Human leukocyte antigen (HLA) system is a gene complex that encodes the major histocompatibility complex (MHC) protein of human, which is located on the cell membrane of human leukocytes. These cell surface proteins are responsible for regulating the human immune system. In vitro HLA stabilization assay is a common method to screen and predict the immunogenicity of T cell epitopes. At present, Creative Biolabs is able to provide high-quality HLA stabilization assay service as a powerful support for tumor-associated antigen discovery.
Introduction of HLA
HLA genes are highly polymorphic, which means that they have many different alleles, enabling them to fine-tune the adaptive immune system. With the identification of the first HLA class II epitope in 1994, the role of CD4+ T cells in anti-tumor immunity has been paid more and more attention. Tumor exons provide comprehensive information on mutation, high expression gene and abnormal splicing, which can be used in individualized tumor immunotherapy. Because the new epitope-specific T cells often have antitumor effect, the mutant tumor antigen T cell epitopes have attracted special attention. The binding and stability of potential T cell epitopes to class I MHC molecules form the basis of their immunogenicity and provide a basic understanding of the factors that determine the cellular immune response. T cell receptor (TCR) regulates T cell-mediated immunity by recognizing short peptide fragments binding to MHCs expressed on the surface of almost all nucleated cell types. The formation of stable MHC class I (MHCI) depends on the binding affinity of peptides to MHC.
Fig.1 MHC peptide binding assays. (Gfeller, 2016)
HLA Stabilization Assay in Creative Biolabs
Peptide induced MHC stability assay (also called HLA stability assay) is one of the simplest and most cost-effective methods to test peptide binding to class I alleles. This method generally uses one or a combination of database searches to identify peptides, so as to synthesize peptides, and further evaluate their ability to bind to the appropriate HLA alleles, usually using defective transporter associated with antigen processing (TAP) cell lines, such as T2 (human) or RMA-S (mouse). In practice, because of the high cost of peptide synthesis and the low frequency of specific memory T cells, which limits the ability to be evaluated in a single assay.
Therefore, Creative Biolabs tries to predict the effect of immune superiority on peptide selection, usually through the combination of computer analysis and experience, to generate a small number of possible candidates. Previous successful experience has shown that without efficient TAP mediated transport of cytosolic peptides to endoplasmic reticulum (ER), the assembled class I complexes are structurally unstable and can only remain on the cell surface instantaneously. However, when RMA-S or T2 is incubated with an exogenous peptide capable of binding to class I, the surface pHMCs can be stabilized and easily detected by flow cytometry against class I mAb.
Fig.2 The competitive peptide binding to HLA complex was measured by flow cytometry. (Delcommenne, 2017)
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References
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