13). Collectively, these observations exposed a potential mechanism for the MHC class II-associated susceptibility and resistance to T1D, whereby I-A g7 would promote the selection of MHCIIpromiscuous, 4.1-like autoreactive T-cell clonotypes, capable of triggering diabetogenesis only in the absence, but not presence of anti-diabetogenic MHC class II molecules, such as I-A b in mice and DQ6 in humans. That is, anti-diabetogenic MHCII would harness the intrinsic, I-A g7 -shaped MHCII promiscuity of these TCRs to generate disease-suppressing autoregulatory T cells (2).
In an attempt to further understand the mechanistic underpinnings of the above phenomena, we sought to identify the peripheral autoantigenic target of the 4.1-TCR in the context of I-A g7 . By screening a broad repertoire of known autoantigens and a degenerate epitope-I-A g7 fusion library in artificial APCs with a 4.1-TCR-transgenic reporter T-cell line, we have identified an agonistic amino acid sequence motif that, in the context of I-A g7 , triggers robust 4.1-TCR signaling. Remarkably, we find that the 4.1-TCR recognizes seven different I-A g7 -binding HIPs that share this motif but result from the fusion of several different chromogranin A and/ or insulin C fragments, including post-translational modifications. These data suggest that 4.1-like MHCII promiscuous TCRs are also antigenically promiscuous, thus suggesting that I-A g7 may contribute to T1D susceptibility by expanding the antigenic repertoire of selected TCRs.

Mice
NOD/ShiLtJ mice were from the Jackson Lab (Bar Harbor, ME, USA). 4.1-NOD and 4.1-NOD.Rag2 -/mice have been described (8). All mice were bred and maintained in a specific-pathogen free (SPF) animal facility, and the experimental procedures were approved by the Ethics Committees for Animal Experimentation from the University of Barcelona and University of Calgary.

pMHCII Tetramers
CHO-S cells expressing peptide-I-A g7 b -Fc-hole/GFP and I-A d a -Fcknob/CFP were grown in a fed-batch culture (up to 1 L) in a shaking incubator (37°C and 8% CO 2 ) for 14 days with a temperature shift to 34°C when cell density reached 5 × 10 6 cells/ml. At day 14 or when cell viability dropped below 60%, cell culture supernatants were harvested and secreted pMHCII heterodimers purified on a protein G affinity column, as previously described (14).

Artificial Antigen-Presenting and TCR-Expressing JurMA Cell Lines
The HEK-293T-I-A g7 cell line was generated by sequential transduction of HEK-293T (ATCC, #CRL-11268) cells with lentivirus encoding for I-A d a =CFP, I-A g7 b =GFP, HLA-DM a/b /GFP, and human CD74 (invariant chain -Ii-) fused to human CD80 (hCD80) with sequential flow cytometry-based cell-sorting guided by CFP fluorescence (reporting I-A d a expression), GFP (reporting DM a/b expression), anti-hCD80 (reporting Ii, expression), and anti-I-A g7 (reporting I-A d a /I-A g7 b heterodimer expression). 4.1-and BDC2.5-JurMA cells [derived from the TCR b -null Jurkat cell line J.RT3-T3.5 (ATCC) and carrying a NFAT-driven luciferase reporter] were generated by transducing JurMA cells with retroviruses encoding monocistronic, P2A-tethered TCR a-TCR b chains.
Plasmids encoding I-A g7 -binding peptides eluted from the CIITA transgenic insulinoma NIT-1 b-cell line (expressing I-A g7 on the cell surface) (15) or Tandem HIPs (5) were produced by VectorBuilder.

