DC cell subset and preparation method and application thereof

Abstract

The invention relates to a DC (dendritic cell) subgroup as well as a preparation method and application thereof. The DC subgroup highly expresses T cell chemotactic related genes and classical myeloid related genes. The DC subgroup has an efficient anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and clinical medicine, and in particular to a DC cell subset and a preparation method and application thereof. Background Art

[0002] Dendritic cells (DC) are the most powerful antigen-presenting cells that can capture and process tumor antigens, and then present antigens to T cells to initiate T-cell-dependent specific anti-tumor immunity, which is also very important for effective immune checkpoint therapy. In the tumor microenvironment, especially in "cold" tumors, immunosuppressive factors often lead to death, insufficient infiltration or impaired function of DCs in the tumor, allowing many types of cancer to escape anti-tumor immunity. Therefore, it is very important to discover a group of more powerful DC cells in the tumor microenvironment and use them as biomarkers of anti-tumor immune response, so as to transform and apply them to help better enhance the efficacy of ICB.

[0003] Regulation of cell death in DCs is critical for maintaining the number of DCs in the tumor microenvironment and affecting anti-tumor immune responses. Studies have reported that DC death is associated with apoptosis via Fas- and perforin-dependent pathways, but the potential role of other forms of cell death in regulating DC survival in the tumor microenvironment remains unclear. Programmed necrosis is a form of regulated necrosis that depends on RIPK3-mediated MLKL phosphorylation, which is usually not expressed in most tumor cells due to RIPK3 epigenetic silencing. In contrast, immune cells (including T cells and DCs) highly express RIPK3. Therefore, studying the potential role of DC cell death in the development of cold tumors is of great significance for enhancing the survival of DCs in the tumor microenvironment and thus better exerting anti-tumor immunity. Summary of the invention

[0004] Problem that the invention aims to solve

[0005] In response to the defects of the prior art, the present application provides a DC cell subset with high T cell chemokines and classical myeloid cell genes, which has the advantages of long survival time, stronger antigen presentation function to promote T cell activation and more effective anti-tumor effect.

[0006] Solutions for solving problems

[0007] In a first aspect, the present invention provides an isolated DC cell subpopulation, wherein the DC cell subpopulation highly expresses T cell chemotaxis-related genes and classical myeloid-related genes.

[0008] Preferably, the DC cell subpopulation further highly expresses one or more of guanosine nucleotide binding-related genes, programmed cell death genes, interferon-induced genes, and antigen presentation-related genes.

[0009] Preferably, the DC cell subpopulation highly expresses T cell chemotaxis-related genes, classical myeloid-related genes, guanylate binding-related genes, programmed cell death genes, interferon-induced genes, and antigen presentation-related genes.

[0010] Preferably, the DC cell subpopulation highly expresses T cell chemotaxis-related genes and classical myeloid-related genes.

[0011] Preferably, the T cell chemotaxis-related gene is selected from Cxcl9 and / or Cxcl10.

[0012] Preferably, the guanosine nucleotide binding-related gene is selected from one or more of Gbp2, Gbp5 and Gbp6.

[0013] Preferably, the classical myeloid-associated gene is selected from one or more of Ly6i, Ly6a and Ly6c2, or the classical myeloid-associated gene is selected from LY6S.

[0014] Preferably, the programmed cell death gene is Zbp1.

[0015] Preferably, the interferon-inducible gene is selected from one or more of Stat1, Ifi47 and Isg15.

[0016] Preferably, the antigen presentation-related gene is selected from Ctss and / or B2m.

[0017] Preferably, the genes highly expressed in the DC cell subpopulation are Cxcl9, Cxcl10, Gbp2, Gbp5, Gbp6, Ly6i, Ly6a, Ly6c2, Zbp1, Stat1, Ifi47, Isg15, Ctss and B2m.

[0018] Preferably, the genes highly expressed in the DC cell subpopulation are CXCL9, CXCL10, GBP2, GBP5, GBP6, LY6S, ZBP1, STAT1, IFI47, ISG15, CTSS and B2M.

[0019] Preferably, the genes highly expressed in the DC cell subpopulation are Cxcl9 and Ly6c2.

[0020] Preferably, the genes highly expressed in the DC cell subpopulation are CXCL9 and LY6S.

[0021] Preferably, the DC cell subpopulation is isolated from a mammal.

