Method for changing HLA (human leukocyte antigen) of cell strain to present peptide, cell strain with changed HLA to present peptide and application of cell strain

Abstract

The invention provides a method for changing a cell strain HLA (human leukocyte antigen) to present peptide, a cell strain with changed HLA to present peptide and application of the cell strain. The method for changing the cell strain HLA to present peptide comprises the following steps. The HLA gene is transduced to a K562 cell strain by lentivirus, so that the cell strain expresses an HLA molecule. Then, the cell strain expressing the HLA molecules is transduced by lentivirus with the AIRE gene, and a gene transcription map and a protein map expressed by the cell strain are changed through the over-expressed AIRE effect, so that the variety of peptides presented by the HLA molecules is changed.

Description

Technical Field

[0001] The present invention relates to a method for changing the HLA peptide presentation of a cell line, a cell line with altered HLA peptide presentation, and uses thereof. Background Art

[0002] Since the effectiveness of AI models is highly dependent on the diversity and representativeness of training data, the generalization ability of the model will be limited when the training data is insufficient to cover all possible biological variability. The present invention ectopically expresses the autoimmune regulator (AIRE) gene to change the cell gene transcription profile and protein profile, thereby increasing the diversity of HLA-presented peptides. AIRE is expressed in the thymic medulla and plays an important role in the negative selection of immune T cells during thymic development. The T cell receptor (TCR) on the surface of each T cell recognizes specific antigens presented by the HLA (Human Leukocyte Antigen) complex on the antigen-presenting cell. TCR is produced by VDJ recombination fragments and has high diversity and antigen specificity. Some T cells recognize self-antigens presented by HLA and induce autoimmune diseases. These T cells must be eliminated through the negative selection mechanism in the thymus during development. Through AIRE regulation, medullary thymic epithelial cells (mTECs) express tissue-specific proteins specific to various tissue cells in the body. When the TCR of T cells that may cause autoimmune diseases binds too strongly to the HLA molecule complex on the surface of mTECs, it induces apoptosis of T cells that may cause autoimmune diseases.

[0003] Cell lines lacking HLA expression (e.g., K562, a human chronic myeloid leukemia cell line) can be used for HLA-related cellular immunology research. AIRE is a transcription factor that can induce the expression of tissue-specific proteins that are typically expressed only in certain cells. Summary of the Invention

[0004] The present invention provides a method for altering HLA peptide presentation in a cell line, a cell line with altered HLA peptide presentation, and uses thereof. Through AIRE, the K562-HLA cell line expresses tissue-specific proteins that are typically expressed only in other tissues of the body, thereby increasing the chances of HLA presenting different peptide sequences.

[0005] The present invention provides a method for altering HLA peptide presentation in a cell line, comprising the following steps: HLA genes are transduced into the cell line using a lentivirus to cause the cell line to express HLA molecules. Subsequently, the cell line expressing HLA molecules is transduced with a lentivirus carrying the AIRE gene. Overexpression of AIRE alters the gene transcription profile, protein types, and expression levels of the cell line, resulting in changes in the types of peptides presented by the HLA molecules.

[0006] In one embodiment of the present invention, the cell line comprises a human chronic myeloid leukemia cell line.

[0007] In one embodiment of the present invention, the human chronic myeloid leukemia cell line includes the K562 cell line.

[0008] In one embodiment of the present invention, the HLA molecule includes an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule.

[0009] The cell line with altered HLA peptide presentation of the present invention is prepared by the above-mentioned method for altering the HLA peptide presentation of a cell line.

[0010] The uses of the cell lines of the present invention include: extracting proteins from the cell lines, separating HLA molecules and their bound peptides from the cell line proteins by immunoprecipitation, and obtaining peptides presented by a single HLA through LC-MS / MS analysis and data analysis; performing RNA analysis on the cell lines by next-generation sequencing (NGS); and using the peptides presented by a single HLA and the RNA analysis as data for establishing an artificial intelligence model to predict peptides presented by HLA molecules. The cell lines described above are cell lines in which the HLA-presented peptides have been altered.

[0011] Based on the above, the present invention provides a method for altering HLA peptide presentation in a cell line, a cell line with altered HLA peptide presentation, and uses thereof. HLA genes are transduced into a cell line lacking HLA molecule expression via lentivirus, causing it to express HLA molecules. AIRE then induces the expression of tissue-specific proteins, previously expressed only in specific cells, thereby altering the overall protein types and expression levels expressed by the cell line. This further alters the types of HLA peptides presented, thereby improving the performance of artificial intelligence models. DETAILED DESCRIPTION

[0012] The following examples are provided for detailed description, but the examples provided are not intended to limit the scope of the present disclosure. In addition, the terms "including", "comprising", "having", etc. used in the text are open-ended terms, that is, meaning "including but not limited to".

[0013] The present invention provides a method for altering HLA peptide presentation in a cell line, comprising the following steps: HLA genes are transduced into a cell line lacking HLA molecule expression using a lentivirus, causing the cell line to express HLA molecules. Subsequently, the cell line expressing HLA molecules is transduced using a lentivirus carrying the AIRE gene. The overexpressed AIRE alters the gene transcription profile, protein types, and expression levels of the cell line, resulting in a change in the types of peptides presented by the HLA molecules.

