Method for screening high activity dendritic cell vaccine using cd39 and tim3

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a method for screening high-activity dendritic cell vaccine by using CD39 and TIM3, which comprises the steps of sorting and / or identifying mature dendritic cells by using biomarkers CD39 and TIM3. The present application takes CD39 and TIM3 as biomarkers for sorting mo-DC, and can screen high-activity mo-DC vaccine. Experimental results show that the mo-DC vaccine obtained by using the method has high expression of biomarkers such as CD11C, HLA-DR and CD197. Meanwhile, the experimental results also show that the T cell proliferation ratio of the CD39+TIM3+DC stimulation group is significantly higher than that of the DC stimulation group, indicating that the DC sorted by using CD39 and TIM3 has strong ability to induce immune response, and is significantly higher than the mo-DC group without screening.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for screening highly active dendritic cell vaccines using CD39 and TIM3. Background Technology

[0002] Monocyte-derived dendritic cell (mo-DC) vaccines have significant value in tumor immunotherapy. They induce CD14+ monocyte differentiation through GM-CSF and IL-4, enabling efficient presentation of tumor antigens (such as neoantigens or tumor lysates), activation of CD8+ cytotoxic T cells and CD4+ helper T cells, and induction of specific anti-tumor immune responses. Clinical studies have shown that mo-DC vaccines can alleviate clinical symptoms in 37% of patients with solid tumors such as breast cancer, and their efficacy can be significantly enhanced when combined with immune checkpoint inhibitors, such as anti-PD-1. However, current mo-DC vaccines lack specific molecular markers and mainly rely on broad-spectrum markers such as CD11C, CD83, and HLA-DR to distinguish cell subpopulations. However, these markers are cross-expressed with monocytes or other DC subpopulations, making it difficult to assess vaccine purity and functional activity.

[0003] In the field of tumor immunotherapy, CD39 and TIM3 have been extensively studied as immunomodulatory molecules, but they have not yet been clearly identified as specific marker molecules for tumor-associated cells (mo-DCs). CD39 is a nucleotide hydrolase that catalyzes the hydrolysis of extracellular ATP to produce adenosine (ADO), inhibiting the activity of T cells and natural killer (NK) cells in the tumor microenvironment and promoting the function of immunosuppressive regulatory (Treg) T cells. Although CD39 is highly expressed in various immune cells, such as Treg cells and myeloid-derived suppressor cells (MDSCs), and plays an immunosuppressive role in tumor-associated macrophages (TAMs), its expression and function in mo-DCs have not been systematically studied. TIM3 is an immune checkpoint receptor initially discovered in exhausted T cells, but it is also expressed in monocytes and macrophages. TIM3 inhibits T cell activation and pro-inflammatory cytokine secretion by binding ligands such as galectin-9 and carcinoembryonic antigen-associated cell adhesion molecules (CEACAM1), and participates in the regulation of immune synapses. However, current research focuses primarily on the function of TIM3 in T cells and the tumor microenvironment. Its expression pattern in mo-DCs and its impact on DC maturation and antigen presentation remain unclear, and there is no evidence to support its role as a specific marker molecule for mo-DCs. Summary of the Invention

[0004] Currently, there are no specific marker molecules for mo-DCs (morphological dendritic cells) in peripheral blood from cancer patients. Commonly used markers such as CD11C, HLA-DR, CD197, PD-L1, CD80, CD83, CD86, and CD40 can be expressed in various immune cells, and their expression in monocytes and mo-DCs is unstable, making it impossible to accurately identify mo-DCs. Furthermore, in vitro induced mo-DCs are unstable, often leading to variations in the expression of these molecules. Therefore, new markers are needed for further screening to obtain high-purity mo-DCs. To this end, the purpose of this invention is to provide a method for screening highly active dendritic cell vaccines using CD39 and TIM3.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for screening highly active dendritic cell vaccines using CD39 and TIM3 includes steps of sorting and / or identifying mature dendritic cells using biomarkers;

[0007] The biomarkers include CD39 and TIM3.

[0008] Furthermore, flow cytometry was used to sort mature dendritic cells expressing the CD39 and TIM3 biomarkers for the preparation of dendritic cell vaccines.

[0009] Furthermore, the biomarkers also include one or more of CD11C, HLA-DR, CD197, PD-L1, CD80, CD83, CD86, and CD40.

[0010] Furthermore, the dendritic cell vaccine is used in oncology.

[0011] Furthermore, mature dendritic cells were prepared using peripheral blood mononuclear cells.

[0012] Furthermore, the preparation of mature dendritic cells from peripheral blood mononuclear cells includes the following steps:

[0013] S100. Peripheral blood mononuclear cells were isolated from in vitro blood samples;

[0014] S200. Peripheral blood mononuclear cells were induced to differentiate using inducing factor I to obtain immature dendritic cells;

[0015] S300. Immature dendritic cells were induced by inducing factor II to obtain mature dendritic cells.