[0022] In a second aspect, the present invention provides a method for preparing a DC cell subset as described in any one of the above, the method comprising the following steps:

[0023] (1) constructing a mammal with a programmed necrosis gene defect or a mammal with a programmed necrosis gene defect in DC cells, establishing a tumor model, and collecting tumor cells at a specific time;

[0024] (2) preparing the tumor cells obtained in step (1) into a single cell suspension, and using cell sorting technology to sort out the DC cell subpopulation from the single cell suspension according to specific surface markers; or,

[0025] (1) collecting bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from a subject and culturing them in vitro, adding GM-CSF and IL-4 to the culture to induce the bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells to differentiate into DC precursor cells, and then adding IFN-γ to the culture system to continue culturing;

[0026] (2) Using cell sorting technology to sort out the DC cell subpopulation from the culture obtained in step (1) based on specific surface markers.

[0027] Preferably, the subject does not express a necroptosis gene.

[0028] Preferably, the programmed necrosis gene is RIPK3 and / or MLKL.

[0029] Preferably, the specific surface marker is Ly6C protein.

[0030] Preferably, the tumor model is selected from breast cancer, skin melanoma, prostate cancer, cervical squamous cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, endometrial carcinoma, sarcoma and mesothelioma.

[0031] In a third aspect, the present invention provides a drug combination, comprising any one of the DC cell subsets described above or the DC cell subsets obtained by the preparation method described above;

[0032] Preferably, the drug combination also includes a PD-1 neutralizing antibody.

[0033] Preferably, the pharmaceutical combination is in the form of a pharmaceutical composition.

[0034] Preferably, the DC cell subset and the PD-1 neutralizing antibody are each in the form of a separate preparation;

[0035] Preferably, the DC cell subset and the PD-1 neutralizing antibody are administered simultaneously or sequentially.

[0036] In a third aspect, the present invention provides use of any of the above-mentioned DC cell subpopulations, or the DC cell subpopulations obtained by the above-mentioned preparation method, or the above-mentioned therapeutic composition in the preparation of a drug for treating tumors.

[0037] Preferably, the tumor is selected from breast cancer, skin melanoma, prostate cancer, cervical squamous cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, endometrial cancer, sarcoma and mesothelioma.

[0038] Effects of the Invention

[0039] The dendritic cells (DC) subpopulation provided by the present invention (hereinafter sometimes referred to as CXCL9 hi ZBP1 hi DC or CXCL9 hi Ly6C hi DC or the DC subpopulation of the present invention), after the programmed necrosis gene (Ripk3 and / or Mlkl) is deficient, its number in the tumor microenvironment of tumor-bearing mice is significantly increased, and tumor growth is significantly inhibited; in mice with DC-specific lack of RIPK3, the number of this DC subpopulation is significantly increased, and tumor growth is significantly inhibited; this DC subpopulation can recruit more T cells and enhance the secretion capacity of CD8+T cell effector factors, thereby increasing the efficacy of immune checkpoint blockade therapy and achieving the purpose of improving the survival of tumor patients; and the high expression of this DC subpopulation gene is positively correlated with the survival of patients with various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The DC cell subpopulation CXCL9 prepared in Example 1 of the present invention hi ZBP1 hi Schematic diagram of the characteristics of each highly expressed gene, where A is a schematic diagram of the proportion of each type of DC cell subsets in the tumor microenvironment, and B is a schematic diagram of CXCL9 hi ZBP1 hi Volcano plots of different DC cell subsets, C to E are CXCL9 hi ZBP1 hi Schematic diagram of each highly expressed gene in DC;

[0041] Figure 2 The DC cell subpopulation CXCL9 prepared in Example 1 of the present invention hi ZBP1 hi Schematic diagram of each highly expressed gene in DC;

[0042] Figure 3 The DC cell subset CXCL9 constructed using Mlk1 gene-deficient mice in Example 1 of the present invention hi ZBP1 hiSchematic diagram of each highly expressed gene in DC;

[0043] Figure 4 CXCL9 in Examples 1 and 2 of the present invention hi ZBP1 hi Schematic diagram of DC cell single cell data and in vitro experimental results, where A is CXCL9 hi ZBP1 hi Single cell sequencing analysis of DC subsets and other DC subsets; B is CXCL9 hi ZBP1 hi Schematic diagram of genes highly expressed in DC; C and D are CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo Schematic diagram of the results of mixed lymphocyte reaction (MLR) experiment of DC subsets; E is CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo Schematic diagram of the results of the cell migration experiment conducted by DC on T cells;

[0044] Figure 5 CXCL9 differentiated from bone marrow cells in Example 3 of the present invention hi ZBP1 hi Schematic diagram of DC cell in vitro experimental results, A is CXCL9 hi ZBP1 hi Schematic diagram of genes highly expressed in DC, B and C are CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo Schematic diagram of the results of mixed lymphocyte reaction (MLR) experiment of DC subsets; D is CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo Schematic diagram of the results of the cell migration experiment conducted by DC on T cells;

