
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02113-6
10.1016/j.jbc.2024.107612
107612
Research Article
A structural basis of T cell cross-reactivity to native and spliced self-antigens presented by HLA-DQ8
Tran Mai T. 1
Lim Jia Jia 1
Loh Tiing Jen 1
Mannering Stuart I. 2
Rossjohn Jamie jamie.rossjohn@monash.edu
13∗
Reid Hugh H. hugh.reid@monash.edu
1∗
1 Infection and Immunity Program & Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
2 Immunology and Diabetes Unit, St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
3 Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff, UK
∗ For correspondence: Jamie Rossjohn; Hugh H. Reid jamie.rossjohn@monash.eduhugh.reid@monash.edu
27 7 2024
9 2024
27 7 2024
300 9 10761221 1 2024
22 6 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4+ T cell clones not only respond to proinsulin C-peptide (PI40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI40-47-IAPP74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognize self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4+ T cell clones reactive to native PI40-54 epitope and multiple HIPs fused at the same N-terminus (PI40-54) to the degradation products of two highly expressed pancreatic islet proteins, neuropeptide Y (NPY68-74) and amyloid polypeptide (IAPP23-29 and IAPP74-80). We observed that five out of the seven selected SKW3 T cell lines expressing TCRs isolated from CD4+ T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI40-54 and HIP epitopes. Crystal structures of two TRAV26-1+-TRBV5-1+ T cell receptors (TCRs) in complex with PI40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI40-54 and HIPs determined that the P2, P7, and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8.

Keywords

Type 1 diabetes
major histocompatibility complex (MHC)
T cell receptor cross-reactivity
hybrid insulin peptide (HIP)
TCR-pMHC complexes
autoimmunity
Abbreviations

HIP hybrid insulin peptide

IAPP islet amyloid polypeptide

PTM post-translational modification

T1D Type 1 diabetes

TCRs T cell receptors

Reviewed by members of the JBC Editorial Board. Edited by Wolfgang Peti
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pmcType 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which insulin-producing pancreatic β-cells are destroyed. Susceptibility to T1D has a strong genetic association with the human leukocyte antigen (HLA) loci of HLA-DRB1 and HLA-DQB1, and in particular with HLA-DR3 (DRB1∗03:01), HLA-DR4 (DRB1∗04:01), HLA-DQ2 (DQA1∗05:01/DQB1∗02:01), and HLA-DQ8 (DQA1∗03:01/DQB1∗03:02). The haplotypes of HLA-DR3-DQ2 (OR ∼ 3.64) and HLA-DR4-DQ8 (OR ∼ 11.37) present the strongest genetic risk (1). While these HLA-DR and HLA-DQ haplotypes confer a greater risk of T1D, they do differ in their contribution to the associated risk (2, 3, 4, 5). For example, the HLA-DQ alleles confer the major T1D-predisposing risk (6, 7). Furthermore, heterozygous HLA-DQ2+/DQ8+ individuals have a much higher risk for T1D than those who express either HLA-DQ8 or HLA-DQ2 alone (8).

HLA-DQ8-mediated susceptibility to T1D has been mapped to the HLA-DQ β-chain polymorphism, βAla57, which forms part of the P9 binding pocket of the antigen binding cleft. Given the strong T1D association with HLA-DQ8, how the T cell response to antigens leads to T1D autoimmune disease remains poorly explored. T1D is characterized by the appearance of antibodies against β-cell antigens many years before the clinical onset of T1D (9, 10). A number of well-established islet autoantigens have been implicated in relation to T1D, namely (pro)insulin, insulinoma antigen-2 (IA-2), heat shock protein (HSP), islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), Glutamic acid decarboxylase 65 (GAD65), Zinc transporter8 (ZnT8), islet amyloid polypeptide (IAPP), and chromogranin A (ChgA) (11). Several processes have been suggested to induce autoimmune and aberrant inflammatory disease including tissue-specific antigens not being expressed in the thymus, weak affinity of a self-peptide for the major histocompatibility complex (MHC) (12), TCR cross-reactivity between self-antigens and neoantigens derived from the post-translational modification (PTM) of self-peptides (12, 13, 14).

PTM of self-peptide antigens adds a further layer of complexity to the recognition of peptides presented by HLA. PTMs are well documented as being associated with autoimmune diseases including citrullination, deamidation, and spliced peptide formation in rheumatoid arthritis, celiac disease, and T1D, respectively (12, 14, 15, 16, 17, 18, 19, 20). Insulin/proinsulin reactive T cells that have escaped during thymic selection may be a result of PTM-neoantigens and/or peptide epitopes not found in the thymus (21). It has previously been shown that diabetogenic CD4+ T cell clones were stimulated by peptide mapping within the proinsulin C-peptide (22). Approximately 26% of infiltrating CD4+ T cell clones isolated from the pancreatic islets of a diseased T1D patient recognized distinct overlapping epitopes of the C-peptide region presented by HLA-DQ8 or the HLA-DQ8 trans-dimer (22). CD4+ T cells from the peripheral blood of patients with recent-onset or long-standing T1D also responded to these proinsulin C-peptide epitopes (22, 23). It was subsequently observed that human CD4+ T cells respond to C-peptide and cross-reacted to spliced peptides, neoantigens formed by the fusion of proinsulin C-peptide and a peptide from other highly expressed β-islet proteins, such as islet amyloid polypeptide (IAPP), to form a hybrid insulin peptide (HIP; PI40-47-IAPP74-80, GQVELGGG-NAVEVLK) that is presented by HLA-DQ8 (15). This observation was compatible with recent studies of CD4+ T cell clones derived from PBMCs of recent-onset T1D patients that were reactive to several HIPs (24, 25, 26, 27). In a longitudinal case study, T cell reactivity to HIPs (including PI40-47-IAPP74-80) was also detected in individuals at risk of developing T1D which was predominantly pro-inflammatory in those people who subsequently developed islet autoantibodies or T1D (28). A recent report, providing further evidence for the association of HIPs with T1D, demonstrated the presence of HIP, (PI1-47- IAPP74-89-PI48-86) autoantibodies in the sera of patients with T1D as well as in ∼80% of patients with high-risk pre-T1D, often observed prior to seroconversion (i.e., sero-positive for antibodies to at least one of: insulin, GAD65, IA-2, or ZnT8) (29). We reported that TCRs from five islet-infiltrating CD4+ T cell clones isolated from the residual pancreatic islets from a deceased organ donor who had T1D, showed responses to self-antigen (PI40-54) with four of the selected five clones displaying cross-reactivity to the HIP presented by HLA-DQ8 (14). Additionally, using an HLA-DQ8-HIP tetramer, HIP-responsive CD4+ T cells were confirmed to be present in the periphery of patients with recent-onset T1D, and the isolated CD4+ T cells showed a bias in TRAV26-1 and TRABV5-1 gene usage (14). A number of HIPs fused by the same portion of insulin C-peptide (PI40-47) with abundant degradation products of pancreatic β-cell proteins were reported to be autoantigens recognized by human CD4+ T cells (30). These HIPs have a common feature of a negatively charged glutamic acid anchor residue at position P1 and P9 in the peptide that is the preferential antigen-binding cleft anchor residues for the corresponding P1 and P9 pockets of HLA-DQ8 (3, 14, 31). Our previous study provided a molecular basis for the biased selection of TRBV5-1+ TCRs in the CD4+ T cell repertoire responsive to HIP (PI40-47-IAPP74-80) presented by HLA-DQ8 (14). However, our understanding of the mechanism by which human CD4+ T cells recognize self-peptide (PI40-54) and cross-react to these HIPs, bound to HLA-DQ8, remained unclear.

Here, we provide insight into TRAV26-1+-TRBV5-1+ TCR recognition of PI40-54 and their cross-reactivity to multiple HIPs (PI40-47-NPY68-74, PI40-47-IAPP74-80 and PI40-47-IAPP23-29) presented by HLA-DQ8 (14). Using alanine scanning mutagenesis within PI40-54 and HIPs (P-3–P12) in T cell stimulation assays, along with crystal structure analyses, we show the residues at P2, P7, and P8 are critical for T cell-cross-reactivity of these peptide antigens. Accordingly, we provide insight into the molecular mechanisms underpinning TCR cross-reactivity toward HLA-DQ8-restricted self and HIP peptides.

Results

Human CD4+ T cells recognized multiple HIPs

Previously we observed that human CD4+ T cell clones from T1D patients exhibited a HIP (PI40-47-IAPP74-80) response compared to unmodified C-peptide (PI40-54), supporting the notion that a naturally occurring hybrid peptide is presented by HLA-DQ8 and recognized by TCRs (14). To explore differential TCR cross-reactivity, we selected two HIPs as they shared an identical partial (N-terminal proinsulin) sequence of the previously studied PI40-47-IAPP74-80 peptide (HIP1; GQVELGGG-NAVEVLK) but differed in the C-terminal portion of the HIP, namely PI40-47-IAPP23-29 (HIP2; GQVELGGG-TPIESHQ) and PI40-47-Neuropeptide Y 68 to 74 (NPY68-74) (HIP3; GQVELGGG-SSPETLI) (For HIPs1-3: P1 to P9 binding register underlined; P1 and P9 anchor residues in bold). A panel of seven TCRs from human PI40-54 and/or HIP1 reactive CD4+ T cell clones we previously described (14) that shared common TRAV26-1 and/or TRBV5-1 TCR gene usage was selected to investigate differential cross-reactivity for PI40-54 and HIPs1-3 (Table S1). Cross-reactivity was assessed using SKW3 T cell lines, transduced with the panel of the PI40-54/HIP1 TCRs, in activation assays conducted in the presence of PI40-54, HIPs, glia-α1 (control), or no peptide-pulsed HLA-DQ8+ BLCL 9031 antigen-presenting cells. The upregulation of the early activation marker CD69 on TCR-transduced SKW3 T cells was measured to assess these responses.

Five out of the seven selected HIP TCR transduced SKW3 T cell lines showed varied responses to PI40-54 and multiple HIPs (Figs. 1 and S1A). TRAV26-1+-TRBV5-1+ SKW3-A5.5 and SKW3-ET650-4 T cells showed very similar patterns of antigen recognition, both eliciting a relatively low response to PI40-54 and HIP3 compared to the relatively higher responses to HIP1 and HIP2 (Figs. 1 and S1). For example, the EC50 for HIP3 versus HIP2 is approximately 10-fold and 3-fold higher with respect to stimulation of SKW3-A5.5 and SKW3-ET650-4, respectively (Fig. S1B). Although sharing the same gene usage, the TRAV26-1+-TRBV5-1+ SKW3-A2.13 cells, in contrast, displayed a distinct pattern of recognition displaying a relatively strong response to PI40-54 and HIP1 and HIP3 (EC50 8.5 ng/ml) and no response to HIP2 (Figs. 1 and S1B). Likewise, the TRBV5-1+ SKW3-A1.9 T cell line had a similar pattern of antigen response to that of TRAV26-1+-TRBV5-1+ SKW3-A2.13 T cells but a much weaker response to HIP3 (EC50 2.9 μg/ml) (Fig. S1B). Finally, TRAV26-1+ SKW3-ET650-2 T cells showed a unique pattern of antigen recognition with a relatively strong response to HIP1 and a weak response to PI40-54 and HIP3 (EC50 4.2 μg/ml) (Figs. 1 and S1B). Accordingly, despite the biased TCR gene usage in the PI40-54 and HIP responding T cell repertoire, alterations in the C-terminal region of these peptides have significant effects on the cross-reactivity observed for individual TCRs thus indicating the roles of non-germline encoded CDR3 loops in fine specificity of antigen recognition.Figure 1 Assessing cross-reactivity of HLA-DQ8-HIP1 restricted TCRs for cross-reactivity to other HIPs. Expression of CD69 on the surface of TCR transduced SKW3 cell lines stimulated 20 h with no peptide BLCL 9031 (gray), with 50 μg/ml or 100 μg/ml peptide-pulsed BLCL 9031; PI40-54 (black), HIP1 (green), HIP2 (blue), HIP3 (red), and glia-a1 (purple). The black dots present the MFI CD69 of average duplicated values from each individual experiment. An average MFI CD69 from four (n = 4) to five (n = 5) independent experiments were performed. The significance of the MFI CD69 values was analysed by comparing the MFI CD69 of BLCL 9031 (no peptide) with PI40-54 or HIPs-pulsed BLCL 9031. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.002, ∗∗∗∗p < 0.0001 and error bars represent ± SEM. p values were determined by one-way ANOVA with Dunnett's multiple comparison testing.

To investigate whether a lack of stability of HLA-DQ8 presenting PI40-54 can diminish the immunogenicity of the PI40-54 peptide, the glycine at P9 was mutated to glutamic acid. The replacement of P9-Gly with P9-Glu in PI40-54 significantly enhanced responses in selected SKW3-TCR T cell lines (A2.13, A3.10, and ET650–4) (Fig. S2). This observation further supports the notion that HIPs, due to their higher affinity, are more immunogenic epitopes because they form more stable complexes with HLA-DQ8 than the unmodified PI40-54 peptide which is in keeping with our previous study (14) and in other systems (19, 32, 33).

TCR binding affinity for HLA-DQ8-HIP2H11C and HLA-DQ8-HIP3L11C

Given that an unstable peptide in complex with HLA-DQ8 may impact TCR binding affinity studies and downstream crystallization trials (14, 34), we re-engineered our constructs to produce HLA-bound peptide (pHLA) with a substituted P9-Glu as well as a "cysteine trap" construct. Specifically, peptides were designed with a P11-Cys that will form a disulfide bond with the adjacent HLA-DQ8 α-chain Cys-72 residue (αI72C mutation), effectively trapping the peptide in the correct binding register. The resultant sequences of the peptides designed in this way were: PI40-54 (QVELGGGPGAESCQ) or just cysteine at P11 in HIP2 (GQVELGGG-TPIESCQ), and HIP3 (GQVELGGG-SSPETCI) (mutated residues in red). As described above and in the previous studies (14, 34) substitution of P9-Gly to glutamic acid in PI40-54 peptide or P11 cysteine substitution did not impact T cell recognition.