Generation of a Combinatorial
Peptide-I-A g7 b Library The expression vector encoding for I-A g7 b (pCMV-GS linker-I-A g7 b ) was PCR-linearized at the N-terminal end of I-A g7 b with the primers "Backbone-F" (5'-TCGACAGCGACGTGGGCGAG-3') and "Backbone-R" (5'-TCCGAGGCTGCTAGCGACCAG-3'). Two additional primers "Insert-F" (5'-GCTAGCAGCCT CGGAGGTNNKCGANNKNNKCTANNKGTANNKNNKGA GGGAGGTGGAGGCGGCTCAGGAGGTGGAAGCGGCG GCTCTGGAGACTCCGAAAGGCATTTCG-3') and "Insert-Rev" (5'-CCACGTCGCTGTCGAAGCGC-3') were used to amplify a 250-bp sequence, encoding the amino acid sequence XRXXLXVXXE-GGGGGSGGGSGGS, flanked by a 15-bp long nucleotide sequence that was homologous to the desired integration site in the linearized pCMV-I-A PCR-amplified products were digested with DpnI (NEB) to remove potential contaminating plasmid, and DNA fragments were purified by GeneElute Gel extraction kit (Sigma) following the manufacturer's instructions. The circular plasmid library was generated via a seamless recombination reaction between the linearized pCMV-I-A g7 b vector and the combinatorial 250-bp sequence, catalyzed by the In-fusion HD reagent (Takara) using 300 ng of vector and a 1:3 vector:insert molar ratio. After pooling three In-fusion reactions, plasmid DNA was precipitated, resuspended in ultrapure water and used to transform electrocompetent DH10B bacteria (ThermoFisher) (exponential protocol; 2 KV, 250 Ω and 25 µF). The plasmid library was then tittered and pools of 500 colony-forming units (CFU)/well grown overnight at 37°C in U-bottomed 96-well plates containing 100 mL of LB media supplemented with 100 mg/ml of ampicillin, in a bacterial shaker at 200 rpm. The bacterial library was preserved as a glycerol stock at −80°C.

4.1-TCR Reactivity to the Combinatorial Peptide-I-A g7 b Library
Plasmid DNA from each library pool was extracted using GenElute ™ Plasmid Miniprep Kit (Sigma) and 300 ng used to transfect pre-seeded 2.5 × 10 4 HEK-293T-I-A d a cells with jetPRIME (Polyplus). After 24 h, 10 5 4.1-TCR JurMA cells were added and co-cultured for 24 h and used to measure luciferase activity. Pools confirmed to elicit above-threshold luciferase activity at least twice were deconvoluted by screening sub-pools of decreasing complexity (60 cfu and 8 cfu/well), followed by single-colony screening.

4.1-TCR Reactivity to Plate-Bound Peptide-I-A g7 Heterodimers
Soluble epitope-I-A g7 -knob-into-hole-based heterodimers were obtained from 72 h cultures of 2.5 × 10 4 HEK-293T cells cotransfected with 200 ng of epitope-GS linker-I-A g7 b -Hole and I-A d a -Knob expression vectors using jetPRIME (Polyplus). Immulon ® 2 high-binding ELISA plates (ThermoFisher) were coated overnight at 4°C with 25 µg/ml avidin (Pierce) followed by incubation at room temperature for 1 h with 10 µg/ml of biotin-SP-AffiniPure goat anti-human IgG-Fc (Jackson Immunoresearch). After washing with PBS, cell culture supernatants collected after 72 h from the soluble pMHCIIexpressing HEK-293T transfectants were added and incubated for 2 h at room temperature. After washing with PBS, 10 5 4.1-or BDC2.5-JurMA cells were added to each well and the cells were cultured for 24 h before measuring pMHCII-induced luciferase activity.

Luciferase Assay
Cells were pelleted and lysed with Cell Culture Lysis Reagent (Promega) for 20 min. Next, 30 ml of lysate/well was transferred to white-opaque flat-bottom 96-well plates (Greiner). Luciferase activity in relative light units (RLUs) was determined after injection of 100 ml/well of luciferase assay reagent (Promega), with a delay of 2 s and acquisition for 10 s with a GloMax ® 96 Microplate Luminometer (Promega).