[0045] Figure 6 Mice were inoculated with tumor cells (prostate cancer RM-1 (1×10 5 / 25μL) cells), on day 14, Ripk3-deficient mice and mice with specific lack of RIPK3 in DC cells, schematic diagram of tumor growth, wherein Figure A is a bar graph of tumor weight of wild-type mice and Ripk3-deficient mice at D14; Figure B is a schematic diagram of the content of CD8T in the tumor microenvironment of wild-type mice and Ripk3-deficient mice; Figure C is a bar graph of Ripk3f / f Mice and Ripk3 f / f CD11c cre Mouse tumor weight bar graph; Figure D is Ripk3 f / f Mice and Ripk3 f / f CD11c cre Schematic diagram of the content of CD8T in the tumor microenvironment of mice; Figure E is a bar graph of tumor weight of mice with Mlk1 deficiency; Figure F is a schematic diagram of the content of CD8T in the tumor microenvironment of mice with Mlk1 deficiency;

[0046] Figure 7 In prostate cancer, CXCL9 is expressed after deficiency of programmed necrosis genes Ripk3 or Mlkl, or after specific knockout of Ripk3 in DCs. hi ZBP1 hi Schematic diagram of DC cells enhancing the therapeutic effect of PD-1;

[0047] Figure 8 In breast cancer and melanoma, CXCL9 is activated after the programmed necrosis gene Ripk3 is defective hi ZBP1 hi Schematic diagram of how DC cells enhance the therapeutic effect of PD-1. DETAILED DESCRIPTION

[0048] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0049] Over the past decade, cancer treatment has evolved from a strategy focused on targeting tumor cells to a more extensive treatment strategy, including targeted activation of immune cells for the purpose of treating tumors. Immune checkpoints are key regulatory molecules present in the immune system. Under normal circumstances, immune checkpoints inhibit the over-activation of immune responses through a "brake" mechanism; however, in the process of cancer, tumor cells use immune checkpoint molecules to escape the attack of the immune system, thereby surviving and proliferating. Immune checkpoint blockade (ICB) therapy targets immune checkpoint molecules that inhibit T cell activation, enhances T cell-mediated anti-tumor immunity, and exerts anti-tumor effects, such as targeting programmed cell death receptor 1 (PD-1, programmed cell death protein 1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which can significantly advance the treatment of a variety of tumors and prolong the survival of patients, especially "hot" tumors with high infiltration of immune cells. However, in some "cold" tumors, such as prostate cancer, pancreatic cancer, and breast cancer, the function of immune cells (such as T cells and dendritic cells) is suppressed, and they cannot or have difficulty infiltrating the tumor site, resulting in a significant reduction in the effectiveness of ICB therapy (such as PD-1 therapy). Therefore, a full understanding of the factors that affect the presence of immune cells and shape the tumor microenvironment (TME) is crucial to improving the treatment outcomes of different types of cancer.

[0050] In view of the above defects, the inventors conducted a large number of basic and applied studies and unexpectedly discovered a dendritic cell (DC) subpopulation with a special phenotype in the tumor microenvironment of tumor-bearing mice with programmed necrosis genes (Ripk3 and / or Mlk1) defects or mice with DC-specific Ripk3 deficiency: CXCL9 hi ZBP1 hi DC. For example, this subgroup highly expresses T cell chemokines such as CXCL9 and CXCL10, highly expresses interferon-related genes, highly expresses classical myeloid cell genes Ly6c2, Ly6i, etc., so it can better recruit tumor-reactive CD8 T cells, have stronger antigen presentation function, and promote T cell activation; in addition, this DC subgroup highly expresses programmed cell death-related molecule ZBP1, and can survive better under the condition of programmed cell death gene (RIPK3 or MLKL) deficiency. This subgroup of cells has a strong anti-tumor ability; and can enhance the efficacy of immune checkpoint blockade therapy. The high expression of this DC subgroup gene is positively correlated with the survival of patients with various tumors. Based on this, the inventors completed the present invention.

[0051] In a first aspect, the present invention provides an isolated DC cell subpopulation, wherein the DC cell subpopulation highly expresses T cell chemotaxis-related genes and classical myeloid-related genes.

[0052] In certain embodiments, the DC cell subpopulation further highly expresses one or more of guanosine nucleotide binding-related genes, programmed cell death genes, interferon-induced genes, and antigen presentation-related genes.

[0053] In certain embodiments, the DC cell subpopulation highly expresses T cell chemotaxis-related genes, classical myeloid-related genes, guanylate binding-related genes, programmed cell death genes, interferon-induced genes, and antigen presentation-related genes.

[0054] In certain embodiments, the T cell chemotaxis-related gene is selected from Cxcl9 and / or Cxcl10.

