T cell receptor cross-reactivity expanded by dramatic peptide-MHC adaptability

Abstract

T cell receptor cross-reactivity allows a fixed T cell repertoire to respond to a much larger universe of potential antigens. Recent work has emphasized the importance of peptide structural and chemical homology, as opposed to sequence similarity, in T cell receptor cross-reactivity. Surprisingly, though, T cell receptors can also cross-react between ligands with little physiochemical commonalities. Studying the clinically relevant receptor DMF5, we demonstrate that cross-recognition of such divergent antigens can occur through mechanisms that involve heretofore unanticipated rearrangements in the peptide and presenting MHC protein, including binding-induced peptide register shifts and extensions from MHC peptide binding grooves. Moreover, cross-reactivity can proceed even when such dramatic rearrangements do not translate into structural or chemical molecular mimicry. Beyond demonstrating new principles of T cell receptor cross-reactivity, our results have implications for efforts to predict and control T cell specificity and cross-reactivity and highlight challenges associated with predicting T cell reactivities.

Figure 1.. Two distinctive classes of peptides recognized by the DMF5 TCR.

(A) Sequence logos of peptides in the GIG and DRG classes of peptides identified through specificity profiling by yeast display. (B) Dendrogram showing the relationships between the GIG (black) and DRG (red) peptide classes. (C) GIG and DRG peptides are predicted to adopt different and conformations when bound to HLA-A2, with the conformations of the GIG peptides closely resembling the MART-1 decamer. (D) Consistent with the structural modeling, the DRG peptides bind weaker to HLA-A2 as demonstrated by differential scanning fluorimetry (see also Supplementary Table 2). The DSF-determined T_m values correlate with well with structure-based energy scores, supporting the conclusion that the differences in peptide binding are attributable to pMHC structural differences between the two peptide classes. Error bars for the T_m measurements reflect DSF fitting error. R² is the coefficient of determination as determined by linear least squares analysis.

Figure 2.. The DRG peptide MMWDRGLGMM adopts a conformation distinct from MART-1 when bound to HLA-A2 and perturbs the structure of the HLA-A2 α2 helix.

(A) Comparison of the crystallographic structures of the MMWDRGLGMM and MART-1 peptides bound to HLA-A2. Compared to MART-1, the MMWDRGLGMM peptide is more bulged and lifted away from the base of the binding groove. The MMWDRGLGMM and MART-1 peptide backbones differ with an RMSD of 2.3 Å when the HLA-A2 peptide binding domains are superimposed. (B) The MMWDRGLGMM peptide perturbs the short arm of the HLA-A2 α2 helix. Compared to the structure with MART-1, the position of pL7 in MMWDRGLGMM forces a shift of Val152, which in turn forces the helix away from the peptide, indicated by the 1.8 Å displacement of Ala150. (C) The MMWDRGLGMM and MART-1 peptide/HLA-A2 complexes present different surfaces for TCR recognition. Peptide surfaces are colored by partial atomic charge.

Figure 3.. The DMF5 TCR does not show a preference for DRG vs. GIG peptides but the classes are recognized via distinct mechanisms.