[0016] Further, in step S200, the inducing factor I is selected from GM-CSF and / or IL-4.

[0017] Further, in step S300, the inducing factor II is selected from one or more of TNF-α, PGE2, IL-1β, IFN-γ and IL-6.

[0018] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0019] The present invention provides a method for screening highly active dendritic cell vaccines using CD39 and TIM3. CD39 and TIM3 are used as biomarkers for sorting mo-DCs, enabling the screening of highly active mo-DC vaccines. Flow cytometry sorting results show that the mo-DC vaccines obtained using this method highly express known mature DC marker molecules CD11C, HLA-DR, CD197, PD-L1, CD80, CD83, CD86, and CD40. T cell proliferation detection results show that the T cell proliferation ratio in the CD39+TIM3+DC stimulation group is significantly higher than that in the DC stimulation group, indicating that CD39+TIM3+ sorted DCs have a strong ability to induce an immune response, and this characteristic is higher than that of unselected mo-DCs.

[0020] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0021] Figure 1 This is a flow cytometry FSC-SSC scatter plot of PBMC provided in Embodiment 1 of the present invention.

[0022] Figure 2 This is a histogram of the flow cytometry analysis results of PBMCs provided in Embodiment 1 of the present invention.

[0023] Figure 3 This is a statistical chart showing the expression of CD39 and TIM3 in mononuclear cells of 15 tumor patients provided in Example 1 of this invention.

[0024] Figure 4 This is a flow cytometry analysis diagram provided in Embodiment 2 of the present invention.

[0025] Figure 5 This is a diagram showing the detection results of different biomarkers on the surface of mo-DC cells provided in Embodiment 2 of the present invention.

[0026] Figure 6 This is a flow cytometry image of D39+TIM3+mo-DC cells sorted by flow cytometry, provided in Embodiment 3 of the present invention.

[0027] Figure 7 This is a diagram showing the detection results of different biomarkers on the surface of mo-DC cells provided in Embodiment 3 of the present invention.

[0028] Figure 8 This is a flow cytometry analysis diagram of different groups of DC-stimulated T cell proliferation provided in Embodiment 4 of the present invention.

[0029] Figure 9This is a statistical chart showing the T cell proliferation rate of different groups of CD39+TIM3+ cells provided in Example 4 of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention. Some terms used in this invention are listed below.

[0031] CD11c is an important surface marker of dendritic cells (DCs), widely expressed in various DC subsets, especially in conventional DCs with strong antigen-presenting capabilities. However, CD11c is not a completely DC-specific marker; it may also be expressed on some macrophages or activated T cells. CD11c participates in the endocytosis process of DCs, promoting antigen processing and presentation to MHC molecules. CD11c targeting often works synergistically with co-stimulatory molecules (such as CD80 / CD86) and cytokines (such as IL-12) to enhance DC maturation and T cell activation.

[0032] HLA-DR: Human leukocyte antigen-DR (MHC class II molecule), responsible for presenting exogenous antigens to CD4+ T cells. The expression level of HLA-DR is positively correlated with the maturity of dendritic cells (DCs). In vaccines, DCs that highly express HLA-DR can more effectively activate CD4+ T cells and enhance adaptive immune responses.

[0033] CD40: A co-stimulatory molecule belonging to the tumor necrosis factor receptor superfamily, it transmits activation signals by binding to CD40L on the surface of T cells; CD40 upregulation can promote the secretion of pro-inflammatory factors such as IL-12 by dendritic cells and enhance CD80 / CD86 expression, thereby promoting T cell activation and Th1 immune polarization; high CD40 expression is a key indicator of vaccine maturity.

[0034] CD80: A member of the B7 family of co-stimulatory molecules, it binds to CD28 on the surface of T cells, providing a second signal for T cell activation; CD80 works synergistically with CD86 to determine the activation or tolerance state of T cells. High expression of CD80 indicates that dendritic cells (DCs) are in a mature state and can effectively induce anti-tumor or antiviral immune responses.

[0035] CD197: The chemokine receptor CCR7 mediates the migration of dendritic cells (DCs) to lymph nodes and binds to CCL19 / CCL21. High expression of CCR7 enables DCs to migrate to lymphoid tissues and enhances antigen presentation efficiency.

[0036] PD-L1: Programmed death ligand 1, its expression on dendritic cells (DCs) is of great significance for T cell activation and anti-tumor immune response.

[0037] CD83: A marker of DC maturation, directly involved in T cell differentiation signaling. High CD83 expression is a sign of complete DC maturation and is positively correlated with T cell activation efficiency.

[0038] CD86: Another member of the B7 family of co-stimulatory molecules, it binds to CD28 earlier than CD80 and plays a key role in the activation of naive T cells. High expression of CD86 is one of the release criteria for DC vaccines, ensuring that the vaccine can effectively activate naive T cells.