[0055] In certain embodiments, the guanosine nucleotide binding-related gene is selected from one or more of Gbp2, Gbp5, and Gbp6.

[0056] In certain embodiments, the classical myeloid-associated gene is selected from one or more of Ly6i (mouse), Ly6a (mouse), and Ly6c2 (mouse).

[0057] In certain embodiments, the classical myeloid-associated gene is selected from LY6S (human).

[0058] In certain embodiments, the programmed cell death gene is Zbp1.

[0059] In certain embodiments, the interferon-inducible gene is selected from one or more of Stat1, Ifi47 and Isg15.

[0060] In certain embodiments, the antigen presentation-related gene is selected from Ctss and / or B2m.

[0061] In certain embodiments, the DC cell subpopulation is derived from mice, and the genes highly expressed by the DC cell subpopulation are Cxcl9, Cxcl10, Gbp2, Gbp5, Gbp6, Ly6i, Ly6a, Ly6c2, Zbp1, Stat1, Ifi47, Isg15, Ctss and B2m.

[0062] In certain embodiments, the DC cell subpopulation also highly expresses the genes Gbp3, Gbp7, and Cxcl2. In certain embodiments, the DC cell subpopulation is derived from humans, and the genes highly expressed by the DC cell subpopulation are Cxcl9, Cxcl10, Gbp2, Gbp5, Gbp6, LY6S, Zbp1, Stat1, Ifi47, Isg15, Ctss, and B2m.

[0063] In certain embodiments, the genes highly expressed by the DC cell subpopulation are Cxcl9 and Ly6c2.

[0064] In certain embodiments, the genes highly expressed by the DC cell subpopulation are CXCL9 and LY6S.

[0065] In certain embodiments, the DC cell subpopulation is isolated from a mammal.

[0066] In the present invention, the mammal can be an animal selected from bovines, equines, felines, canines, lagomorphs, porcines, camelids, rodents and primates, including but not limited to cows, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, gorillas) and humans, preferably cows, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys and humans, more preferably humans or mice.

[0067] The present invention provides a method for preparing the DC cell subsets described above.

[0068] In a second aspect, the present invention provides a method for preparing a DC cell subset as described in any one of the above, the method comprising the following steps:

[0069] (1) constructing a mammal with a programmed necrosis gene defect or a mammal with a programmed necrosis gene defect in DC cells, establishing a tumor model, and collecting tumor cells at a specific time;

[0070] (2) The tumor cells obtained in step (1) are prepared into a single cell suspension, and the DC cell subpopulation is sorted out from the single cell suspension according to specific surface markers using cell sorting technology.

[0071] In the present invention, the method for constructing a gene-deficient mouse is not particularly limited, and a method known in the art can be used. For example, the method for constructing a Ripk3 gene-deficient mouse in the present invention is carried out with reference to the literature Sudan He et al., Cell, 137, 1100-1111 (2009). f / f CD11c creMouse), first refer to the literature Xiaoliang Yu et al., Blood, 2023Mar 2; 141(9): 1070-1086 to construct Ripk3 f / f Mouse, Cd11c was constructed according to Jackson lab (URL: https: / / www.jax.org / strain / 008068) cre Mouse, Ripk3 f / f Mouse and Cd11c cre Mouse hybridization to obtain Ripk3 f / f CD11c cre Mice (mice with Ripk3 deficiency in dendritic cells). The method for constructing Mlk1 gene-deficient mice was carried out according to the literature Dianrong Li et al., eLIFE, 2017Aug 15; 6: e27692.

[0072] In certain embodiments, the method comprises the following steps:

[0073] (1) collecting bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from a subject and culturing them in vitro, adding GM-CSF and IL-4 to the culture to induce the bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells to differentiate into DC precursor cells, and then adding IFN-γ to the culture system to continue culturing;

[0074] (2) Using cell sorting technology to sort out the DC cell subpopulation from the culture obtained in step (1) based on specific surface markers.

[0075] In certain embodiments, step (1) is specifically:

[0076] Bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells are collected from the subject and cultured in vitro, GM-CSF and IL-4 are added to the culture to induce the bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells to differentiate into DC precursor cells, and then IFN-γ is added to the culture system on the 6th day and cultured for 15 to 30 hours (e.g., 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, etc.);

[0077] The medium was half-changed on the 3rd and 5th days; the concentration of GM-CSF was 10-30 ng / ml, the concentration of IL-4 was 1-10 ng / ml, and the concentration of IFN-γ was 50-150 ng / ml.

[0078] In certain embodiments, the concentration of GM-CSF is 15-25 ng / ml, for example, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, etc.