Next, we used surface plasmon resonance (SPR) to compare the level of cross-reactivity towards HLA-DQ8-HIP2H11C and HLA-DQ8-HIP3L11C for five HIP-reactive TCRs (A5.5, ET650–4, A2.13, A1.9, and ET650–2). SPR confirmed that there was no significant difference in the binding affinity of C-peptide responsive TCRs (A2.13, A1.9, A5.5, A5.8, and A3.10) toward HLA-DQ8-PI40-54G9EL11C compared to HLA-DQ8-PI40-54L11C (Fig. S3). The affinities determined for these TCRs correlated with the cellular studies, where the level of cross-reactivity of TCR A5.5 and ET650-4 toward HLA-DQ8-HIP2H11C and HLA-DQ8-HIP3L11C was similar. While. TCR A2.13 and A1.9 showed a relatively strong and moderate binding affinity toward HLA-DQ8-HIP3 with KD values of 4.5 (±0.6) μM and 36.4 (±3.7) μM, respectively. In contrast, TCR ET650-2 showed a weaker response to HLA-DQ8-HIP2H11C (KD > 189 ± 24.2 μM) than HLA-DQ8-HIP3L11C (KD > 116.9 ± 10.4 μM) (Fig. 2 and Table S2). This indicates that the P6–P12 residues in peptides may influence TCR response thus reaffirming CDR3 regions impact the affinity of the TCR interaction with HLA-DQ8-HIPs.Figure 2 Affinity measurements of TCRs for HLA-DQ8-HIPs. The binding affinity of TCR A5.5, ET650-4, A2.13, A1.9, and ET650-2 to HLA-DQ8-HIP2H11C (A), HLA-DQ8-HIP3L11C (B). The left and the right columns in (A and B) are measured response curves of single dilution series for each TCR and curve fits for TCRs -HLA-DQ8-HIPs. The equilibrium dissociation constant KD values using a single ligand-binding model. Each TCR sample was analysed in duplicate, and three to four (n = 3 to n = 4) (as indicated) independent experiments were performed, and measurements were combined after normalizing each equilibrium response curve against the calculated response maximum. Data are mean ± standard error of the mean (SEM). NB, no binding of TCR.

Molecular basis of TRAV26-1+-TRBV5-1+ TCR A2.13 recognition PI40-54 peptide and cross-react to HIPs presented by HLA-DQ8

To understand the molecular basis for cross-reactivity, we selected two TRAV26-1+-TRBV5-1+ TCRs (A2.13 and ET560–4) which exhibited differential reactivity to HLA-DQ8-HIPs (Table S2). C-peptide-reactive TCR A2.13 was derived from the pancreas of a deceased T1D organ donor, while HIP1-reactive TCR ET650-4 was isolated from PBMCs of a patient with recent onset T1D, and both shared a similar motif (LxRE/DTxTF) in the CDR3β loop (Table S1).

We first determined the structures of TRAV26-1+-TRBV5-1+ TCR A2.13 in complex with HLA-DQ8-PI40-54G9EL11C and HLA-DQ8-HIP3L11C (Fig. 3 and Table S3) and compared it to the TCR A2.13-HLA-DQ8-HIP1L11C complex (14). The ternary complexes of TCR A2.13 showed a common TCR docking polarity over pHLA, with roughly similar angles (64 o–67 o) (Figs. 3A and B and S4, A–C) and the average buried surface area (BSA) raging from 2040 to 2250 Å2, whereby, the TCR Vβ-chain was dominant to the binding with values ranging from 55%–62%, while the Vα-chain was 38%–43% to BSA contribution (Fig. 3, A and B and Table S4). Indeed, the TCR A2.13 β-chain adopted similar positions and conserved recognition patterns across the three structures (Fig. S4, A–D). As such, a conserved interaction network between the TCRβ-framework segment (Arg66β and Asn67β) with HLA-DQ8 α-chain (Gln57α, Thr61α, Ala64α, and Val65α), and between CDR1β (Arg37β) and CDR2β (Phe57β) with HLA-DQ8α (His68α) was observed (Fig. S4E, Table S5, and S6).Figure 3 Structures of TCR A2.13 recognition to DQ8-PI40-54G9EL11C and DQ8-HIP3L11C. Cartoon representation of TCR A2.13 engages to HLA-DQ8-PI40-54G9EL11C (A left), HLA-DQ8-HIP3L11C (B left). The HLA-DQ8α- and β-chains are coloured in neon and purple, respectively. The TCR A2.13 α and β chains are coloured in sky-blue and brown, respectively. The CDR loops 1α, 2α, 3α, α-framework, 1β, 2β, 3β and β-framework are highlighted in red, purple, pink, green, gold, magenta, blue, and dark green, respectively. A and B right, Atomic footprints and docking angles of TCR A2.13 on pHLA. The TCRs’ Vα- and V β-chain center of mass positions are represented in black dots. HLA-DQ8 α- and β-chain are coloured in light-gray, and peptides are in dark gray. TCR footprint colours are in accordance with the nearest TCR contact residues. The pie chart presents the relative contribution of each CDR loop, α-framework, and β-framework of TCR A2.13 to the pHLA interface. The CDR loops of TCR A2.13 formed similarly conserved contacts with the (C) PI40-54G9EL11C (cyan), (D) HIP1L11C (PI40-47-IAPP74-80; cyan-orange) (PDB 6XCP) and (E) HIP3L11C (PI40-47-NPY68-74; cyan-dark gray).

Regarding TCR A2.13 interactions with the HLA-DQ8-PI40-54G9EL11C complex, the α-framework, CDR1α, and CDR3α loops recognized P-2-Gln and P2-Leu, while the CDRβ loops interacted with peptide residues spanning P5 to P12 (Fig. 3C). The germline-encoded Arg37β (CDR1β) oriented upward to contact P10-Ser and P12-Gln via H-bond and vdW interactions, respectively (Fig. 4, A and B left). Similarly, Phe57β and Ser58β (CDR2β) formed interactions with both P8-Ala and HLA-DQ8α (Ala64α and His68α) (Fig. 4, C and D left and Table S5). Glu109β and Arg110β (CDR3β) sat atop the peptide and formed vdW interactions with P5-Gly and P6-Pro residues (Fig. 3C and Table S5). Furthermore, the hypervariable CDR3β loop extensively contacted both HLA-DQ8α- and β-chains (Fig. S5A left and Table S5).Figure 4 Mechanism of self-peptide recognition and cross-reactivity to HIPs of TRAV26-1+-TRAV5-1+TCR A2.13.A, Superposition of the germline encoded CDR1β Arg37β (gold) of TCR A2.13 in complex pMHC. B, comparison of interactions between the Arg37β with (right) HLA-DQ8- PI40-54G9EL11C (cyan), (middle) HLA-DQ8-HIP1L11C (orange) and (left) HIP3L11C (dark gray). C, superposition of the CDR2β loop of TCR A2.13. D, conserved interactions between the CDR2β loop with P8 and HLA-DQ8α-chain residues of (right) HLA-DQ8- PI40-54G9EL11C, (middle) HLA-DQ8-HIP1L11C and (left) HIP3L11C complexes. The HLA-DQ8α- and β-chains are coloured in neon and purple, respectively. the PI40-54G9EL11C and HIPs colours as described above. Water molecule is presented in orange sphere. H-bonds and vdW are shown as black dashed lines and orange dashed lines, respectively.

