Fluorine substitutions in an antigenic peptide selectively modulate T cell receptor binding in a minimally perturbing manner

T cell receptor (TCR) recognition of antigenic peptides bound and presented by major histocompatibility complex (MHC) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide/MHC molecules weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Further, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. Here, we studied how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the HTLV-1 Tax11-19 peptide (Tax) enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with A6, independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, in probing the mechanism of fluorine’s effect on TCR binding, our data were most consistent with fluorine’s unique “polar hydrophobicity,” a finding which should impact other attempts to alter molecular recognition with fluorine

Ethyl 5-[6-(furan-2-yl)-1,2,4-triazolo[3,4-b][1,3,4]thia­diazol-3-yl]-2,6-di­methylnicotinate

In the title compound, C17H15N5O3S, the plane of the triazolo–thia­diazole system forms dihedral angles of 15.68 and 4.46° with the planes of the pyridine and furan rings, respectively. In the mol­ecule, there is an intra­molecular C—H⋯N inter­action. The crystal structure also contains other inter­molecular inter­actions, such as C—H⋯O hydrogen bonds, π–π stacking (centroid–centroid distances = 3.746 and 3.444 Å), non-bonded S⋯N [3.026 (2) Å] and C—H⋯π inter­actions

Deciphering the unusual HLA-A2/Melan-A/MART-1-specific TCR repertoire in humans.

The Melan-A/MART-1(26-35) antigenic peptide is one of the best studied human tumor-associated antigens. It is expressed in healthy melanocytes and malignant melanoma and is recognized by CD8(+) T cells in the context of the MHC class I molecule HLA-A*0201. While an unusually large repertoire of CD8(+) T cells specific for this antigen has been documented, the reasons for its generation have remained elusive. In this issue of the European Journal of Immunology, Pinto et al. [Eur. J. Immunol. 2014. 44: 2811-2821] uncover one important mechanism by comparing the thymic expression of the Melan-A gene to that in the melanocyte lineage. This study shows that medullary thymic epithelial cells (mTECs) dominantly express a truncated Melan-A transcript, the product of misinitiation of transcription. Consequently, the protein product in mTECs lacks the immunodominant epitope spanning residues 26-35, thus precluding central tolerance to this antigen. In contrast, melanocytes and melanoma tumor cells express almost exclusively the full-length Melan-A transcript, thus providing the target antigen for efficient recognition by HLA-A2-restricted CD8(+) T cells. The frequency of these alternative gene transcription modes may be more common than previously appreciated and may represent an important factor modulating the efficiency of central tolerance induction in the thymus

A molecular switch abrogates glycoprotein 100 (gp100) T-cell Receptor (TCR) targeting of a human melanoma antigen

Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination

UV-induced ligand exchange in MHC class I protein crystals

High-throughput structure determination of protein−ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, “conditional”, peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This “in crystallo” exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC β2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families

T-Cell Promiscuity in Autoimmune Diabetes

OBJECTIVE—It is well established that the primary mediators of β-cell destruction in type 1 diabetes are T-cells. Nevertheless, the molecular basis for recognition of β-cell–specific epitopes by pathogenic T-cells remains ill defined; we seek to further explore this issue

Predicting Peptide Binding Affinities to MHC Molecules Using a Modified Semi-Empirical Scoring Function

The Major Histocompatibility Complex (MHC) plays an important role in the human immune system. The MHC is involved in the antigen presentation system assisting T cells to identify foreign or pathogenic proteins. However, an MHC molecule binding a self-peptide may incorrectly trigger an immune response and cause an autoimmune disease, such as multiple sclerosis. Understanding the molecular mechanism of this process will greatly assist in determining the aetiology of various diseases and in the design of effective drugs. In the present study, we have used the Fresno semi-empirical scoring function and modify the approach to the prediction of peptide-MHC binding by using open-source and public domain software. We apply the method to HLA class II alleles DR15, DR1, and DR4, and the HLA class I allele HLA A2. Our analysis shows that using a large set of binding data and multiple crystal structures improves the predictive capability of the method. The performance of the method is also shown to be correlated to the structural similarity of the crystal structures used. We have exposed some of the obstacles faced by structure-based prediction methods and proposed possible solutions to those obstacles. It is envisaged that these obstacles need to be addressed before the performance of structure-based methods can be on par with the sequence-based methods

Increased peptide contacts govern high affinity binding of a modified TCR whilst maintaining a native pMHC docking mode

NaturalT cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A�0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3b loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134TCR made increased contacts with theTax peptide compared with the A6wt/A2- Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134TCR CDR3b loop.This peptide-focused enhancedTCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies