Method for simultaneously detecting neoantigen immunogenicity and neoantigen specificity TCR (T cell receptor)

Abstract

The invention discloses a method for simultaneously detecting neoantigen immunogenicity and a neoantigen specificity TCR (T cell receptor). A neoantigen polypeptide is added to peripheral blood mononuclear cells (PBMCs) to obtain antigen-stimulated cells, and compared with a traditional method, the method has the advantages that the time required for obtaining antigen-specific cells is shortened,the whole process of verifying the neoantigen immunogenicity and obtaining a TCR CDR3 sequence only needs 13 days, and compared with the 40-day treatment process of the traditional method, the methodsaves the time cost greatly. Compared with a traditional MHC-peptide tetramer technology, the method is low in cost, a special tetramer needs to be prepared for one antigen polypeptide in the MHC-peptide tetramer technology, the cost of the sequencing method is remarkably reduced, and compared with a traditional ELISPOT method with the test flux only reaching 10 pieces per batch, the sequencing method is not limited in test flux, higher in efficiency, high in test result accuracy and wide in applicability.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and cellular immunology, and relates to a method for determining the immunogenicity of neoantigens, in particular to a method for simultaneously detecting immunogenicity of neoantigens and specific TCR of neoantigens. Background technique [0002] T lymphocytes (T lymphocytes), referred to as T cells, are lymphatic stem cells derived from bone marrow. After differentiation and maturation in the thymus, they are distributed to immune organs and tissues throughout the body through lymphatic and blood circulation to exert immune functions. T cells can specifically recognize neoantigens expressed by tumor cells and presented to the major histocompatibility complex (MHC) molecules on the cell surface through T cell antigen recognition receptors (T cell receptors, TCRs), and produce Antigen-specific T cells recognize and kill tumor cells to complete the body's anti-tumor cell immunity. Tumor...

AI Technical Summary

Problems solved by technology

Before preparing the tetramer, it is also necessary to know which type of HLA molecule the neoantigen polypeptide is presented by. Usually, whether there is affinity between the neoantigen polypeptide and HLA is the result of the prediction of the bioinformatics algorithm, which often has a certain deviation from the real situation. Moreover, not every typed HLA molecule can be prepared. In addition, the cost of preparing tetramers is high, and it is easy to degrade and cannot be stored for a long time. It needs to be prepared and used immediately

[0006] Therefore, the traditional MHC-peptide tetramer flow cytometric analysis technology has the following technical problems: (1) The pre-stimulation time is long, and the content of neoantigen-specific T cells in peripheral blood mononuclear cells (PBMC) is low. Stimulate T cells for more than 30 days; (2) Dendritic (DC) cells are required for pre-stimulation, and DC cells are monocytes isolated from PBMCs in the presence of granulocyte-macrophage colony-stimulating factor ( GM-CSF) and interleukin 4 (IL-4) induced differentiation, it usually takes 7 days to grow from monocytes to mature DC cells, and the time period is longer

It is also necessary to consume a certain amount of PBMC to obtain DC cells; (3) The preparation of tetramers is difficult and expensive, and it needs to be prepared and used immediately, and it is not easy to store; (4) The HLA restriction of the peptide must be known before the experiment. Secondly, the combination of MHC molecules and peptides prepared is uncertain, and stable MHC-peptide tetramers cannot be produced.

The above problems lead to high time-consuming and high economic costs of the traditional MHC-peptide tetramer technology, which is not conducive to the large-scale and standardized implementation of neoantigen immunogenicity verification

Images