The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.
Naturally acquired immune responses against human cancers often include CD8 ؉ T cells specific for the cancer testis antigen NY-ESO-1. Here, we studied T cell receptor (TCR) primary structure and function of 605 HLA-A*0201/NY-ESO-1 157-165-specific CD8 T cell clones derived from five melanoma patients. We show that an important proportion of tumor-reactive T cells preferentially use TCR AV3S1/BV8S2 chains, with remarkably conserved CDR3 amino acid motifs and lengths in both chains. All remaining T cell clones belong to two additional sets expressing BV1 or BV13 TCRs, associated with ␣-chains with highly diverse VJ usage, CDR3 amino acid sequence, and length. Yet, all T cell clonotypes recognize tumor antigen with similar functional avidity. Two residues, Met-160 and Trp-161, located in the middle region of the NY-ESO-1 157-165 peptide, are critical for recognition by most of the T cell clonotypes. Collectively, our data show that a large number of ␣ TCRs, belonging to three distinct sets (AVx/BV1, AV3/BV8, AVx/BV13) bind pMHC with equal antigen sensitivity and recognize the same peptide motif. Finally, this in-depth study of recognition of a self-antigen suggests that in part similar biophysical mechanisms shape TCR repertoires toward foreign and self-antigens.antigen recognition ͉ cytolytic T lymphocytes ͉ melanoma ͉ T cell receptors ͉ tumor immunity T he specificity of CD8 ϩ cytolytic T lymphocyte (CTL) responses relies on the interaction of clonotypically distributed antigen receptors (TCR) on the surface of the effector cell with small immunogenic peptide fragments displayed at the surface of a target cell by self-MHC class I molecules (pMHC) (1). Binding of the heterodimeric ␣-chains of the TCR to the pMHC complex is a key step leading to T cell activation and cell killing. The ␣ TCRs bind agonist pMHCs with relatively low affinity (K d Х1-100 M) through complementary determining regions (CDR) present on their variable domains (2). The mature TCR repertoire is shaped by both positive and negative intrathymic selection, leading to an estimated 2.5 ϫ 10 7 different TCR clonotypes in the human peripheral T cell pool (3). A relatively small number of CTL precursor cells is normally selected in response to the antigenic stimulus and comprises cells bearing several TCRs (Ϸ10 to Ϸ50 clonotypes) differing from each other yet having the ability to recognize the same pMHC complex. The recent development and availability of multimerized MHC-peptide complexes combined to TCR spectratyping with high-throughput DNA sequencing allowed the study of the development of antigen-driven CD8 ϩ T lymphocyte response to chronic antigenic exposure, e.g., in viral infection (CMV, EBV, HIV) or in cancer patients. The impact of TCR diversity on recognition of single antigenic pMHC complexes has been extensively investigated, and the relative contributions of each TCR -chain have been addressed in several models. In a few systems, strong biases in the TCR repertoire selection of antigenspecific T cells, resulting in the preferentia...
Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157–165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, KD = ∼1 − 5 μM. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function, in line with the “half-life” model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.
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