[0014] In this embodiment, the cell line may include a human chronic myeloid leukemia cell line, such as the K562 cell line, but the present invention is not limited thereto. Other human cell lines that can be cultured in the laboratory may also be used, such as the Expi293, HEK293, A375, KG-1, or B721.221 cell lines. The HLA molecule may include an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule.

[0015] In this embodiment, the method for transducing HLA genes into cell lines lacking HLA molecule expression is lentiviral vector transduction, followed by culturing in a cell culture medium containing puromycin after infection and removing uninfected cells.

[0016] In this example, a cell line expressing HLA molecules was infected with a lentivirus carrying the AIRE gene. After establishing a cell line carrying HLA and AIRE molecules, the cell line was cultured in a cell culture medium containing blasticidin S and uninfected cells were removed.

[0017] In this example, ectopic expression of AIRE altered the gene transcription profile of the cell line. RNAseq analysis revealed a 2-fold or greater increase in 648 genes and a 2-fold or greater decrease in 288 genes. Proteomic analysis revealed an additional 606 proteins in cells expressing AIRE. This demonstrates that AIRE alters the gene transcription and protein expression profiles of the original cell line.

[0018] The present invention provides a cell line with altered HLA peptide presentation, prepared by the aforementioned method for altering HLA peptide presentation in a cell line. In this embodiment, the cell line may include a human chronic myeloid leukemia cell line, such as the K562 cell line, but the present invention is not limited thereto; other human cell lines that can be subcultured in the laboratory may also be used. HLA molecules may include HLA-A, HLA-B, or HLA-C molecules. Analysis of HLA peptide presentation revealed that HLA-A, B, and C molecules in cells expressing AIRE presented 2,141, 1,613, and 447 novel peptide sequences, respectively.

[0019] The present invention provides a method for using a cell line in which the HLA-presenting peptide has been altered, comprising: extracting protein from the cell line, separating the HLA molecule and its bound peptide from the cell line protein using immunoprecipitation, and obtaining a single HLA-presented peptide through LC-MS / MS analysis and data analysis; performing RNA analysis on the cell line using next-generation sequencing (NGS); and using the single HLA-presented peptide and RNA analysis as data for establishing an artificial intelligence model to predict peptides presented by HLA molecules.

[0020] In this example, RNA analysis of K562 cell lines expressing and not expressing AIRE was performed using next-generation sequencing (NGS). The results showed that the RNA expression of 648 genes was significantly increased in the cell line expressing AIRE.

[0021] The following experimental examples illustrate how the aforementioned cell lines with altered HLA peptide presentations of the present invention can improve the performance of artificial intelligence models. However, the following experimental examples are not intended to limit the present invention.

[0022] Experimental example

[0023] Experimental Example 1-A0201

[0024]

[0025] Experimental Example 2-B1301

[0026] For the experimental results of Experimental Example 2, please refer to Table 1 below.

[0027] Table 1

[0028]

[0029] Experimental Example 3-C1601

[0030] For the experimental results of Experimental Example 3, please refer to Table 2 below.

[0031] Table 2

[0032]

[0033] The above experiments demonstrate that AIRE can alter the total types and levels of RNA and protein expressed by cell lines, further altering the types of peptides presented by HLA. This increases the chances of HLA presenting different peptide sequences, helping to improve the performance of AI models.

[0034] In summary, the present invention provides a method for altering HLA peptide presentation in a cell line, a cell line with altered HLA peptide presentation, and uses thereof. HLA genes are transduced into a cell line lacking HLA molecule expression via lentivirus, causing it to express HLA molecules. AIRE then induces the expression of tissue-specific proteins, previously expressed only in specific cells, thereby altering the overall protein types and expression levels expressed by the cell line. This further alters the types of peptides presented by HLA, increasing the chances of HLA presenting different peptide sequences, thereby helping to improve the performance of artificial intelligence models.

Claims

1. A method for altering HLA peptide presentation in a cell line, characterized in that: include: Transducing the HLA gene into a cell line lacking HLA molecule expression using a lentivirus, so that the cell line expresses the HLA molecule; as well as The cell line expressing the HLA molecule is infected with a lentivirus carrying the AIRE gene. Through the overexpression of AIRE, the gene transcription profile, protein type and expression level expressed by the cell line are changed, resulting in a change in the type of peptide presented by the HLA molecule.

2. The method for changing the HLA presentation of a cell line according to claim 1, characterized in that: The cell lines include human chronic myeloid leukemia cell lines.

3. The method for changing the HLA presentation of a cell line according to claim 2, characterized in that: The human chronic myeloid leukemia cell line includes the K562 cell line.

4. The method of claim 1, wherein: HLA molecules include HLA-A molecules, HLA-B molecules, or HLA-C molecules.

5. A cell line with an altered HLA peptide presentation, characterized in that: The peptide is prepared by the method for changing the HLA presentation of a cell line according to any one of claims 1 to 4.

6. A use of a cell line, characterized in that: The cell line is the cell line with an altered HLA peptide presentation according to claim 5, comprising: After protein extraction from the cell line, the HLA molecules and their bound peptides are separated from the cell line proteins by immunoprecipitation, and peptides presented by a single HLA are obtained through LC-MS / MS analysis and data analysis; Performing RNA analysis on the cell lines by next generation sequencing; and The peptides presented by the single HLA and the RNA analysis are used as data required for establishing an artificial intelligence model, and the artificial intelligence model is used to predict the peptides presented by HLA molecules.

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