In comparison to HLA-DQ8-PI40-54G9EL11C recognition, TCR A2.13 interacted with HLA-DQ8-HIP1L11C marginally differently (Fig. 3D). The P-2-Gln in HIP1 adopted an upward confirmation, as a result, the TCR A2.13 α-chain formed different interactions from that observed in TCR A2.13-HLA-DQ8-PI40-54G9EL11C structure (Fig. 3D). Furthermore, the germline-encoded Arg37β (CDR1β) oriented toward the peptide to contact with P8-Val and mainchain of P9 and P11 as well as HLA-DQ8α (His68α) (Fig. 4, A and B middle). Furthermore, the germline-encoded CDR2β (Phe57β) tilted ∼1.2 Å way from the peptide and made limited interactions with the HLA-DQ8α-chain (Fig. 4, C and D middle). In addition, the CDR3β formed less numerous interactions with the HLA-DQ8β-chain (Fig. S5A middle).

In the TCR A2.13-HLA-DQ8-HIP3L11C ternary complex, (Fig. 3E), the TCR A2.13 α-chain interactions with P-2-Gln and P2-Leu observed in the HLA-DQ8-PI40-54G9EL11C complex, were conserved. However, the position of the germline-encoded Arg37β (CDR1β) is different from that observed in TCR A2.13 bound to the HLA-DQ8-PI40-54G9EL11C or HLA-DQ8-HIP1L11C complexes. Indeed, the Arg37β positioned closer to the HIP3L11C peptide forming numerous interactions with the HIP3 as well as with both HLA-DQ8α- and β-chains (Fig. 4, A and B right and Table S6).

Collectively, structures of the TRAV26-1+-TRBV5-1+ A2.13-HLA-DQ8-peptide complexes indicated that the germline-encoded residues CDR1β Arg37 and CDR2β Phe57 may be key factors modulating T cell cross-reactivity.

Molecular basis of TRAV26-1+-TRBV5-1+ ET650-4 TCR cross-reactivity to HIPs presented by HLA-DQ8

We next determined the structures of the TCR ET650-4 bound to HLA-DQ8-HIP1L11C and HLA-DQ8-HIP2H11C at 2.4 Å and 2.9 Å resolution, respectively (Fig. 5 and Table S3). The TCR ET650-4 docked at the same angle 74o on HLA-DQ8-HIP1L11C or HLA-DQ8-HIP2H11C, each with large BSA values of ∼2390 Å2. The TCR α-chain and β-chain equally contributed at the pHLA interface with BSA ≈ 49%–51% and 48%–51%, respectively (Fig. 5, A and B and Table S4). Despite the different C-terminal peptide sequences (from P6 to P12) in HIP1 and HIP2, TCR ET650-4 exhibited identical recognition patterns on both structures (Fig. 5, A and B). TCR ET650-4 bound to HLA-DQ8-HIP1L11C and HLA-DQ8-HIP2H11C with identical CDR loop orientations and structures that revealed a conserved recognition with HIP1L11C and HIP2H11C (Fig. S6A). Detailly, the α-framework (Lys3α), the CDR1α (S29α and Asn36α), and the CDR3α (Ala109α, Ile110α, Glu111α, and Gln114α) loops formed H-bonds and substantial vdW interactions with the N-terminus of both peptides from P-2-Gln to P2-Leu, respectively. Furthermore, Arg109β (CDR3β) formed interactions with the main chain P6-Asn and P7-Ala (in HIP1) or the main chain P6-Asn and side chain P7-Pro (in HIP2) (Fig. 5, C and D, Tables S7, and S8). In addition, P8-Val (HIP1L11C) or P8-Ile (HIP2H11C) was positioned under and adjacent to the CDR1β (Arg37β) and formed vdW interaction with this residue. Although HIP2H11C contains a longer side chain isoleucine at P8 versa P8-Val in HIP1L11C, the Arg109β (CDR3β) also formed a conserved interaction with this residue with an altered orientation (Fig. 5, C and D, Tables S7, and S8). Specifically, the Arg70β in the HLA-DQ8β-chain had an upward conformation forming a network of H-bond contacts with the α-framework (Tyr40α, His55α) and vdW interactions with the CDR1α (Tyr38α) and CDR3β (G111β, Asp112β, and T113β) loops with HLA-DQ8β Arg70β observed in both structures (Fig. S6B, Tables S7, and S8).Figure 5 Structures of TCR ET650-4 recognition to DQ8-HIP1L11C and DQ8-HIP2H11C. Cartoon representation of TCR ET650-4 engages to HLA-DQ8-HIP1L11C (A left), HLA-DQ8-HIP2H11C (B left). The HLA-DQ8α- and β-chains are coloured in neon and purple, respectively. The TCR ET650-4 α and β chains are coloured in blue and gray, respectively. The CDR loops 1α, 2α, 3α, α-framework, 1β, 2β, 3β and β-framework are highlighted in red, purple, pink, green, gold, magenta, blue, and dark green, respectively. A and B right, surface presentation of TCR footprints and docking angles on pHLA. The TCRs’ Vα- and V β-chain centre of mass positions are represented in black dots. HLA-DQ8 α- and β-chain are coloured in light-grey, and peptides are in dark gray. TCR footprint colours are in accordance with the nearest TCR contact residues. The pie chart presents the relative contribution of each CDR loop, α-framework, and β-framework of TCR ET650-4 to the pHLA interface. The CDR loops formed similarly conserved contacts with the (C) HIP1L11C (PI40-47-IAPP74-80; cyan-orange) and (D) HIP2H11C (PI40-47-IAPP23-29; cyan-pink).

Collectively, a structural comparison of TCR ET650-4 bound to HLA-DQ8-HIP1L11C and HLA-DQ8-HIP2H11C highlighted that the non-germline encoded Arg109β residue in the CDR3β loop is critical for antigen specificity.