Expanded cells were harvested and cocultured, in a U-bottom 96-well plate, with freshly isolated and red blood cell-lysed 10 5 NOD splenocytes and 0.0001-10 µg/ml peptide in complete T-cell medium supplemented with 30 IU/ml of hIL-2. Cell culture supernatants were collected after 48 h and IFNg concentrations were determined by ELISA using ELISA MAX ™ Deluxe Set Mouse IFN-g (BioLegend) according to the manufacturer's instructions. Synthetic soluble peptides (purity >80%) were obtained from GenScript ® .

The 4.1-TCR Does Not Recognize Known T1D-Associated Autoantigens
We generated an HEK-293T-based artificial APC line (16) expressing transgenic I-A d a , I-A g7 b , H2-DM a/b , and invariant chain (CD74 or Ii) to enable the processing and presentation of transiently expressed antigens in the context of I-A g7 ( Figure 1A). We also generated a JurMA cell-based responder T-cell line (17) expressing mCD4, and the alpha and beta chains of the 4.1-TCR or the BDC2.5-TCR [as a positive control, recognizing the BDC2.5mi mimotope in the context of I-A g7 (18)]. This T-cell line carries a NFAT-driven luciferase reporter that is expressed upon TCR ligation ( Figure 1A).
We first sought to use this approach to ascertain the ability of the 4.1-TCR to recognize previously described T1Drelevant autoantigens (19,20), including pro-insulin (INS), chromogranin A (ChrA), islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), glutamic acid decarboxylase 65 (GAD65), zinc transporter 8 (ZnT8), and islet tyrosine phosphatase 2 (I-A2). This was done by transiently transfecting the artificial APC described above with CMV promoter-driven expression vectors encoding fusions of the first 80 amino acids of murine invariant chain 1-80 (Ii 1-80 ) to each of the above autoantigens (16) (to promote antigen processing and presentation) as well as a BDC2.5mi mimotope-coding sequence at the C-terminal end as an internal positive control (pCMV-Ii-autoantigen-2.5mi) ( Figure 1A). Whereas all of the antigen-encoding plasmids described above triggered luciferase activity in BDC2.5-JurMA cells (via recognition of the C-terminal BDC2.5mi epitope encoded in the various plasmids, in the context of I-A g7 ), none triggered luciferase activity in 4.1-JurMA cells ( Figure 1B).
We next considered the possibility that the 4.1-TCR might recognize a HIP. We therefore generated an expression construct encoding li 1-80 fused to 16 previously published HIP-sequences carrying left and right arms derived from Insulin C and Chromogranin A, Amylin, IAPP, or Secretogranin 2, respectively (5). We also tested two additional expression vectors encoding 63 potential (unpublished) HIPs (Supplementary Figure S1). Whereas the BDC2.5-JurMA cell line responded to all these HIP-encoding constructs and the OVA-BDC2.5mi fusion positive control, 4.1-JurMA cells did not respond to any ( Figure 1D).

A Novel Epitope Discovery Approach
We next sought to develop and test a more systematic, sensitive, higher-throughput approach for T-cell epitope discovery, based on combinatorial epitope display, which could be used in combination with the artificial APC/TCR-transduced JurMA cell system described above. We cloned combinatorial peptide-coding sequences immediately upstream of a flexible 13-residue linker-and I-A g7 b −coding sequences into an expression vector. The peptide-linker fusions carrying short arms of homology with the desired plasmid integration site were generated by PCR using degenerate primers and then cloned in-frame (using the Infusion HD reagent) without intervening nucleotides immediately upstream of I-A g7 b in the PCR-linearized expression vector with homologous integration sites (Figure 2A). We fixed the P1, P4, P6, and P9 I-A g7 -anchor residues (P1=R, P4=V, P6=L, and P9=E) in the peptide-coding sequence, but used degenerate "NNK" codon sequences for potential TCR contact residues (P-1, P2, P3, P5, P7, and P8), where "N" can be any nucleotide and "K" is either a G or a T. By reducing codon degeneracy, we sought to maximize the likelihood that each amino acid will be represented at each open position, while minimizing the chances of introducing stop codons. These plasmids were then transfected into HEK-293T cells constitutively expressing an I-A d a transgene (293.I-A d a ). In this design, the various peptidelinker-I-A g7 b protein molecules were expected to heterodimerize with the transgenic I-A d a chain to form stable peptide-loaded MHCII complexes on the artificial APC's surface.
