A class-mismatched TCR bypasses MHC restriction via an unorthodox but fully functional binding geometry

Abstract

MHC restriction, which describes the binding of TCRs from CD4⁺ T cells to class II MHC proteins and TCRs from CD8⁺ T cells to class I MHC proteins, is a hallmark of immunology. Seemingly rare TCRs that break this paradigm exist, but mechanistic insight into their behavior is lacking. TIL1383I is a prototypical class-mismatched TCR, cloned from a CD4⁺ T cell but recognizing the tyrosinase tumor antigen presented by the class I MHC HLA-A2 in a fully functional manner. Here we find that TIL1383I binds this class I target with a highly atypical geometry. Despite unorthodox binding, TCR signaling, antigen specificity, and the ability to use CD8 are maintained. Structurally, a key feature of TIL1383I is an exceptionally long CDR3β loop that mediates functions that are traditionally performed separately by hypervariable and germline loops in canonical TCR structures. Our findings thus expand the range of known TCR binding geometries compatible with normal function and specificity, provide insight into the determinants of MHC restriction, and may help guide TCR selection and engineering for immunotherapy.

Fig. 1. Electron density for key regions of the TIL1383I complex.

Densities are from a 2F_o-F_c composite OMIT map contoured at 1σ, highlighting the TIL1383I CDR loops and peptide in the TCR-peptide/MHC structure. The color scheme used here is maintained through the following figures.

Fig. 2. Structural overview of the TIL1383I-Tyr_370D/HLA-A2 complex.

a TIL1383I binds Tyr_370D/HLA-A2 counterclockwise compared to traditional TCR-peptide/MHC complexes, placing the germline CDR loops in unusual positions. The left panel shows the overview of the TIL1383I complex. The right panel shows a view of the CDR loops over the peptide/HLA-A2 complex. Circles show the positions of the centers of mass of the TCR variable domains. b Histograms of crossing angles of TCRs bound to class I (top) or class II (bottom) peptide/MHC complexes. The value for TIL1383I, indicated in orange, is more than two standard deviations above the means for both classes. The values at the edges (>120°) are for recently described TCRs that bind with reverse polarity. c Quantitative geometrical analysis indicates that, despite binding atypically, TIL1383I still resembles a class I-restricted TCR in how it accesses the Tyr_370D peptide. The position of TIL1383I is indicated by the star, also highlighted by the green arrow. The plots indicate the rotation (θ), tilt (ϕ), and distance (r) of the TCR variable domain COM relative to the MHC peptide-binding groove COM. d The class I-restricted character of TIL1383I revealed in panel c emerges from how the hypervariable CDR3 loops focus on the center and C-terminal bulge of the Tyr_370D peptide. e Modeling the structure of the TIL1383I-Tyr_370D/HLA-A2 complex and the canonically binding HCV1406-NS3/HLA-A2 complex into the structure of the intact TCR/CD3 complex along with the structure of CD8 bound to HLA-A2 shows an approximate 55° difference in the placement of the CD8 coreceptor relative to the CD3 subunits. Data for panels b and c are provided as a Source Data file.

Fig. 3. Contacts between TIL1383I and the HLA-A2 α1 helix, α2 helix, and the Tyr_370D peptide.

a Gln72 and Arg75 on the HLA-A2 α1 helix participate in a complex network of electrostatic interactions involving Glu101 in the TIL1383I CDR3β loop, as shown in the left panel. An unusual salt bridge is also formed between Arg34 in CDR1β and Glu19 at the edge of the HLA-A2 β-sheet platform. The right image illustrates the contacts between the HLA-A2 α1 helix and the TCR. Linewidths are proportional to the number of contacts, enumerated above each line. b TCR contacts to the HLA-A2 α2 helix are shifted towards the helix short arm and the connecting linker region. Images are as in panel a, with the left emphasizing the reliance on electrostatic interactions and the right showing contacts made by the CDR loops to helix. c TCR contacts to the peptide are distributed from pTyr1 to pGln8, with pMet6 inserted into a deep pocket formed by CDR3α and CDR3β.