(A) SPR binding data for DMF5 recognition of the GIG (black) or DRG (red) peptides. K_D values range from 6 μM for the MART-1 GIG peptide to 200 μM for the SMAGIGIVDV GIG peptide (see also Supplementary Table 2). Unlike the pMHC T_m values, the K_D values do not segregate by peptide class. Each curve indicates a global analysis of two independent replicates as described in the Methods. (B) Functional recognition by DMF5-expressing T cells also does not segregate by class and correlates well with TCR binding affinity. The left panel shows IFNγ release for recognition of each peptide, with TCR-transduced PBMCs co-cultured with peptide-pulsed T2 cells. Data for DRG peptides is red. T2 indicates presenting cells without peptide; Tax is a negative control peptide (LLFGYPVYV). Dots show the values from three independent experiments; bars indicate averages. The right panel shows a plot of ΔG° vs. the mean of the cytokine release experiments. Error bars for IFNγ are SEM from the three experiments shown in the left panel; error bars for ΔG° are propagated K_D errors. R² is the coefficient of determination as determined by linear least squares analysis. (C) Although DMF5 does not distinguish between recognition of DRG and GIG peptides in binding or function, the peptide classes are recognized with different kinetics. As shown for MART-1 and SMLGIGIVPV in the left panels, GIG peptides are recognized with fast on-rates and fast off-rates. DRG peptides are recognized with slower on and slower off rates, as shown for LMFDRGMSLL and MMWDRGLGMM in the right panels. Thick red lines indicate fits to dissociation phases. Rates are determined from global analysis of the number of separate injections shown in each panel (see Supplementary Figure 2 for full datasets). (D) The differential impact of the HLA-A2 A150P mutation confirms HLA-A2 α2 helix shift is important for DMF5 recognition of MMWDRGLGMM but not MART-1. As shown by SPR, the A150P mutation has a minor impact on DMF5 binding to MART-1 (ΔΔG° of 0.4 kcal/mol), but a substantial impact on binding to MMWDRGLGMM (ΔΔG° > 2 kcal/mol). Each curve indicates a global analysis of two independent replicates as described in the Methods.

Figure 4.. DMF5 recognizes the GIG peptides with common structural solutions.

The TCR-pMHC structure of DMF5 bound to the SMLGIGIVPV peptide is almost identical to the structure of DMF5 bound to MART-1. When the HLA-A2 peptide binding domains are superimposed, all atoms of the CDR loops in the MART-1 and SMLGIGIVPV complexes differ with a RMSD of only 0.9 Å. The SMLGIGIVPV peptide forms many of the same interactions with DMF5 as it does MART-1, including hydrogen bonds from peptide positions 2 and 3 to Gln30 in CDR1α, and both complexes incorporate the same key water molecule that bridges the peptide center and the TCR. Blue dashed lines in the panels indicate hydrogen bonds.

Figure 5.. DMF5 recognizes the MMWDRGLGMM DRG peptide very differently, inducing a peptide register shift and C-terminal extension while returning the HLA-A2 α2 helix to its usual conformation.

(A) Illustration of the register shift and C-terminal extension in the MMWDRGLGMM peptide induced upon TCR binding. The side chain of pArg5 moves by 7 Å, the backbone at pGly8 is pressed 5 Å into the base of the binding groove, and the side chain of pMet10 is displaced by 8 Å upon shifting out of the HLA-A2 F pocket. (B) The changes in the MMWDRGLGMM peptide seen upon TCR binding bring its conformation closer to but not coincident with the conformation of the GIG peptides, and the TCR-exposed surfaces remain highly distinct. Peptide surfaces are colored by partial atomic charge. (C) The HLA-A2 α2 helix returns to a more traditional geometry in the register-shifted MMWDRGLGMM complex, with Lys146, Ala150, and Val152 all moving back towards the center of the binding groove.

Figure 6.. The GIG and register-shifted DRG surfaces are recognized by DMF5 through small TCR side chain rearrangements and differential use of interfacial water.

(A) In the MMWDRGLGMM complex, the DMF5 TCR is positioned 2 Å closer to the peptide N-terminus, but compared to the GIG complexes there are no CDR loop rearrangements. Gln30 of CDR1α forms similar hydrogen bonds with the peptide N-terminal half (blue dashed lines) in the MMWDRGLGMM interface as it does in the GIG interfaces. (B) In the MMWDRGLGMM interface, side chain re-arrangements in Asn91α and Asn33β occur to optimize electrostatic and steric complementarity, and new water molecules are incorporated to meet hydrogen bonding needs. (C) Steric clashes between Gly101/Thr102 of CDR3β and pArg5, and Phe100 of CDR3α and pAsp4 drive the conformational change in the MMWDRGLGMM peptide upon TCR binding, as shown in this superimposition of the unbound MMWDRGLGMM peptide into the DMF5-MMWDRGLGMM/HLA-A2 complex.