We first tested the sensitivity of this approach by measuring the ability of HEK-293T. I-A d a cells transfected with decreasing ratios of BDC2.5mi-I-A b -coding sequences into an expression vector. To build the constructs carrying combinatorial peptide-coding sequences, peptide-linker fusions carrying short arms of homology to the desired plasmid integration site were generated by PCR using degenerate primers and then cloned in-frame to the PCR-linearized expression vector upstream of I-A g7 b , without intervening nucleotides, using the In-fusion HD reagent. We fixed the P1, P4, P6, and P9 I-A g7 -anchor residues (P1=R, P4=V, P6=L, and P9=E) in the peptide-coding sequence but used degenerate "NNK" codon sequences for potential TCR contact residues (P-1, P2, P3, P5, P7, and P8). 1:4,096 and 0:1, respectively) to activate BDC2.5-JurMA cells. As shown in Figure 2B, dilutions lower than 1:1,024 elicited clearly detectable responses.

Identification of 4.1-TCR Agonists
Having defined the sensitivity threshold of this approach, and to maximize coverage without compromising sensitivity, we screened pools of 500 peptide-linker-I-A g7 b clones per well distributed in ten 96-well plates, thus representing a total of 5 × 10 5 peptides ( Figure 2C). As cutoff luciferase activity values, we used the average of the normalized relative light units for all the screened pools plus three standard deviations. Pools eliciting responses above the cutoff value (each containing 500 clones) were deconvoluted by subcloning and re-screening until single positive clones were identified in each pool. Of the 17/960 positive pools that were identified, we deconvoluted the top 10 ( Figure 2C). A limitation of these stringent experimental conditions is that it allows for the identification of epitopes with the highest MHC/TCRbinding affinities/avidities, potentially excluding their lower-avidity counterparts. Furthermore, although not investigated herein, it is possible that screening of bacterial pools containing fewer than 500 clones/pool might increase the sensitivity of this approach.
We next compared the agonistic activity of the top 10 peptidelinker-I-A g7 b plasmids and the corresponding synthetic peptides. As expected, sensitivity was greater with the plasmids than with the peptides, as exogenously added peptides must be able to efficiently displace endogenous, HEK-293T-derived epitopes from surface I-A g7 , to elicit cognate T-cell responses. Nevertheless, these experiments allowed the validation of two homologous peptide sequences (A5D and B2B), differing only at positions P-1 and P3, as being strong 4.1-TCR agonists ( Figure 2D). Of these, B2B had slightly higher agonistic activity on 4.1-JurMA cells than A5D ( Figure 3A) and was therefore chosen for further experimentation.
This approach deviates significantly from the strategy used to identify the BDC2.5mi mimotope for the BDC2.5-TCR (18,21), which involves the screening of a peptide-I-A g7 library expressed on baculovirus-infected fly cells by several rounds of FACS-based sorting using soluble TCR tetramers. In the approach described herein, we combine the use of an easy to maintain, highly sensitive reporter cell line with an efficient artificial antigen presentation system that employs the highly transfectable HEK-293T cell line. These features make this screening approach more efficient, higher throughput, and less technically demanding, as expression of soluble TCRs is not a trivial undertaking.