[0079] In certain embodiments, the concentration of GM-CSF is 18-22 ng / ml.

[0080] In certain embodiments, the concentration of IL-4 is 2 to 9 ng / ml, for example, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, etc.

[0081] In certain embodiments, the concentration of IL-4 is 3-8 ng / ml.

[0082] In certain embodiments, the concentration of IFN-γ is 60-120 ng / ml, for example, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng / ml, 105 ng / ml, 110 ng / ml, 115 ng / ml, 120 ng / ml, etc.

[0083] In certain embodiments, the in vitro culturing is performed in RPMI-1640 medium.

[0084] In certain embodiments, the RPMI-1640 culture medium comprises: 5-15 wt% FBS, 0.5-2 wt% penicillin-streptomycin, 0.5-2 wt% sodium pyruvate and 1-3 mM β-mercaptoethanol.

[0085] In certain embodiments, the RPMI-1640 culture medium comprises: 8-12 wt% FBS, 0.5-1.5 wt% penicillin-streptomycin, 0.5-1.5 wt% sodium pyruvate and 1.5-2.5 mM β-mercaptoethanol.

[0086] In certain embodiments, the subject does not express a necroptosis gene.

[0087] In certain embodiments, the programmed necrosis gene is RIPK3 and / or MLKL.

[0088] In certain embodiments, the specific surface markers may be selected based on the species of the subject (e.g., human, mouse, dog, etc.) and the ratio of the target cells to be sorted and enriched to the final separated cells (e.g., 50%, 60%, 70%, 80% or more than 90%) to select a combination of one or more markers, for example, for mice, Ly6C protein may be selected. For humans, Ly6S protein may be selected.

[0089] In certain embodiments, the tumor model is selected from breast cancer, skin melanoma, prostate cancer, cervical squamous cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, endometrial carcinoma, sarcoma, and mesothelioma.

[0090] In certain embodiments, the tumor model is selected from breast cancer, skin melanoma, prostate cancer.

[0091] In the bone marrow cell utilization or preparation method of the present invention, the method of collecting peripheral blood and separating mononuclear cells is not particularly limited, and methods known in the art can be used, for example, an extracorporeal circulation method or equipment can be used (for example, a human mononuclear cell separator can be used to circulate 3000 to 6000 mL of peripheral blood).

[0092] In the preparation method of the present invention, the method for removing red blood cells from anticoagulated blood is not particularly limited, and methods known in the art can be used, for example, density gradient centrifugation can be used.

[0093] The cell sorting technology adopted in the preparation method of the present invention is well known in the art, and can be completed by methods or equipment commonly used in the art, and is not subject to any restrictions in the present invention. As long as the technology, method and equipment that can utilize surface markers to sort cells can be used in the present invention. For example, magnetic sorting technology or flow cytometry can be adopted. The specific implementation method of cell sorting technology such as magnetic sorting technology or flow cytometry can be found in many scientific literatures, or implemented according to the instructions or recommended experimental procedures of the equipment or instrument manufacturers, and those skilled in the art are fully capable of obtaining these specific implementation methods or experimental procedures.

[0094] In a third aspect, the present invention provides a drug combination, comprising any one of the DC cell subsets described above or a DC cell subset obtained by the above preparation method.

[0095] The DC cells of the present application obtained by in vitro culture were cultured for six days, pre-stimulated with IFN-γ, then stimulated with LPS, and loaded with OVA-peptide, and stimulated with Cpg ODN 1826 and Poly I: C. When the tumor in the tumor mouse model reached a certain volume, the above DC vaccine (1 to 8 million) was injected for treatment. The tumor growth treated with DCs deficient in programmed necrosis genes (RIPK3 and / or MLKL) was significantly inhibited.

[0096] In certain embodiments, the drug combination further comprises a PD-1 neutralizing antibody.

[0097] In certain embodiments, the number of DC cell subsets in the drug combination is selected according to the species of the subject. For example, for mice, the number of DC cell subsets in the combination is 1 to 8 million.

[0098] In certain embodiments, the drug combination is in the form of a pharmaceutical composition.

[0099] In certain embodiments, the DC cell subset and the PD-1 neutralizing antibody are each in the form of separate formulations.

[0100] In certain embodiments, the DC cell subset and the PD-1 neutralizing antibody are administered simultaneously or sequentially.

[0101] In the present application, the sources of IgG and PD-1 neutralizing antibodies are not limited. For example, in the embodiments of the present invention, IgG and PD-1 neutralizing antibodies are derived from Bio-X Cell.

[0102] In a third aspect, the present invention provides use of any of the above-mentioned DC cell subpopulations, or the DC cell subpopulations obtained by the above-mentioned preparation method, or the above-mentioned therapeutic composition in the preparation of a drug for treating tumors.