Fig. 4. TIL1383I avoids common or shared TCR-HLA-A2 contacts.

a TIL1383I does not interact with the charge of the Arg65 sidechain on the HLA-A2 α2 helix when bound to Tyr_370D/HLA-A2, nor does it use Tyr52 in CDR2β to contact HLA-A2. The solvent-exposed surface areas of the Arg65 and Tyr52β sidechains are shown, emphasizing their lack of participation in the interface. b There are no shared Vβ-HLA-A2 contacts between TIL1383I and two other TCRs that share TRBV10-3 owing to the different placements of the CDR1β and CDR2β loops. c HLA-A2 is not serving as a molecular mimic of a class II protein, as without substantial rearrangements, the binding geometry of TIL1383I would result in significant interatomic steric clashes with the elevated β1 helix of a class II protein, shown by the red lines when the structure of HLA-DQ2.5 is aligned with HLA-A2 in the TIL1383I-Tyr_370D/HLA-A2 complex.

Fig. 5. Specificity within the TIL1383I-Tyr_370D/HLA-A2 interface.

a TIL1383I shows high specificity for the Tyr_370D peptide, as shown by a functional analysis of a positional scanning library of the peptide. Cells of the heat map indicate the extent of IL-2 released when TCR-transduced Jurkat cells were mixed with peptide-pulsed HLA-A2⁺ T2 cells. The color scale is on the right, with red indicating a loss of T-cell recognition. b Assessment of the positional scanning Tyr_370D library by a novel TCR-binding assay with UV-exchanged peptide/HLA-A2 complexes. Cells of the heat map indicate the change in TCR-binding free energy relative to the wild-type peptide. The color scale is again on the right, with red indicating the largest reduction in binding free energy. There is good concurrence between the functional and binding analysis of the library. c Assessment of the UV-exchanged peptide/HLA-A2 library panel b using differential scanning fluorimetry to measure T_m values. Cells of the heat map indicate the T_m. The color scale is again on the right, with red indicating the largest reduction in T_m. Most peptide variants have little impact on T_m and thus peptide binding. d, e Structural determinants of high specificity for Asp3 through Met6 of the Tyr_370D peptide in the interface with TIL1383I, showing how the interface would be intolerant to most substitutions at these peptide positions. f The binding data in panel b was used to generate a theoretical TCR-binding score for the 1.28 billion nonamers that retain methionine at position 2 and valine at position 9. The wild-type peptide has a score of 1.0, and the variant peptide scores have scores that are Gaussian-distributed around 0.5. There are 1.57 million peptides with a score of 0.8 or higher. The right panel shows sequence logos for the peptides in the top two deciles, along with the number of peptides. Data for panel f are provided as a Source Data file.

Fig. 6. Experimental validation of the TIL1383I specificity analysis.

In total, 72 peptides from the top 5% (scores ≥ 0.95) in the specificity analysis in Fig. 5f were selected for evaluation in co-culture experiments measuring IL-2 production. Peptides selected included the 24 highest-scoring peptides with two amino acid changes relative to the native Tyr_370D peptide, the 24 highest-scoring peptides with three amino acid changes, and the 24 highest-scoring peptides with four amino acid changes. Five peptides at 50% (scores = 0.5, 6–7 amino acid changes) were also selected, as were the five lowest-scoring peptides (bottom 20%, scores = 0.19, 7 amino acid changes). The sequence of each peptide is listed. Amino acid changes relative to the Tyr_370D peptide are in bold red font. The number of amino acid substitutions is given in the “# subs” column. IL-2 produced normalized to the amount produced by the WT peptide is colored red (maximum) to white (zero), along with the percent. The score from the fingerprint analysis is indicated by the “score” column. The estimated ΔG° (in kcal/mol) and K_D for TCR binding, computed from the score as described in the Methods, are also indicated. “neg CTRL” indicates a negative control peptide unrelated to Tyr_370D.