Definition of the Agonistic B2B I-A g7 -Binding Register
We next sought to identify the B2B I-A g7 binding register(s) that most efficiently trigger(s) 4.1-TCR signaling. To do this, we used plate-bound, knob-into-hole-based B2B-linker-IA g7 ab monomers secreted from transiently-transduced HEK-293T cells. As described previously, this design results in increased production yields and pMHCII structural stability than those using leucine zipper-heterodimerization domains (14,22). Although the peptidelinker-I-A g7 b library that was used to identify the B2B mimotope used four pre-defined anchor residues, it remained possible that the B2B sequence was preferentially displayed to the 4.1-TCR on alternative binding registers, such as those in which B2B uses the negatively charged Glu or Asp at P7 and P8, respectively, to anchor itself on I-A g7 's pocket 9. This was addressed by producing three different B2B-linker-I-A g7 pMHCIIs in which the B2B sequence was forced to bind to I-A g7 on three different registers via the strategic introduction of register-fixing Cysteine residues on the I-A g7 a chain and the C-terminus of B2B (cys-trap) (14,22). We then compared the luciferase activity in 4.1-JurMA cells challenged with platebound monomers.
As shown in Figure 3B, the construct displaying B2B on register 3 (the Glu at B2B's P9 docking into I-A g7 's pocket 9) had superior agonistic activity than those displaying it on registers 1 and 2 (B2B docked into I-A g7 's P9 pocket via the Glu at P7 and the Asp at P8, respectively). This was subsequently confirmed by staining 4.1-JurMA cells with pMHCII tetramers displaying B2B on register 3 ( Figure 3C).

Using the B2B Motif to Identify 4.1-TCR Ligands
To further validate P2, P3, P5, P7, and P8 as 4.1-TCR-contact residues in B2B anchored on I-A g7 via register 3, we compared the luciferase activity of B2B-like peptides carrying alanine substitutions at these positions. These experiments revealed strong independent contributions of each of these residues to the agonistic activity of B2B ( Figure 3D). Of note, replacement of the Asp at P8 for a Glu reduced B2B's agonistic activity, suggesting an important contribution for Asp at P8 on 4.1-TCR agonism.
Database searches using a degenerate amino acid sequence carrying B2B's 4.1-TCR contact residues and permissive I-A g7 anchor residues did not yield naturally occurring beta cellspecific autoantigenic targets, suggesting the possibility that the 4.1-TCR might recognize a post-translationally generated and/or modified epitope, such as a HIP. We thus looked for potential HIPs sharing B2B's 4.1-TCR contact motif XLGXEXE(D/E)X. We focused on HIPs identified in beta cell granules via mass spectrometry (MS) (5,23,24), as well as potential new HIPs in which the candidate left and right arms had been previously documented via MS (25). Briefly, we fused these MSdocumented left and right peptides in silico and performed a motif search fitting the motif described above, also incorporating in the search other conserved residues for each position. The potential left and right arms included peptides derived from ChrA, Secretogranin-1 (Scg1), Secretogranin-2 (Scg2), Secretogranin-3 (Scg3), Secretogranin-5 (Scg5), IAPP, Ins1, Ins2, Pancreatic Prohormone (Ppy), Peptide YY (Pyy), Urocortin-3 (Ucn3), Neuroendocrine Convertase 1 (Pcsk1), and Neuroendocrine Convertase 2 (Pcsk2). We also considered HIPs fitting the 4.1-TCR-recognition motif upon deamidation of glutamine residues.
Using the above criteria, we identified 13 potential HIP agonists for the 4.1-TCR ( Figure 4A and Supplementary Table S2), two of which have been previously identified via MS (HIP 55: QLELGGEVEDPQV and HIP 30: LQTLALEVEDPQV) (25). In all these HIPs, the left arm is donated by the Insulin C-peptide, truncated at seven different carboxyterminal residues (ranging from residues 69 to 85), whereas the right arm corresponds to five different naturally occurring proteolytic products of either ChgA (n = 4) or InsC 57-63 ( Figure 4A and Supplementary Table S2). None of the other hybrid peptides that were probed, composed of all random combinations of in silico generated right and left arms from prevalent polypeptides in beta cell granules and crinosomes (including, pro-Ins1, pro-Ins2, ChrgA, IAPP, and Scg1) including variants carrying sequential truncations of one amino acid residue (a total of 7.5 million combinations), conformed to the agonistic motif described above.
Experiments using pMHCII tetramers for the seven HIPs indicated that these HIPs can be subclassified into two potential subsets with differences in functional avidity mirrored by differences in physical pMHCII-binding avidity to 4.1-JurMA cells ( Figure 5A) and CD4+ T cells from 4.1-NOD.Rag2 −/− mice ( Figure 5B). One of these subsets includes peptides such as HIP 39 and HIP 32 Q9E that exhibit high overall functional and physical avidity. The other subset, including the peptides HIP 15, HIP 18, HIP 30, HIP 30 Q10E, and HIP 32, is recognized with low functional and/or physical avidity. Intrinsic particularities of the different peptides within these two subgroups may account for an imperfect correlation in physical vs. functional avidity. For example, HIP 32 has a Q at P9, which is less favorable than a negatively charged residue at P9 (D in HIP 15). It is possible that, at high concentrations of peptide, the functional assay favors a response against HIP 32 over HIP 15, whereas the presence of D at P9 in HIP15 enhances binding to I-A g7 , thus favoring tetramer staining. Furthermore, although HIP 30 Q10E was more potent than its wild-type counterpart HIP 30 in the IFNg secretion assay, HIP 30 I-A g7 tetramers displayed higher physical binding avidity to the 4.1-TCR. This observation suggests that the deamidation Q10E in HIP 30 results in the introduction of a stronger I-A g7 anchor residue, shifting the register from agonistic to non-agonistic. This change would be expected to have more impact on tetramer staining (requiring a unique register binding) than in the IFNg secretion assay where register multiplicity occurs at high concentration of soluble peptide.

DISCUSSION
In the work reported herein, we describe a novel autoantigen discovery approach to define the fine autoantigenic specificities of defined TCRs. We validated this approach by identifying the fine autoantigenic specificity of the 4.1-TCR, a highly diabetogenic, I-A g7 -restricted but MHCII-promiscuous TCR whose developmental biology recapitulates MHCII allelic associations with T1D susceptibility or resistance. Previous, albeit unpublished, work using beta cell-specific cDNA expression libraries, combinatorial peptide libraries, and known recombinant autoantigens, such as insulin, phogrin, IGRP, IAPP, and ZNT8 among others, failed to yield an antigenic target for this TCR, thus precluding further dissection of the mechanisms underpinning its MHC promiscuity and diabetogenicity. Here, we used a radically different approach to address this knowledge gap. We transfected pools of cDNAs from a random epitope-GS linker-I-A g7 b chain fusion library into I-A d a chain-transgenic artificial APCs and screened the transfectants for recognition by a 4.1-TCR-transgenic T-cell line carrying an NFAT-driven luciferase reporter. Pool deconvolution, I-A g7 -binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif [XL(G/A)XEXE (D/E)X] that was shared by seven agonistic HIPs. These HIPs comprised fusions of Insulin C, truncated at seven different carboxyterminal residues, with five different naturally occurring proteolytic products of either ChgA or InsC 57-63 , including post-translationally modified variants. Collectively, our data validate a highly sensitive MHCII-restricted epitope discovery approach and demonstrate that the 4.1-TCR is also autoantigen-promiscuous, suggesting that I-A g7 may contribute to diabetogenesis by "expanding" the antigenic repertoire of at least some selected TCRs.