[0103] In certain embodiments, the tumor is selected from breast cancer, skin melanoma, prostate cancer, cervical squamous cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, endometrial carcinoma, sarcoma, and mesothelioma.

[0104] The method of the present invention is described below by specific examples. It should be understood that these examples are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited by the scope of the following examples; the implementation conditions used in the examples can be further adjusted according to specific requirements, and the unspecified implementation conditions are usually the conditions in conventional experiments. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0105] Example 1

[0106] Ripk3 gene-deficient mice were constructed with reference to Sudan He et al., Cell, 137, 1100-1111 (2009).

[0107] Orthotopic prostate tumor models were established in wild-type and necroptosis-deficient mice (8-week-old male mice):

[0108] Prostate cancer cells RM-1 (1×10 5 ) were injected into the prostate of C57BL / 6J male mice at about 8 weeks old, thus successfully establishing an orthotopic prostate tumor model.

[0109] When the tumor grows to day 14, the mice are killed and the tumor is isolated from the prostate. The isolated tumor is first cut into 2-3 mm 3 The small pieces were digested with DMEM medium containing type I collagenase (1.5 mg / mL) (Gibco), hyaluronidase (1.5 mg / mL) (Sigma) and DNase I (20 U / ml) for 30 minutes at 37° C. After digestion, the obtained mixture was filtered through a 70 μm cell strainer to obtain a single cell suspension, which was washed twice with flow staining buffer (PBS containing 2% serum and 1 mM EDTA).

[0110] After washing, CD45 magnetic beads (MojoSort TM Mouse CD45 Nanobeads, 480028, Biolegend) were used to enrich CD45 in the tumor microenvironment from single cell suspensions. + Through bioinformatics analysis of DCs in immune cells, it was found that a group of DCs had a significantly increased cell ratio after RIPK3 deficiency (see Figure 1 In addition, this DC subset highly expresses T cell chemotaxis-related genes such as Cxcl9 and Cxcl10, guanine nucleotide binding-related genes such as Gbp2, Gbp5, and Gbp6, classical myeloid-related genes Ly6i, Ly6a, and Ly6c2, and programmed necrosis-related gene Zbp1 (see Figure 1 BC), high expression of interferon-induced genes such as Stat1, Ifi47 and Isg15 (see Figure 1 D), high expression of Ctss, B2m and other antigen presentation related genes (see Figure 1 E), which we named CXCL9 hi ZBP1 hi ( Figure 1 AB), otherwise it is named CXCL9 lo ZBP1lo DC.

[0111] Further, this example also refers to the literature Xiaoliang Yu et al., Blood, 2023 Mar 2; 141 (9): 1070-1086 to construct Ripk3 f / f Mouse, Cd11c was constructed according to Jackson lab (URL: https: / / www.jax.org / strain / 008068) cre Mouse, Ripk3 f / f Mouse and Cd11c cre Mouse hybridization to obtain Ripk3 f / f CD11c cre Mice (mice with specific lack of RIPK3 in DC). Orthotopic prostate tumors were constructed in the same way as above, and tumors were collected at D14 and made into single cell suspensions. Flow cytometry and bioinformatics analysis also found that CXCL9 highly expressed CXCL9 and Ly6C. hi ZBP1 hi DC subsets ( Figure 2 ).

[0112] Furthermore, this example also refers to the literature Dianrong Li et al., eLIFE, 2017Aug 15; 6: e27692 to construct Mlk1 gene-deficient mice. Orthotopic prostate tumors were constructed according to the above method, and tumors were collected at D14 and made into single cell suspensions. Flow cytometry and bioinformatics analysis also found that CXCL9 highly expressed CXCL9 and Ly6C. hi ZBP1 hi DC subsets ( Figure 3 ).

[0113] Example 2

[0114] Using bioinformatics analysis, single-cell sequencing was analyzed and CXCL9 was found hi ZBP1 hi Compared with other DC subsets, DC has stronger lymphocyte chemotaxis, can better activate T cells, has stronger antigen presentation ability, and is more sensitive to IFN response ( Figure 4 (A).