[0039] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0040] Example 1

[0041] The expression levels of CD39 and TIM3 in peripheral blood mononuclear cells (PBMCs) of cancer patients were detected as follows:

[0042] PBMCs were isolated and obtained from 15 tumor patients, and PBMCs (1×10⁻⁶) were collected. 6 Each PBMC was stained with 5 μg of CD39 and TIM3 flow cytometry antibodies, and the expression of CD39 and TIM3 in PBMCs was detected by flow cytometry. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown;

[0043] Figure 1 Scatter plot of FSC-SSC during flow cytometry of PBMC;

[0044] Figure A, with FSC-A (forward scattered light area) as the X-axis and SSC-A (side scattered light area) as the Y-axis, shows the distribution of cell populations in PBMCs. The figure distinguishes between lymphocytes (56.7%) and monocytes (23.7%) using gating. Figure B, with FSC-A as the X-axis and FSC-H (forward scattered light height) as the Y-axis, was used to screen for single cells. The figure shows that single cells account for 98.1%.

[0045] The arrows indicate that the cell population selected from Figure A is further analyzed as a single cell in Figure B.

[0046] Figure 2Histogram of flow cytometry analysis results for PBMCs;

[0047] Figure A shows the detection results of CD39 on the cell surface, and Figure B shows the detection results of TIM3 on the cell surface.

[0048] Figure 3 The figure shows the statistical distribution of CD39 and TIM3 expression in monocytes from 15 tumor patients. As can be seen from the figure, about 99% of monocytes express CD39 and about 10% of cells express TIM3.

[0049] Example 2

[0050] Mature mo-DCs were obtained by inducing peripheral blood monocytes in vitro, as follows:

[0051] PBMCs were isolated from peripheral blood of cancer patients using low-density gradient centrifugation. They were cultured for 5–7 days in a medium containing GM-CSF (1000 U / mL) and IL-4 (500 U / mL) to induce differentiation into immature dendritic cells (DCs). Then, TNF-α (50 ng / mL), IL-1β (10 ng / mL), PGE2 (1 μg / mL), and 5 μg flow cytometry antibody were added for labeling and staining, and the cells were cultured for 48 hours. The maturation status of the induced mo-DCs was then assessed. Figure 4 As shown, mo-Dc accounts for approximately 60.1%.

[0052] Further detection of mo-DC cell surface biomarkers CD11C, HLA-DR, CD40, CD80, CD197, PD-L1, CD83, and CD86 was performed, and the results are as follows: Figure 5 As shown;

[0053] Figure A shows the detection results of CD11C, a biomarker on the surface of mo-DC cells.

[0054] Figure B shows the detection results of HLA-DR, a biomarker on the surface of mo-DC cells;

[0055] Figure C shows the detection results of CD40, a biomarker on the surface of mo-DC cells;

[0056] Figure D shows the detection results of CD80, a biomarker on the surface of mo-DC cells;

[0057] Figure E shows the detection results of CD197, a biomarker on the surface of mo-DC cells;

[0058] Figure F shows the detection results of PD-L1, a biomarker on the surface of mo-DC cells;

[0059] Figure G shows the detection results of CD83, a biomarker on the surface of mo-DC cells;

[0060] Figure H shows the detection results of CD86, a biomarker on the surface of mo-DC cells;

[0061] pass Figure 5 It can be seen that in in vitro induced mo-DCs, the detection values ​​of some immune biomarkers are low, such as CD40 at approximately 63.6%, CD80 at approximately 59.5%, PD-L1 at approximately 60.4%, and CD83 at approximately 56.6%. mo-DCs with low detection values ​​of these biomarkers usually have low activity and weak antigen presentation ability, which seriously affects the efficacy of mo-DC vaccines.

[0062] Example 3

[0063] The screening of in vitro induced mo-DCs using CD39 and TIM3 markers was performed as follows:

[0064] 5 μg of CD39 and TIM3 flow cytometry antibodies were used to label and induce mo-DCs in vitro. CD39+TIM3+ mo-DCs were then sorted using flow cytometry. The results are as follows: Figure 6 As shown.

[0065] Collect 1×10 cells from the sorted cells 6 Each sample was labeled with CD11C, HLA-DR, CD197, PD-L1, CD80, CD83, CD86, and CD40 respectively using flow cytometry antibodies. Flow cytometry was used to detect these biomarkers, and the results are as follows: Figure 7 As shown;

[0066] Figure A shows the detection results of CD11C, a biomarker on the surface of mo-DC cells.