Studies on the developmental biology of the TCR cloned from the NOD islet-derived CD4+ T-cell clone NY4.1, in TCR-transgenic NOD mice and H-2-congenic or I-A-and I-E-transgenic NOD mice, revealed several unique features. First, transgenic expression of the 4.1-TCR could rapidly accelerate diabetes in both RAGcompetent and RAG-deficient NOD mice (unlike the BDC2.5-TCR) (8). Second, when expressed in NOD mice carrying both proand anti-diabetogenic MHCII molecules, this TCR underwent negative selection or Treg cell re-programming upon recognizing unknown peptides in the context of anti-diabetogenic MHC class II molecules on hematopoietic APCs (2,(9)(10)(11)(12)(13). Although 4.1-NOD and 4.1-NOD.Rag2 −/− mice spontaneously develop a highly accelerated form of diabetes, the bulk 4.1-CD4+ population of these animals is hypo-responsive to TCR stimulation as compared to bulk CD4+ T cells from NOD mice. We believe that this "anergic" state is induced by peripheral antigenic cross-reactivity to other, perhaps more abundant mimics of the HIPs, a subject that we are currently investigating.
Unlike I-A g7 , 4.1-TCR tolerogenic MHCII molecules could not promote positive selection of 4.1-CD4+CD8+ thymocytes when selectively expressed on cortical thymic epithelial cells, indicating that positive and negative selection of the 4.1-TCR are driven by completely different peptide-MHCII complexes (pro-and antidiabetogenic, respectively), exposed to 4.1-thymocytes by different thymic cell types (cortical thymic epithelial cells and hematopoietic APCs, respectively) at different stages of thymocyte development (2,(9)(10)(11)(12)(13). Studies in I-A transgenic mice carrying chimeric I-A alleles confirmed that the tolerogenic effects of protective MHCII on 4.1-TCR-induced diabetogenesis mapped to residues 56-67 of the I-Ab chain (2,12,13). These observations exposed a potential mechanism for the MHC class II-associated susceptibility and resistance to T1D, whereby anti-diabetogenic MHC class II alleles (e.g., I-A b in mice and DQ6 in humans) would exploit the MHCII promiscuity of disease-initiating, 4.1-like CD4+ T-cell specificities to abort the pathogenic activity of their pro-diabetogenic counterparts (e.g., I-A g7 in mice and DQ2/DQ8 in humans). This hypothesis implied that CD4+ T-cell specificities contributing to the initiation and/or amplification of diabetogenesis might be inherently MHCIIpromiscuous and therefore capable of promoting/enhancing disease only in the absence, but not presence of anti-diabetogenic MHC class II molecules (2).
Among the HIPs tested in this study, HIP 30 shares the same contributing left-peptide fragment with HIPs recognized by the diabetes-triggering T-cell clones BDC-2.5 and BDC-6.9. This fragment is either linked to the N-terminus of insulin C-peptide (HIP 30), a ChgA peptide (BDC-2.5), or an IAPP-peptide (BDC-6.9). The presence of all three HIPs has been confidently verified through mass spectrometric analyses of NOD islets (5,23). It was previously shown that this C-peptide fragment is the dominant C-peptide cleavage product that is co-secreted with insulin from perfused rat pancreas in a glucose-sensitive manner, implying a specific proteolytic reaction within insulin granules that leads to the generation of this cleavage product (24). A proteolytic transpeptidation reaction at this C-peptide location may thus facilitate the formation of these and other disease-relevant HIPs in beta cells.
