Method for preparing HLA-A24:02 APC cells and application thereof
By constructing an HLA-A24:02 expression vector and infecting K562 cells, HLA-A24:02 APC cells were prepared, solving the problem of obtaining HLA-A24:02 subtype DC cells. This achieved efficient and low-cost antigen presentation, which is suitable for immune research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 赣州市人民医院
- Filing Date
- 2026-04-07
- Publication Date
- 2026-06-05
AI Technical Summary
Dendritic cells (DCs) account for a very low percentage of human peripheral blood, making it difficult to meet the large-scale needs of scientific research and applications. Existing technologies are also insufficient to efficiently obtain HLA-A24:02 subtype-specific engineered APC cells.
An expression vector for HLA-A24:02 was constructed, and K562 cells were infected by lentiviral packaging. mCherry-positive cells were sorted by flow cytometry, and the expression of HLA-A24:02 was detected. The cells were then co-incubated with the antigen peptide and finally co-cultured with PBMCs. The secretion of IFN-γ was detected by ELISApot, and HLA-A24:02 APC cells were prepared.
APC cells, which can replace HLA-A24:02 subtype DC cells, are provided to achieve the same antigen presentation function as natural HLA-A24:02 subtype DC cells, reducing cell acquisition costs and the impact of individual differences, simplifying the detection process, and improving cell purity and functional stability.
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Figure CN122146790A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell preparation technology, and more specifically, to a method for preparing HLA-A24:02APC cells and its application. Background Technology
[0002] Antigen-presenting cells (APCs) are core cells in the body's immune system responsible for presenting antigens to T cells and activating T cell function. Dendritic cells (DCs), as classic APCs, play a crucial role in immune activation. However, the proportion of DCs in human peripheral blood is extremely low, and the yield of DCs obtained through isolation and induction is also low, making it difficult to meet the large-scale needs of scientific research and applications. Therefore, we have made improvements and proposed a method for preparing HLA-A24:02 APC cells and its application. Summary of the Invention
[0003] This invention provides a method for preparing HLA-A24:02APC cells, comprising: Step 1: Construct the expression vector for HLA-A24:02; Step 2: The vector is packaged into lentivirus and used to infect K562 cells; Step 3: Flow cytometry sorting of mCherry-positive K562 cells; Step 4: HLA antibody detection of HLA-A24:02 expression on K562 cells; Step 5: Load antigen peptides onto K562-HLA-A24:02; Step 6: K562-HLA-A24:02 was co-cultured with PBMCs, and IFN-γ was detected by ELISpot.
[0004] As a preferred technical solution of this application, step 1 specifically includes: The A2H and β2M subunits of HLA-A24:02 were linked together using a linker, a signal peptide sequence was added to the N segment, and an mcherry tag was added to the C segment to form a vector for fusion expression of HLA-A24:02.
[0005] As a preferred technical solution of this application, in step 2, lentivirus packaging uses 293T cells, and the packaging system includes pSPAX2, pMD2.G and HLA-A24:02 expression vector, with a molar ratio of 2:1:1. Virus fluid is collected after transfection and culture for 48-72 hours.
[0006] As a preferred technical solution of this application, in step 3: mCherry-positive cells are successfully infected K562 cells, which are separated by flow cytometry.
[0007] As a preferred technical solution of this application, step 4 includes: If the sorted K562-HLA cells are bound to the HLA antibody (W6 / 32) and the result is positive, it proves that HLA is successfully expressed on the surface of K562 cells.
[0008] As a preferred technical solution of this application, step 5 includes: using K562-HLA cells as APC cells and co-incubating them with the antigen peptide.
[0009] As a preferred technical solution of this application, the antigenic peptide is a tumor-associated antigenic peptide, selected from one or more of MAGE-A1, MAGE-A3, and NY-ESO-1.
[0010] As a preferred technical solution of this application, in step 6, the cells are co-cultured with PBMCs to stimulate T cell immune responses. The secretion of IFN-γ is detected by ELISPOT. The results show that K562-HLA cells loaded with antigen peptides can effectively stimulate T cells to secrete IFN-γ.
[0011] An HLA-A24:02APC cell was prepared using a method for preparing HLA-A24:02APC cells.