[0067] Figure B shows the detection results of HLA-DR, a biomarker on the surface of mo-DC cells;

[0068] Figure C shows the detection results of CD197, a biomarker on the surface of mo-DC cells;

[0069] Figure D shows the detection results of CD86, a biomarker on the surface of mo-DC cells;

[0070] Figure E shows the detection results of CD83, a biomarker on the surface of mo-DC cells;

[0071] Figure F shows the detection results of CD80, a biomarker on the surface of mo-DC cells;

[0072] Figure G shows the detection results of PD-L1, a biomarker on the surface of mo-DC cells;

[0073] Figure H shows the detection results of CD40, a biomarker on the surface of mo-DC cells;

[0074] pass Figure 7 It can be seen that the expression of the above biomarkers in CD39+TIM3+mo-DC is above 95%, and is significantly higher than that of CD11C, HLA-DR, CD40, CD80, CD197, PD-L1, CD83 and CD86 biomarkers expressed by unselected mo-DC cells directly induced in vitro. This indicates that CD39 and TIM3 can be used as biomarkers for screening highly active and mature mo-DCs.

[0075] Example 4

[0076] Using a DC-stimulated T cell proliferation model, CD39 was detected. + TIM3 + The difference in T cell proliferation capacity induced by mo-DC before and after sorting was as follows:

[0077] Cell Trace solution using fluorescent dyes TM After T cells were stained with Violet (5mM) to label them, they were seeded into 96-well U-shaped culture plates (1×10⁻⁶ m²). 6 Add CD39+TIM3+screened DC (CD39+TIM3+screened DC) (1×10) to the hole at the same time. 5 (pieces / hole) or DC (1×10) 5 Cells were added to each well (number per well). An equal volume of culture medium was added to the control group (CTRL), along with T cell proliferation medium, CD3 / CD28 magnetic beads, and IL-2 (10 μg / ml) (Immunocal Cult™-XFT cell expansion medium, stem cells). After co-culturing at 37°C for 72 h, all cells were collected. All T cells were labeled with CD3 flow cytometry antibody, and CellTrace in T cells was analyzed. TM Violet expression ratio, results as follows Figure 8 and Figure 9 As shown;

[0078] Figure 8 This is a flow cytometry analysis of T cell proliferation stimulated by DCs in different groups. The horizontal axis in the figure represents CellTrace. TM Violet fluorescence intensity, with the vertical axis representing PB450-A fluorescence intensity, the scatter points in the figure representing cells, and the color intensity indicating the number of cells.

[0079] Figure A shows the CTRL group, displaying cells without specific treatment, CellTrace. TMViolet and PB450-A showed low fluorescence intensity and low cell numbers;

[0080] Figure B shows the CD39+TIM3+screened DC group, CellTrace. TM Violet and PB450-A fluorescence intensity were significantly enhanced, and the number of cells was significantly increased.

[0081] Figure C shows the DC group, CellTrace. TM The fluorescence intensity of Violet and PB450-A was between that of the control group and the screening group, and the number of cells also increased.

[0082] Figure 9 The graph shows the statistical distribution of T cell proliferation rates in different groups. A p ≤ 0.01 indicates high statistical significance.

[0083] Figure 8 and Figure 9 The results showed that the T cell proliferation rate in the CD39+TIM3+sreened DC stimulation group was significantly higher than that in the DC stimulation group, indicating that DCs sorted using the biomarkers CD39 and TIM3 have a strong ability to induce immune responses, and this characteristic is higher than that of DCs not screened by the biomarkers CD39 and TIM3.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening highly active dendritic cell vaccines using CD39 and TIM3, characterized in that, This includes steps for sorting and / or identifying mature dendritic cells using biomarkers; The biomarkers include CD39 and TIM3.

2. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 1, characterized in that, Mature dendritic cells expressing the biomarkers CD39 and TIM3 were sorted by flow cytometry for the preparation of dendritic cell vaccines.

3. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 1, characterized in that, The biomarkers also include one or more of CD11C, HLA-DR, CD197, PD-L1, CD80, CD83, CD86 and CD40.

4. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 1, characterized in that, The dendritic cell vaccine is used in cancer diseases.

5. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 1, characterized in that, Mature dendritic cells were prepared using peripheral blood mononuclear cells.

6. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 5, characterized in that, The preparation of mature dendritic cells from peripheral blood mononuclear cells includes the following steps: S100. Peripheral blood mononuclear cells were isolated from in vitro blood samples; S200. Peripheral blood mononuclear cells were induced to differentiate using inducing factor I to obtain immature dendritic cells; S300. Immature dendritic cells were induced by inducing factor II to obtain mature dendritic cells.

7. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 6, characterized in that, In step S200, the inducing factor I is selected from GM-CSF and / or IL-4.

8. The method for screening highly active dendritic cell vaccines using CD39 and TIM3 as described in claim 6, characterized in that, In step S300, the inducing factor II is selected from one or more of TNF-α, PGE2, IL-1β, IFN-γ and IL-6.

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