[0115] The tumor model was constructed according to the method of Example 1, and Ly6C was used as a marker for flow cytometry sorting to prepare CXCL9 from DCs in the tumor microenvironment (Tumor-infiltrating DCs, TIDCs). hi ZBP1 hi DC and CXCL9 lo ZBP1lo DC( Figure 4 B). The obtained CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo DCs were subjected to mixed lymphocyte reaction (MLR) assay. First, CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo DCs were stimulated with OVApeptide. After 5 hours, OVApeptide was washed off with PBS and mixed with CD8+T cells isolated from the spleen of OT-1 mice at a ratio of 1:20. Three days later, OVApeptide was stimulated again. Four hours later, intracellular factor staining was performed. It was found that CXCL9 hi ZBP1 hi DC vs CXCL9 lo ZBP1 lo DC can promote CD8T cells to secrete more cytokines, such as IFN-γ, TNF-α, Perforin, etc., which can promote the proliferation of CD8T cells. hi ZBP1 hi DC has stronger antigen presenting ability and can better activate T cells ( Figure 4 C and D).

[0116] The tumor model was constructed according to the method of Example 1, and Ly6C was used as a marker for flow cytometry sorting to prepare CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo DC, cell migration assay. First, CXCL9 hi ZBP1 hi DC and CXCL9 lo ZBP1 lo DCs were stimulated with LPS and R848, and the supernatants were taken and placed in the lower layer of the transwell chamber. CD8 T cells obtained by magnetic bead enrichment and separation in the spleen of wild-type mice were placed in the upper layer of the transwell chamber. Six hours later, the CD8 T cells that migrated from the lower chamber were counted, and CXCL9 was found. hi ZBP1 hi DCs can recruit more CD8 T cells, thus proving that CXCL9 hi ZBP1 hi DC has a stronger ability to attract CD8T cells ( Figure 4 Middle E).

[0117] Example 3

[0118] Mouse bone marrow cells were isolated from Ripk3 gene-deficient mice and cultured in RPMI-1640 medium (containing 10 wt% FBS, 1 wt% penicillin-streptomycin, 1 wt% sodium pyruvate and 2 mM β-mercaptoethanol) containing GM-CSF (20 ng / ml) and IL-4 (5 ng / ml). The medium was half-changed on D3 and D5 to obtain bone marrow-derived dendritic cells (BMDCs). IFN-γ (100 ng / ml) was stimulated on D6 for 24 hours, and Ly6C was used as a marker for flow cytometry sorting to obtain CXCL9 from bone marrow-derived DCs (BMDCs). hi ZBP1 hi DC and CXCL9 lo ZBP1 lo DC subsets ( Figure 5 (A).

[0119] According to the method of Example 2, MLR and cell migration experiments were performed respectively, and the same conclusion was obtained, that is, CXCL9 hi ZBP1 hi DC vs CXCL9 lo ZBP1 lo DC can promote CD8T cells to secrete more cytokines, such as IFN-γ, TNF-α, etc., which can promote the proliferation of CD8T cells and recruit more CD8T cells ( Figure 5 (B to D).

[0120] Furthermore, in 8-week-old tumor-bearing (prostate cancer) mice, it was found that mice with programmed necrosis gene defects such as RIPK3 deficiency had CXCL9 hi ZBP1 hi The proportion of DC subsets increased significantly, and tumor growth was significantly inhibited. + The T ratio increased significantly (e.g. Figure 6 Similarly, in Ripk3 f / f CD11c cre In mice, in which RIPK3 was specifically knocked out in DC cells, tumor growth was also significantly inhibited, and CD8 + The T ratio also increased significantly (e.g. Figure 6 In Mlk1-deficient mice, CXCL9 hi ZBP1 hi The proportion of DC subsets increased significantly, and tumor growth was significantly inhibited. + The T ratio increased significantly (e.g. Figure 6 E to F in the figure).

[0121] Therefore, the CXCL9 provided in this application hi ZBP1 hi DCs can be obtained by differentiation and culture from the tumor microenvironment and bone marrow cells of Ripk3-deficient mice, and CXCL9 hi ZBP1 hi DC has stronger antigen presenting ability and stronger ability to chemotactic lymphocytes, thereby better activating T cells, enabling them to exert their effector functions and ultimately inhibiting tumor growth.

[0122] Example 4

[0123] Eight-week-old wild-type and RIPK3-deficient male mice were inoculated with prostate cancer RM-1 (1×10 6 / 75μL) cells, and during the growth of the tumor, the tumor diameter was measured continuously. The mouse tumor volume was calculated using the formula: V = 0.5 × long diameter × short diameter × short diameter. When the tumor volume reached 100mm 3 At about 1:10 pm, control IgG (clone 2A3, 10 mg / kg) and PD-1 neutralizing antibody (clone29F.1A12, 10 mg / kg) were administered every three days. The tumor volume of wild-type control mice grew to 1500 mm 3 After about 24 hours, the mouse tumors were harvested and single-cell suspensions were prepared. Flow cytometry was used to detect the expression of CXCL9 in the tumor microenvironment. hi ZBP1 hi The proportion of DCs, the proportion of CD8T cells, and the ability of CD8T cells to secrete IFN-γ. The results showed that the combination of RIPK3 deficiency and PD-1 neutralizing antibody further inhibited tumor growth compared with PD-1 neutralizing antibody alone. hi ZBP1 hi The proportion of DCs, the proportion of CD8T cells, and the ability of CD8T cells to secrete IFN-γ were significantly improved compared with PD-1 neutralizing antibody treatment alone ( Figure 7 Thus, CXCL9 hi ZBP1 hi DC populations can promote the efficacy of PD-1 neutralizing antibodies.

