Structural And Immunological Study On Modified Antigens Presented By MHC Class I

Major histocomaptibility complex (MHC) is a tightly linked cluster of genes in vertebrate, whose products are the cell surface molecules that plays roles in intercellular recognition and in discrimination between self and nonself. The particular set of MHC molecules as antigen-presenting structures expressed by an individual influences the repertoire of antigens to which that T cells can repond. Therefore, the MHC partly determines the immune response of an individual to antigens of infectious organisms, and it has been implicated in the susceptibility to disease and in the development of autoimmunity. In humans, the MHC is referred to as the human leukocyte antigen (HLA) complex.Polypeptides produced by antigen processing and proteasomal degradation of intracellular proteins serve as cytotoxic T lymphocyte (CTL) epitopes presented by MHC class I molecules, which play a critical role in effective cellular immunity. Eukaryotic proteins bearing various post-translational modifications (PTMs) can generate a class of modified antigens, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for T-cell receptor (TCR)-mediated recognition. Evidence suggests that peptides containing PTMs, including formylation, deimination, glycosylation, acetylation, phosphrylation, cysteinylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition. Indeed, most naturally occurring peptides bearing PTMs can be discriminated from their unmodified homologs specifically by T cells. In some cases, quantitative and qualitative changes in PTM that occur during infection, inflammation, cellular transformation, death and aging result in the display of new MHC-associated neoantigens. The significance of PTMs upon epitopes and their use as vaccine candidates for the immunotherapy of cancer and autoimmune diseases have been increasingly appreciated.As one of the most common PTMs of eukaryotic proteins, Nα-terminal acetylation generates a class of Nα-acetylpeptides that are presented by MHC class I at the cell surface. Although such PTMs play a pivotal role in adjust the proteolysis, the molecular basis for the presentation and T-cell recognition of Na-acetylpeptides retains largely unknown. Herein, we determined a high-resolution crystallographic structure of HLA-B39complexed with an Nα-acetylpeptide derived from natural cellular processing, and also the complexes of B39with unmodified peptides. Unlike the NH2-free PI residues of unmodified peptides, the hydroxyl side chain of the acetylated Pl-Ser inserts into pocket A of the antigen-binding groove, and the Na-linked acetyl protrudes out of the groove for the T-cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide, but also notably switches the residues in the α1-helix of B39, which may impact the T-cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from human and murine revealed universal diminished stabilities. The CTL responses stimulated by synthetic Nt-acetylated HLA-A2restricted epitopes in HLA-A2.1/Kb transgenic mice suggested immunogenic enhancement after the Nt-acetylation of epitope. These findings provided the first insight into the mode of Nα-acetylpeptide-specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.With above understanding, to investigate the CTL-based cellular immunity against Nt-acetylated antigen of influenza virus, we used influenza A virus as the object of study, started with the prediction and identification of Nt-acetylated CTL epitopes derived from influenza virus, thereby evaluated their immunogenicities. In this study, we firstly identified an Nt-acetylated viral CTL epitope which stimulates specific T-cell responses in HLA-A24+healthy donors. This result contributed to the category of MHC-bound CTL epitopes, and provided support to understand the PTM-dependent T celluar immunity.Besides Nt-acetylation, phosphorylation as a signature of cancer, malignant transformation, and apoptosis has been regarded increasingly. And phosphorylation at two proximal sites is a common format in phosphoproteome. Thus, we solved the structures of a couple of diphosphorylated peptides (a10mer and a9mer) complexed with HLA-B27, respectively. These data clarified not only the particular features of diphosphopeptides other than peptides containing a single phosphate displayed by MHC class I, but also the molecular basis of the presentation of self antigens which serve as neoepitopes after the multiple altering of PTMs and protein slicing. In addition, we showed the phosphosite-dependent effect of phosphates on peptide binding affinity by using circular dichroism spectroscopy. Our work provided the first-ever structural and thermodynamic insights into the presentation of diphosphopeptides by MHC class I, and highlighted a potential influence of such special modification event on antigenic identity and TCR engagement. Multisite phosphopeptides represent a library of new potential drug and vaccine candidates against autoimmune disease and tumor require further exploration.Moreover, based on previous reports, we surveyed the celluar immunogenicity of seven HLA-A2restricted phosphopeptides via inoculation of HLA-A2transgenic mice with peptides, and objectively evaluated the value of phosphopeptides which be applied as potential tumor vaccines. Furthermore, we also used5’-RACE system to obtain the sequences of phosphopeptide-specific TCR repertoires, which provided methodological approaches for further study in this area.In summary, in this study we made structural and immunological investigations on Nt-acetylated and phosphorylated peptides presented by MHC class I molecules using the method combination of immunoinformatics, mouse-model, cell biology, and structural crystallography. Our results revealed the aspect and mechanism of antigens that function as’multi-altered self’ through the PTMs of adding groups and protein slicing, and suggested the potential influence of the PTMs on antigen presentation and T-cell recognition. The application values of such antigens containing PTMs in immunotherapy and vaccine design are discussed.