[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: In the scheme of this application: 1. This invention constructs an APC cell (K562-HLA-A24:02) that can replace HLA-A24:02 subtype DC cells, filling the gap in engineered APC cells specifically for the HLA-A24:02 subtype. This cell can achieve the same antigen presentation function as natural HLA-A24:02 subtype DC cells, providing a dedicated tool cell for immune research targeting this HLA subtype; 2. This invention constructs APC cells using the K562 cell line as a base. K562 cells can be permanently passaged and expanded in vitro without the need for repeated isolation and induction of DC cells from human peripheral blood. This reduces the amount of peripheral blood used, lowers the dependence on raw materials for cell acquisition and costs, and avoids the influence of individual differences in peripheral blood sources on experimental results. Attached Figure Description
[0013] Figure 1 The flowchart of the HLA-A24:02APC cell preparation method provided in this application; Figure 2 A schematic diagram of the K562-HLA-A24:02 cells provided in this application; Figure 3 A schematic diagram of the expression vector for HLA-A24:02 provided in this application; Figure 4Flow cytometry images of mCherry fluorescence expression in K562 cells and K562-HLA-A24:02 cells provided in this application; Figure 5 Flow cytometry diagram of HLA molecule expression on the surface of K562 cells and K562-HLA-A24:02 cells provided in this application; Figure 6 The image shows the ELISPOT assay results for the K562-HLA-A24:02APC cell antigen presentation function provided in this application. Detailed Implementation
[0014] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
[0015] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.
[0016] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.
[0017] Example 1, please refer to Figures 1-6 A method for preparing HLA-A24:02APC cells, comprising: Step 1: Construct the expression vector for HLA-A24:02; specifically, the A2H and β2M subunits of HLA-A24:02 are linked together using a linker, a signal peptide sequence is added to the N segment, and an mcherry tag is added to the C segment to form a vector for fusion expression of HLA-A24:02, as described above. Figure 3 The A2H and β2M subunits are linked by a linker, ensuring their spatial conformational stability and guaranteeing the normal function of the HLA-A24:02 molecule. The N-terminal signal peptide guides the HLA-A24:02 molecule to be directionally transported to the cell surface, meeting the functional requirements of antigen presentation. The C-terminal mcherry tag provides an intuitive label for subsequent flow cytometry sorting and expression verification, eliminating the need for additional labeling reagents, simplifying the detection process, and reducing the interference of exogenous reagents on cell function. Step 2: Lentiviral packaging of the vector to infect K562 cells; 293T cells were used for lentiviral packaging, and the packaging system included pSPAX2, pMD2.G, and HLA-A24:02 expression vector in a molar ratio of 2:1:1. Viral fluid was collected after 48-72 hours of culture following transfection. 293T cells are commonly used lentiviral packaging cells, with advantages of high transfection efficiency and high viral yield. The 2:1:1 molar ratio of pSPAX2 (helper packaging plasmid) and pMD2.G (enveloping plasmid) with the target vector optimizes viral packaging efficiency, ensuring the integrity of viral particles and infectivity. The 48-72 hour culture time after transfection ensures that viral particles fully mature and are released into the culture medium, maximizing the collection of effective viral fluid and providing a sufficient source of virus for subsequent K562 cell infection. Step 3: Flow cytometry sorting of mCherry-positive K562 cells; mCherry tags have the characteristics of strong fluorescence signal, good stability and no cytotoxicity. Positive cells can be accurately identified and sorted by flow cytometer. Compared with traditional screening methods (such as antibiotic screening), flow cytometry sorting is faster and has higher purity. It can directly obtain a high proportion of successfully infected cells and avoid the problems of cell damage and long screening cycle in the antibiotic screening process. Step 4: HLA antibody detection of HLA-A24:02 expression on K562 cells; sorted K562-HLA cells bind to HLA antibody (W6 / 32). If the result is positive, it proves that HLA is successfully expressed on the surface of K562 cells. Figure 5 ; Figure 5 The red curve represents the FITC fluorescence signal of parental K562 cells after incubation with W6 / 32 antibody (negative control), and the blue curve represents the FITC fluorescence signal of K562-HLA-A24:02 cells after incubation with W6 / 32 antibody. The results indicate that HLA-A24:02 molecules were successfully expressed on the surface of K562-HLA-A24:02 cells. Step 5: Loading antigen peptides onto K562-HLA-A24:02 cells; The HLA-A24:02 molecules expressed on the surface of K562-HLA-A24:02 cells can specifically bind to the antigen peptides to form antigen peptide-HLA complexes, which are key to activating T cell immune responses; The loading process does not require complex instruments and reagents, and only co-incubation is needed to achieve the binding of antigen peptides, making the operation convenient. Different antigen peptides can be replaced according to experimental needs to adapt to different immune activation scenarios. Step 6: K562-HLA-A24:02 was co-cultured with PBMCs, and IFN-γ was detected by ELISpot.
[0018] Furthermore, in step 3: mCherry-positive cells are successfully infected K562 cells, which are sorted by flow cytometry, referring to... Figure 4 ; Figure 4 The left image shows uninfected K562 parental cells (mCherry negative control), with an mCherry positivity rate of 1.22% in the Q3 region; Figure 4 The right image shows K562-HLA-A24:02 cells after lentiviral infection and sorting. The mCherry positivity rate in the Q3 region is 88.9%, indicating that the HLA-A24:02 fusion gene is successfully expressed in K562 cells. Furthermore, step 5 includes: using K562-HLA cells as APC cells and co-incubating them with the antigen peptide.