Influence of hypervariable CDR3 loop on TCR cross-reactivity

Next, we investigated why TCR A2.13 and ET650-4 showed relatively low (KD > 92.7 μM) and high (KD = 11.2 μM) affinities toward HLA-DQ8-HIP1L11C, respectively (Table S2). A comparison of the TCR A2.13 and TCR ET650-4 bound HLA-DQ8-HIP1L11C structures showed that the TCR ET650-4 Vα- and β-chains docked at slightly different positions over HLA-DQ8-HIP1L11C, particularly, the CDR3α loop had a distinct conformation (Fig. S7A). A longer CDR3α loop of the TCR ET650-4 enabled it to lay over the peptide to form substantial interactions with the N-terminus (P-3-Gly to P2-Leu) of the HIP1L11C and the HLA-DQ8 α-chain (Figs. 5C, 3B and S7B).

Although the CDR3β loop of TCRs ET650-4 and A2.13 share a very similar motif (LxRD/ETxY), the CDR3β loop of TCR ET650-4 contains Arg109β in contrast to Glu109β in TCR A2.13 and is also one residue longer than A2.13 (Table S1). The differences in the CDR3β loop of the two TCRs led to different interactions between TCR ET650-4 with the HLA-DQ8-HIP1L11C complex compared to that observed in the A2.13-HLA-DQ8-HIP1L11C structure (Figs. 5C, 3B and S7C). Namely, Arg109β of TCR ET650-4 tilted toward a negatively charged patch formed by HLA-DQ8-HIP1L11C (Fig. S8) which forced the HLA-DQ8 Arg70β in an upward orientation compared to the downward conformation observed in A2.13-HLA-DQ8-HIP1L11C structure (Fig. S7D). Additionally, the Arg110β (CDR3β loop) of TCR ET650-4 reached over and interacted with the HLA-DQ8 α-chain causing Asn67β and Arg66β (β-framework) to have a different orientation compared to that observed in the TCR A2.13-HLA-DQ8-HIP1L11C complex (Fig. S7C). Moreover, The CDR2β loop of TCR ET650-4 did not show peptide contact (Fig. S7E) which may be explained by the different docking positions that the TCR β-chain adopted. Thus, this structural study of TCR A2.13 and ET650-4 bound to HLA-DQ8-HIPlL11C provided evidence that the CDR3 loops are key factors in determining the level of TCR cross-reactivity for these highly related peptide antigens.

Fine specificity of C-peptide and HIP recognition by CD4+ T cells

To investigate the fine specificity of these CD4+ T cell clones, we measured their response toward PI40-54, HIP1, and HIP3 in which each amino acid residue was sequentially mutated. We selected four TCR transduced SKW3-T cell lines (A2.13, ET650–4, A3.10, and ET650–5) as those TCRs exhibited variable binding affinities towards PI40-54 and HIPs (Table S2). Since PI40-54 lacked the preferred acidic P9 anchor residue and also because a single alanine mutation may introduce an unstable pHLA complex, which in turn may impact T-cell responses, the synthetic PI40-54G9E peptide was used for these experiments. As shown above, no difference was observed in the affinity of the selected TCRs for HLA-DQ8 presenting either native PI40-54L11C or PI40-54G9EL11C peptides (Fig. S3). A functional study revealed PI40-54G9E peptide induces a significantly higher stimulation of reactive TCRs compared to native PI40-54 presented by HLA-DQ8, suggestive of the formation of a more stable pHLA complex with the mutant variant (Fig. S2).

To understand the binding energies associated with these residues, the residues in PI40-54G9E, HIP1, and HIP3 were subjected to single alanine scanning substitution at each position (P-3–P12) except P1-Glu, P7-Ala (HIP1), P8-Ala (PI40-54G9E), and P9-Glu. Whilst residues P-3 - P5 are shared across PI40-54 and HIPs, they differ from P6–P12. To determine which residues are important for TCR cross-reactivity, we made single residue substitutions from P6 to P12 in the PI40-54G9E peptide to that of the corresponding position of HIP1 (except P11Leu which is shared between PI40-54G9E and HIP1) and assessed the effect of these substitutions on T cell stimulation (Table S9 (underlined residues) and Fig. S9).

For the PI40-54G9E peptide, the mutagenesis study revealed that alanine substitution of P2-Leu, P3-Gly, P5-Gly, or P6-Pro decreased SKW3-A2.13 T cell recognition. Alanine substitution at P-2-Gln, P-1-Val, P2-Leu, P3-Gly, P6-Pro, or P7-Gly almost abolished or significantly decreased the response of SKW3-ET650-4 T cells (Fig. 6).Figure 6 CD69 upregulation of TCR transduced SKW3-T cell lines response to WT and mutated peptides. A, the measurement of CD69 expression on surface of the transduced SKW3-T cell clones (A2.13, ET650–4, A3.10, and ET650–5) were cultured without or with 50 μg/ml of synthesised PI40-54G9E- or mutated PI40-54G9E-pulsed BLCL 9031 (top panel), HIP1- or mutated HIP1-pulsed BLCL 9031 (middle panel), HIP3 or mutated HIP3-pulsed BLCL 9031 (bottom panel). The black dots present the MFI CD69 of average duplicated values from each individual experiment from four (n = 4) independent experiments. The residues subjected to single alanine scanning substitution and mutated residue are underlined and coloured in red, respectively. Substituted residues that significantly inhibited or enhanced the T cell response are highlighted in red or blue, respectively. B, the data presented in (A) displayed as the fold increase in CD69 expression. The fold increase in MFI CD69 expression was calculated by dividing the average MFI CD69 from each sample by the average MFI CD69 from BLCL 9031 (no peptide). The significance of the MFI CD69 values was analysed by comparing the MFI CD69 of WT peptide-pulsed BLCL 9031 with single mutated peptide-pulsed BLCL 9031. Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.002, ∗∗∗∗p < 0.0001 and error bars represent ± SEM. p values were determined by one-way ANOVA with Dunnett's multiple comparison testing.

Next, we analyzed the impact of sequential alanine substitution in HIP1 on T cell recognition. Substitution of P-1-Val, P2-Leu, P5-Gly, or P6-Asn to alanine significantly diminished the response of SKW3-A2.13 cells. Notably, increased stimulation (2 folds) was observed for the SKW3-A2.13 T cell line when P8-Val was exchanged for alanine. Similarly, alanine mutation at P-2-Gln, P-1-Val, P2-Leu, P3-Gly, or P8-Val abrogated stimulation of SKW3-ET650-4 T cells. Likewise, P-1-Val, P2-Leu, P6-Asn, or P8-Val to alanine substitution almost completely abolished the response of SKW3-A3.10 and SKW3-ET650-5 T cells (Fig. 6).

Next, we analyzed alanine substitution in HIP3 and observed the SKW3-A2.13 T cell line had a similar response to that observed for the mutagenesis of the PI40-54G9E peptide. Alanine substitution at L2-Leu, P3-Gly, or P5-Gly significantly decreased the response of SKW3-A2.13 T cells. Similarly, alanine mutation at P-2-Gln, P-1-Val, P2-Leu, P3-Gly, or P8-Pro significantly inhibited recognition by SKW3-ET650-4 T cells. Remarkably, alanine substitution at P5-Gly or P7-Ser of HP3 significantly increased 1.5-fold and threefolds the stimulatory response of SKW3-ET650-4 T cells, respectively (Fig. 6).