Our finding that the 4.1-TCR is not only promiscuous for MHCII (2,(9)(10)(11)(12)(13) but also promiscuous for a broad repertoire of I-A g7 -binding HIPs that cannot be presented to the 4.1-TCR by anti-diabetogenic MHCII molecules such as I-A b (our unpublished data) provide fresh new insights into the potential mechanistic underpinnings of the positive and negative T1D-MHCII associations. HIPs result from the fusion of pro-insulin and chromogranin A, pro-insulin, or IAPP in beta cell secretory granules and/or crinosomes (5,23). The low water content and high concentration of donor polypeptides in these compartments afford an optimal environment for HIP formation that cannot be replicated in the thymic cell types responsible for central tolerance. As a result, HIPs have emerged as natural ligands of autoreactive CD4+ T cells in both NOD mice (5,26) and humans (27,28). In turn, this implies that the peptide(s) responsible for central tolerance of 4.1-like thymocytes in the context of antidiabetogenic MHCII on hematopoietic APCs must be different, as suggested by the inability of the HIPs described herein to trigger 4.1-TCR signaling in the context of I-A b . Ongoing research seeks to identify 4.1-TCR agonists in the context of I-A d and I-A b , to ascertain the nature of the tolerogenic MHCII complexes responsible for T1D protection. Whatever the nature of these epitopes, this ability of a single TCR to engage different peptide and MHCII combinations suggest that the docking geometry of this TCR onto different pMHCII complexes may be plastic. Resolution of the x-ray crystal structures of these different complexes should provide evidence for or against this possibility.
The antigenic promiscuity of the 4.1-like TCRs may be a feature enabled by the pro-diabetogenic I-A g7 molecule, but "forbidden" for TCRs restricted by Asp-b57+ anti-diabetogenic MHCII molecules. Thus, it is possible that the unique structural features of I-A g7 (and DQ2/DQ8) enable this. It is possible that the low-avidity HIPs may not be adequately recognized by naïve 4.1-like CD4+ T cells and perhaps even antagonize T-cell activation by their high avidity counterparts or promote Treg deviation when presented by tolerogenic, immature DCs. Furthermore, it is reasonable to suspect that 4.1-like CD4+ Tcell activation (from naïve to effector) involves a multistep succession of activation events sustained by different HIPs, whereby productive recognition of the low-avidity HIPs might require pre-activation of the T cells by their high-avidity counterparts. An interesting consequence of this model is that changes in the composition of the agonist and partial agonist HIP pool would impact the effector/regulatory balance of the 4.1like CD4+ T-cell subset. Whether or not HIP composition, including presence of deamidated versions of HIPs that promote antigenicity, change with age remains to be determined but is also a possibility.
Finally, it is possible that other HIP-reactive, I-A g7 -restricted TCR specificities may also recognize multiple HIPs, including some of the new HIPs described herein. Although the left and right arms of the HIPs described herein have been identified via MS (including the full sequence of HIP 30), assessment of their pathophysiological significance (i.e., whether they are processed and presented in vivo) will require confirmation of their expression in islet cells or associated APCs. This will be intimately linked to the refinement and development of new HIP identification methods from complex samples, including insulin secretory granules and crinosomes. Current MS methods may not be sensitive enough to identify disease-relevant HIPs existing at low concentrations. This may account for the detection of a fairly high number of described proteolytic beta cell products in these HIP-forming beta cell structures that have not yet been found in HIPs. In this regard, our epitope identification method will help facilitate the evaluation of MS data and other proteomic techniques for the presence of the candidate epitopes.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by University of Calgary and University of Barcelona Animal Care Committees.

AUTHOR CONTRIBUTIONS
DP executed the experimental work., PSo generated expression vectors encoding I-A g7 , BDC2.5-TCR, and 4.1-TCR. TD provided candidate HIP peptide sequences. PSe designed the antigen discovery approach. PSe and PSa coordinated and supervised the study's execution and wrote the manuscript with DP. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the Ministerio de Economia y Competitividad of Spain (MINECO, RTI2018-093964-B-I00), Generalitat de Catalunya (SGR and CERCA Programmes), the Canadian Institutes of Health Research (CIHR), and the ISCIII and FEDER (PIE14/00027, PI15/0797). DP and PSo were supported by predoctoral studentships from FPU (MINECO). PSe was an investigator of the Ramon y Cajal re-integration program and was supported by a JDRF Career Development Award. The JMDRC is supported by Diabetes Canada.