[0019] Furthermore, the antigenic peptide is a tumor-associated antigenic peptide, selected from one or more of MAGE-A1, MAGE-A3, and NY-ESO-1; MAGE-A1, MAGE-A3, and NY-ESO-1 are all common tumor-associated antigens that are highly expressed in various tumor cells. Their corresponding antigenic peptides can be presented by HLA-A24:02 molecules, activating tumor-specific T cells and exerting anti-tumor immune effects.
[0020] Furthermore, in step 6, K562-HLA cells were co-cultured with PBMCs to stimulate T cell immune responses. IFN-γ secretion was assessed using ELISPOT. The results showed that loading the antigen peptide onto K562-HLA cells effectively stimulated T cell IFN-γ secretion. Figure 6 ; Figure 6 In the study, the blank control group was the PBMC-only group, and the PHA group was the positive control; the K562-HLA+PBMC group was the negative control without antigen peptide loading; the K562-HLA+antigen peptide 1+PBMC group and the K562-HLA+antigen peptide 2+PBMC group were the antigen peptide loading experimental groups. The results showed that the number of IFN-γ secretion spots in the antigen peptide loading experimental groups was significantly higher than that in the negative control group, indicating that K562-HLA-A24:02APC cells can effectively present antigens and activate T cell immune responses.
[0021] Example 2: An HLA-A24:02APC cell was prepared by the HLA-A24:02APC cell preparation method.
[0022] According to another aspect of the present invention, the use of HLA-A24:02APC cells as alternatives to HLA-A24:02 subtype DC cells as antigen-presenting cells is provided.
[0023] The sequence of HLA-A24:02 is: MATGSRTSLLLAFGLLCLPWLQEGSAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPCIVKWDRDMGGGSGGGGSGGGSGGGGSGSHSMAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPR FIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTMHMTHHPISDHEATLRCWALGFYPAEITL TWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKVRRVRGSGATNFSLLKQAGDVEENPGPMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK 。
[0024] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, an electrical connection, or a connection that allows communication between them; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.
[0025] Obviously, the embodiments described above are merely some embodiments of the present invention, not all embodiments. The accompanying drawings show preferred embodiments of the present invention, but do not limit the patent scope of the present invention. The present invention can be implemented in many different forms; rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the patent protection scope of this invention.
Claims
1. A method for preparing HLA-A24:02APC cells, characterized in that, include: Step 1: Construct the expression vector for HLA-A24:02; Step 2: The vector is packaged into lentivirus and used to infect K562 cells; Step 3: Flow cytometry sorting of mCherry-positive K562 cells; Step 4: HLA antibody detection of HLA-A24:02 expression on K562 cells; Step 5: Load antigen peptides onto K562-HLA-A24:02; Step 6: K562-HLA-A24:02 was co-cultured with PBMCs, and IFN-γ was detected by ELISpot.
2. The method for preparing HLA-A24:02APC cells according to claim 1, characterized in that, Step 1 specifically includes: The A2H and β2M subunits of HLA-A24:02 were linked together using a linker, a signal peptide sequence was added to the N segment, and an mcherry tag was added to the C segment to form a vector for fusion expression of HLA-A24:
02.
3. The method for preparing HLA-A24:02APC cells according to claim 1, characterized in that, In step 2, lentivirus packaging was performed using 293T cells. The packaging system included pSPAX2, pMD2.G, and HLA-A24:02 expression vector, with a molar ratio of 2:1:
1. Viral fluid was collected after transfection and culture for 48-72 hours.
4. The method for preparing HLA-A24:02APC cells according to claim 1, characterized in that, In step 3: mCherry-positive cells are successfully infected K562 cells, which are then sorted by flow cytometry.
5. The method for preparing HLA-A24:02APC cells according to claim 1, characterized in that, Step 4 includes: If the sorted K562-HLA cells are bound to the HLA antibody (W6 / 32) and the result is positive, it proves that HLA is successfully expressed on the surface of K562 cells.
6. The method for preparing HLA-A24:02APC cells according to claim 1, characterized in that, Step 5 includes: using K562-HLA cells as APC cells and co-incubating them with the antigen peptide.
7. The method for preparing HLA-A24:02APC cells according to claim 6, characterized in that, The antigenic peptide is a tumor-associated antigenic peptide, selected from one or more of MAGE-A1, MAGE-A3, and NY-ESO-1.
8. The method for preparing HLA-A24:02APC cells according to claim 1, characterized in that, In step 6, K562-HLA cells were co-cultured with PBMCs to stimulate T cell immune responses. The secretion of IFN-γ was detected by ELISPOT. The results showed that loading antigen peptides onto K562-HLA cells could effectively stimulate T cells to secrete IFN-γ.
9. An HLA-A24:02APC cell, characterized in that, It is prepared by the HLA-A24:02APC cell preparation method according to any one of claims 1-8.
10. The use of HLA-A24:02APC cells according to claim 9 as alternatives to HLA-A24:02 subtype DC cells as antigen-presenting cells.