For the substitution of individual residues from P6 to P12 of the PI40-54G9E peptide to the corresponding HIP1 residue, we found that the substitution of P6-Pro to P6-Asn in PI40-54G9E peptide significantly decreased or abrogated stimulation of the SKW3-A2.13, SKW3-ET650-4, and SKW3-A3.10 T cells (Fig. S9). Alanine substitutions at P7-Gly in PI40-54G9E reduced the response of the SKW3-A2.13 or SKW3-ET650-4 T cells, respectively. Interestingly, the substitution of P8-Ala to P8-Val or P10-Ser to P10-Val significantly enhanced four folds or one fold the reactivity of the SKW3-A3.10 T cells, respectively (Fig. S9).

In keeping with our analysis of the contacts formed between TCR A3.10 (14), A2.13, and ET650-4 from P10 to P12 in HLA-DQ8-PI40-54G9EL11C/-HIP1L11C/-HIP3L11C, we observed that alanine or cysteine mutation at these positions had no significant effect on TCR recognition and further supports our conclusion that the introduced disulfide bridge between P11-Cys and HLA-DQ8αI72C did not significantly influence TCR recognition (Fig. 6 and Tables S5–S7). Overall, the alanine scanning on the C-terminus of PI40-54G9E and HIP-specific T cell clones revealed that position P2, P5, P6, P7, and P8 are crucial for antigen specificity of TCRs whereas P2, P7 and P8 were important for determining the level of cross-reactivity for a given TCR-pHLA combination.

Discussion

Given the vastly higher number of potential antigens compared to the available TCRs after thymic selection, T cell immunity has evolved under such selection pressure to provide a comprehensive coverage of the antigenic space whereby TCRs can recognise more than one pMHC (35). The cross-reactivity of TCRs to multiple antigens presented by MHC II was observed by functional and structural studies of TCR-pMHC II complexes. These studies revealed a broad mechanism that has provided a greater understanding of T cell cross-reactivity (36, 37, 38). Molecular mimicry has been proposed as a mechanism, whereby T cells target particular self-antigens which mimic pathogen- or microbial-derived peptides observed in multiple sclerosis (MS) and celiac disease (CD) (39, 40). Moreover, ‘Hotspot molecular mimicry’ or altered TCR-pMHC II docking orientation provided a model for T cells reactivity, whereby TCRs are highly focused on a small area of the peptide (41, 42). Furthermore, the conformational plasticity of CDR Loops is proposed to reflect a mechanism that facilitates cross-reactive T cells (37, 38).

As such, general mechanisms underlying T cells cross-reactivity have been studied in autoimmune diseases, namely MS, CD, and rheumatoid arthritis (RA). However, the mechanism by which human CD4+ T cells recognize self-peptide (PI40-54) and cross-react to the neoantigens (HIPs) bound to MHC II in T1D is unknown. Here, we found that five of seven selected CD4+ T cell clones recognized multiple HIPs and, in particular, two TCRs, A2.13 and ET650-4, showed distinct recognition of HIPs, despite sharing the same TRAV26-1/TRBV5-1 gene usage. We provide insight into the molecular basis of how the TCR A2.13 and ET650-4 recognized native C-peptide (PI40-54) and cross-reacted to HIPs presented by HLA-DQ8.

The cross-reactivity of TCR A213 depended on the germline-encoded CDR1β and CDR2β loops, with Arg37β and Phe57β being key factors modulating T cell cross-reactivity. The specific TCR A2.13 recognition of HLA-DQ8-HIP1 and HLA-DQ8-HIP3L11C was mediated by the orientation of the Arg37β to form interactions with P8 and HLA-DQ8 residues. As a consequence of the longer side-chain at P8-Val in HIP1L11C (P8-Ala in PI40-54G9EL11C and P8-Pro in HIP3), the TCR A2.13 β-chain lost contact with the HLA-DQ8α-chain, consequently, a low binding affinity of TCR A2.13 toward HLA-DQ8-HIP1L11C was observed. This was evidenced by the alanine mutagenesis study whereby alanine substitution of P8-Val in HIP1 significantly enhanced the response of A2.13 T cells. Although, the P-2-Gln in the TCR A2.13-HLA-DQ8-HIP1 complex had a different conformation to that observed in the TCR A2.13-HLA-DQ8-PI40-54G9EL11C- and -HIP3L11C complexes, the alanine scanning studies demonstrated that P-2-Gln was not a critical residue for TCR A2.13 recognition.

Structural studies of TRAV26-1+-TRBV5-1+ TCR ET650-4 complexes suggested different mechanisms for how this TCR cross-reacts to HIPs. Unlike TRAV26-1+-TRBV5-1+ TCR A2.13, the recognition of TCR ET650-4 to HLA-DQ8-HIPs was characterized by biased TRAV26-1 gene usage, which is the same as the TRAV26 biased usage observed in both HLA-DQ2.5-gluten and HLA-DQ8-gluten-specific CeD T cell clones, namely TRAV26-1+-TRBV7-2+ TCRs D2, JR5.1 and S16 (19). Given TCR A2.13 and ET650-4 share the same TRAV/TRBV gene usage, it is self-evident that differences in sequence and length of the CDR3 loops of TCR ET650-4 and A2.13 are the main influence on differences observed in their respective recognition of pHLA. Several structural studies have revealed that the CDR3α and the CDR3β loops play a major role in binding and cross-reactivity, indeed, the CDR3 loops undergo conformational rearrangements and dominantly interact with pMHC (18, 19, 37, 43, 44). The flexibility of the CDR3 loops has been also observed in the structural comparison of unliganded versus liganded of the same TCRs with different pMHC (45, 46, 47).

Additionally, we and others have previously reported that a non-germline-coded arginine residue in the CDR3α or CDR3β loop, especially at position 5 (CDR3β Arg109β) is critical in mediating recognition with pHLA in celiac disease (CeD) (48, 49, 50, 51). Furthermore, the common feature of the Arg109β (CDR3β) slotting to a negatively charged patch on the pHLA-DQ8 surface was also observed in the TRAV20+/TRBV9+ TCR T15-HLA-DQ8.5-glia-γ1 crystal structure (50). Consequently, the Arg109β positioned away from the peptide, while the negatively charged Glu109β (CDR3β) of TCR A2.13 sit atop, which can accommodate a longer side P8-Ile in HIP2. Furthermore, an analogous interface with HLA-DQ8-HIP1L11C and HLA-DQ8-HIP2H11C was observed for the TCR ET650-4, which exhibited an alternative conformation the Arg109β (CDR3β) to accommodate the P8-Ile in HIP2H11C. Collectively, this observation suggests that the non-germline encoded Arg109β have a prominent function in determining pMHC specificity in T1D.

Moreover, mutagenesis studies on the PI40-54G9E and HIP epitopes indicated that the P-2-Gln, P-1-Val, P2-Leu, and P8-Pro (in HIP3) or the longer side-chain residue Val at position P8 (in HIP1) are critical for recognition by the ET650-4 T cell clone. While, P2-Leu, P5-Gly, and a smaller side chain residue Ala (in PI40-54G9E) or Pro (in HIP3) at P8 are crucial for the TCR A2.13 recognition. This observation is consistent with the structural data showing that these were major TCR contact sites. Additionally, the SPR study showed a low binding affinity of TCR ET650-4 toward HLA-DQ8-HIP3L11C (KD = 65 μM). The substitution of P7-Ser for alanine in HIP3 substantially enhanced the T-cell stimulation. Comparison of the peptide sequence of PI40-54 and HIPs revealed that PI40-54, HIP1, and HIP2 peptides contain a smaller side-chain residue at position P7 (P7-Gly in PI40-54G9E, P7-Ala in HIP1, and P7-Pro in HIP2), suggesting a smaller side chain residue at P7 is crucial for TCR ET650-4 reactivity.

Although the TCR A2.13 and ET650-4 had no contacts between the TCR and the peptide at P3, the space created by having a P3-Gly accommodated HLA-DQ8-Phe58β to form interactions with the peptide and TCR. The decreased response observed for the P3-Ala mutant may be due to an altered HLA-DQ8 Phe58β conformation impacting TCR recognition.

The peptide mutagenesis-T cell stimulation data also revealed the crucial role of residue P2 and P8 for cross-recognition by TCR TRAV26-1+ ET650-5 and TRBV5-1+ TCRs A3.10. The responses of the SKW3 T cells expressing these TCRs were abolished when the P8-Val of HIP1 was substituted with P8-Ala. Especially, a longer side chain, such as Val, at P8 is critical for the TCR A3.10 recognition. This was demonstrated by the alanine mutagenesis study whereby the response of SKW3 TCR A3.10 was also significantly enhanced when P8-Ala of PI40-54G9E was substituted for P8-Val.

The structures of TCR A2.13 and ET650-4 bound to HLA-DQ8-PI40-54 and -HIPs presented here provide a framework for understanding how autoreactive T cells can cross-react with self-peptide and their PTM versions like HIP albeit with significantly different affinities for their respective pHLA.

Experimental procedures

Synthetic peptides

Peptides were synthesized by GL Biochem using Fmoc chemistry. Peptides were purified by RP-HPLC to at least 95% purity and lyophilized. Peptides were reconstituted in water to a concentration of 5 mg/ml, aliquoted, and stored at −20 °C.

T cell activation assay

For TCR transduced SKW3 T cell lines (SKW3-TCR), response to antigens was determined using flow cytometry to assess the level of upregulation of the T cell activation marker CD69. One hundred thousand BLCL 9031 (HLA-DR4+/HLA-DQ8+) cells, used as APCs, were incubated at 37 °C, 5% CO2 for 24 h with 50 μg/mg of synthesized C-peptide (PI40-54; GQVELGGGPGAGSLQ), PI40-54G9E (GQVELGGGPGAESLQ), HIP1 (PI40-47-IAPP74-80: GQVELGGGNAVEVLK), HIP2 (PI40-47-IAPP23-29: GQVELGGGTPIESHQ), and HIP3 (PI40-47-NPY68-74: GQVELGGGSSPETLI) and glia-α1 (PSGEGSFQPSQENPQ: control) (mutation in bold, IAPP74-80, IAPP23-29 and NPY68-74 sequence in italics) in 96 well round-bottomed plates. To confirm HLA-DQ8-restriction, 2 μg/ml HIP3 (SKW3-A2.13) or 50 μg/ml (all other SKW3-TCR cell lines) HIP2 or HIP3 pulsed BLCL 9031 cells were blocked with a final concentration of 30 μg/ml anti-HLA-DQ monoclonal antibody (clone SPV-L3) for 3 h before stimulation (i.e. before the addition of SKW3-TCR cell lines). Functional studies of TCR-transduced SKW3- T cell lines were conducted as described in our previous study (14). Briefly, 1 × 105 transduced SKW3-TCR cells or SKW3 parental cells (control) were added to each well and incubated at 37 °C, 5% CO2 for 20 h. The cells were then washed twice with FACS buffer (Phosphate-buffered saline, 10% Fetal Calf Serum (FCS; Merck)) at 350g, 5 min, stained a mixture of 1:100 diluted V450 mouse anti-human CD3 (clone UCHT1, BD Biosciences) and APC Mouse Anti-Human CD69 (clone FN50, BD Biosciences) for 1 h on ice in dark. The cells were then washed twice with PBS buffer (350g, 5 min) to remove excess antibodies followed by live/dead cell staining with Zombie NIR (Biolegend) for 30 min at RT in the dark. After live/dead cell staining, the cells were washed 6 times with FACS buffer and analyzed via flow cytometry (LSR II; BD Biosciences; BD FACSDiva-8.0.1 software). For the alanine scan experiments, each synthesized peptide PI40-54G9E, HIP1, and HIP3 was added to a final concentration of 50 μg/ml.

The flow cytometry gating strategy for the cellular activation assay- (Gate 1): SKW3-TCR lymphocytes; 10,000 events from single cells (Gate 2) were acquired from SKW3-TCR lymphocytes; from Gate 2, live cells were isolated (Gate 3); The double GFP+ RFP+ cells (Gate 4) were acquired from the live cell population and then displayed as pseudocolor plots or histograms of CD69 versus forward scatter (Fcs-H) from the cells with positive GFP+ RFP+. Using these settings, the median fluorescence intensity (MFI) corresponds to the relative level of CD69 expressed on the SKW3-TCR.TCR cell lines were determined using Flowjo v10.6.0 (FlowJo), and then Prism 10 (GraphPad Software). Four independent experiments were conducted, and all samples were performed in duplicates. Statistical significance was determined using p-values generated by t test or one-way ANOVA multiple comparison of the MFI of no-peptide-stimulated SKW3-TCR T cells or peptide-stimulated SKW3-TCR T cells and versa the MFI of peptide/mutated-peptide-stimulated SKW3-TCR T cells.

TCR expression, refolding, and purification

The extracellular domains with an engineered disulfide linkage in the constant domains of TCR α- or β-chain were expressed in Escherichia coli BL21 (DE3). TCR expression and purification were performed as described in our previous study (14, 52). In brief, the αβ TCRs were purified separately from inclusion bodies and, subsequently, refolded as described previously (14). Briefly, TCR α- and β-chains were refolded in a solution containing 5 M Urea Refolding Buffer (5 M Urea, 100 mM Tris-HCl pH 8, 480 mM L-arginine-HCl, 2 mM Na-EDTA, 0.2 mM PMSF, 0.5 mM Oxidized Glutathione, 5 mM Reduced Glutathione) for 72 h at 4 °C. The samples were dialyzed three times into 10 mM Tris pH 8.0 for 1 day. The refolded TCRs were purified on DE52 AIE Cellulose columns (Cytiva), followed by size exclusion (HiLoad 16/600 Superdex 200pg column; Cytiva), hydrophobic interaction (HiTrapTM Phenyl HP column; Cytiva) and anion exchange (HiTrapTM Q HP column; Cytiva) chromatography.

HLA-DQ8-HIPs expression and purification

The extracellular domains of HLA-DQ8 α- and β-chains (HLA-DQA∗03:01 HLA-DQB1∗ 03:02) containing fos/jun leucine zipper were cloned into pZIP3 vector (19, 32, 50, 53). HLA-DQ8-peptide containing P11C mutations (Cysteine Trap) the HLA- DQA∗03:01 gene encoded an I72C mutation for covalent bond formation with the peptide. Gene segments (IDT) encoding the PI40-54G9EL11C (GQVELGGGPGAESCQ), HIP1, (PI40-47-IAPP74-80: GQVELGGGNAVEVCK), HIP2 (PI40-47-IAPP23-29: GQVELGGGTPIESCQ) and HIP3 (PI40-47-NPY68-74: GQVELGGGSSPETCI) (binding register underlined, mutation in bold, IAPP74-80, IAPP23-29 and NPY68-74 sequence in italics) were engineered, expressed, and purified as described previously (14, 19). Briefly, the HLA-DQ8-peptide monomers were expressed via the baculovirus-mediated insect cell expression system (Trichoplusia ni BTI-TN-5B1-4 cells; Thermo Fisher Scientific). The soluble protein was concentrated, and buffer exchanged from the culture medium via tangential flow filtration (TFF) (Cogent M1, Merck) into 10 mM Tris, pH8.0, and 500 mM NaCl, followed by immobilized metal ion affinity (Ni-NTA Superflow; Qiagen), Size exclusion (HiLoad 16/600 Superdex 200pg column; Cytiva) and anion exchange (HiTrapTM Q HP column; Cytiva) chromatography.

Surface plasmon resonance

SPR measurements and analysis were carried out as described previously (14, 19). SPR experiments were all conducted on the Biacore T200 system (Cytiva). Briefly, 2000 RU of biotinylated HLA-DQ8-CLIP (reference), HLA-DQ8-HIP2HL11C, DQ8-HIP3L11C, HLA-DQ8-PI40-54L11C, and DQ8-PI40-54G9EL11C were immobilized in separate flow channels of a BIAcore streptavidin sensor chip (Cytiva). Dilution of serial concentration from 200 μM to 0 μM of each TCR was passed over the surface of the chip in SPR buffer (20 mM HEPES pH7.5, 150 mM NaCl, and 0.005% surfactant P20) with a flow rate of 10 μl/min for 60 s. Three independent experiments with replicates were performed for each TCR. The equilibrium dissociation constant KD values were analyzed using Prism 10 (GraphPad software).

Crystallization, data collection, and processing

Crystallisation trials were implemented as described in the previous study (14). In brief, crystallization was performed by the handing-drop vapor-diffusion method using a 1:1 ratio of a protein to mother liquor at 20 °C. TCR A2.13 and HLA-DQ8-PI40-54G9EL11C or HLA-DQ8-HIP3L11C complex was co-complexed at ∼7 mg in 10 mM Tris pH8 and 150 mM NaCl for 5h at room temp (RT). The A2.13-HLA-DQ8-PI40-54G9EL11C complex crystallized in 0.2 M potassium sodium tartrate (K Na tartrate), 24% w/v PEG 3350. The A2.13-HLA-DQ8-HIP3L11C complex crystallized in 0.2 M K Na tartrate, 14% w/v PEG 20,000 with seeding and homemade additive C8 silver bullet (following the manufacturer’s recipes, Hampton Research). Similarly, the TCR ET650-4 and HLA-DQ8-HIP1L11C or HLA-DQ8-HIP2H11C complex were co-complexed as described above. The ET650-4-HLA-DQ8-HIP1L11C complex crystallized in 0.2 M K Na tartrate, 15% w/v PEG 20,000 with seeding and homemade additive C8 silver bullet. The ET650-4-HLA-DQ8-HIP2H11C complex crystallized in 0.2 M potassium dihydrogen phosphate (KH2PO4), 15% w/v PEG 20,000 with seeding and additive 30 mM MnCl2. The crystals were transferred into mother liquor 0.2 M KH2PO4, 13% w/v PEG 3350 supplemented with 30% glycerol and frozen in liquid N2. Datasets were collected at Australian Synchrotron’s microfocus beamline (MX2) using an Eiger x16M detector (54). the program XDS and Aimless of CCP4 8.0 package were used for data processing (55), followed by molecular replacement in PHASER using a separate search model of TCR and HLA-DQ8 from the previously published structure of TCR A2.13-HLA-DQ8-HIPL11C (PDB ID: 6XCP). The model building and refinement were conducted using Coot version 0.9 (56) and Phenix program version 1.19.2 to 4158 (57, 58), and PDB structure validation server was used for crystal structure validation. IMGT unique numbering system was used to number TCR variable domains (59). PyMol version 2.1 (http://www.pymol.org/) was used to generate all structural figures

Statistics

Tabulated data were analyzed in Graphpad Prism 10 (Graphpad Software). Each data set was assessed for normality using the Shapiro-Wilk normality test. Differences between columns were analyzed by two-tailed Student’s t test for normally distributed data. Differences between groups were analyzed using one-way ANOVA with Dunnett’s post-tests for normally distributed data.

Data availability

The structures were deposited in the PDB database; A2.13-HLA-DQ8-PI40-54G9EL11C, PDB 8VCX; A2.13-HLA-DQ8-PI40-47-NPY68-74L11C, PDB 8VCY; ET650-4-HLA-DQ8-PI40-47-IAPP74-80L11C, PDB 8VD0; ET650-4-HLA-DQ8-PI40-47-IAPP23-29H11C, PDB 8VD2, HLA-DQ8-PI40-54G9EL11C, PDB 8VDD; and HLA-DQ8- PI40-47-IAPP74-80L11C, PDB 8VDU.

Supporting information

This article contains supporting information (14, 22).

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Supplemental Figures Tables

Acknowledgments

We thank the staff at the Monash Macromolecular crystallisation facility and the Australian Synchrotron for assistance with crystal screening and data collection.

Author contributions

H. H. R. and J. R. conceptualization, H. H. R. supervision, H. H. R. and M. T. T.writing–original draft, H. H. R., J. R. writing–review & editing. J. R. funding acquisition; J. R. project administration; J. R. and S. I. M. resources; T. J. L., M. T. T., and J. J. L. investigation; M. T. T. formal analysis.

Funding and additional information

This work was supported by grants from the 10.13039/501100000925 National Health and Medical Research Council of Australia (NHMRC, Project Grant No. APP1123586 , S. I. M. and H. H. R.) J. R. is supported by an 10.13039/501100000925 NHMRC investigator award.
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