[0124] Ripk3 was constructed according to the method in Example 1 f / f CD11c creMice (mice with specific lack of RIPK3 in DC) were used to construct a prostate cancer cell RM-1 subcutaneous tumor model and the above-mentioned PD-1 neutralizing antibody administration treatment regimen was used. It was found that the combination of specific lack of RIPK3 in DC and PD-1 neutralizing antibody further inhibited tumor growth and CXCL9 hi ZBP1 hi The proportion of DC and CD8T increased significantly ( Figure 7 D and E).

[0125] According to the method in Example 1, Mlk1 gene knockout mice were constructed, and a prostate cancer cell RM-1 subcutaneous tumor model was constructed. According to the above-mentioned PD-1 neutralizing antibody administration treatment regimen, it was found that the combination of Mlk1 gene deletion and PD-1 neutralizing antibody further inhibited tumor growth, and the proportion of CD8 T cells and the ability of CD8 T cells to secrete IFN-γ were significantly increased ( Figure 7 F to H).

[0126] The above data indicate that after defective necroptosis, CXCL9 in the tumor microenvironment of prostate cancer hi ZBP1 hi The proportion of DCs will increase significantly, thereby enhancing the therapeutic effect of PD-1 neutralizing antibodies.

[0127] Example 5

[0128] Eight-week-old wild-type and Ripk3-deficient female mice were selected and breast cancer cells EO771 (2×10 5 / 75μL) was injected orthotopically into the third pair of mammary fat pads, and tumor measurement and PD-1 neutralization treatment were as described in Example 4. It was found that in the breast cancer tumor model, Ripk3 deficiency and PD-1 neutralizing antibody combined treatment can make the breast cancer model respond to PD-1 neutralizing antibody treatment, significantly inhibiting tumor growth and tumor weight, and RIPK3 deficiency can increase CXCL9 in the breast cancer tumor microenvironment hi ZBP1 hi The proportion of DC cells ( Figure 8 A and B).

[0129] In the melanoma tumor model, melanoma cells B16-F10 (2×10 5 / 75μL) was injected into the right thigh of wild-type and programmed necrosis key protein Ripk3-deficient female mice, and tumor measurement and PD-1 neutralization treatment regimen were as described in Example 4. It was found that in the melanoma tumor model, the combination of Ripk3 deficiency and PD-1 neutralizing antibody treatment can make the melanoma tumor model respond to PD-1 neutralizing antibody treatment, significantly inhibiting tumor growth and tumor weight, and Ripk3 deficiency can increase the expression of CXCL9 in the melanoma tumor microenvironment hi ZBP1 hi The proportion of DC cells ( Figure 8 C and D).

[0130] The above data show that after defective necroptosis, CXCL9 in the tumor microenvironment is upregulated in a variety of tumor models, such as prostate cancer, breast cancer, and melanoma. hi ZBP1 hi The proportion of DCs will increase significantly, thereby enhancing the therapeutic effect of PD-1 neutralizing antibodies.

[0131] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. An isolated DC cell subset, characterized in that: The DC cell subsets highly express T cell chemotaxis-related genes and classical myeloid-related genes.

2. The DC cell subset according to claim 1, characterized in that The DC cell subset further highly expresses one or more of guanosine nucleotide binding-related genes, programmed cell death genes, interferon-induced genes, and antigen presentation-related genes; Preferably, the DC cell subpopulation highly expresses T cell chemotaxis-related genes, classical myeloid-related genes, guanylate binding-related genes, programmed cell death genes, interferon-induced genes, and antigen presentation-related genes.

3. The DC cell subset according to claim 2, characterized in that The T cell chemotaxis-related gene is selected from Cxcl9 and / or Cxcl10; And / or, the guanosine nucleotide binding-related gene is selected from one or more of Gbp2, Gbp5 and Gbp6; and / or, the classical myeloid-associated gene is selected from one or more of Ly6i, Ly6a and Ly6c2, or, the classical myeloid-associated gene is selected from LY6S; And / or, the programmed cell death gene is Zbp1; And / or, the interferon-induced gene is selected from one or more of Stat1, Ifi47 and Isg15; And / or, the antigen presentation related gene is selected from Ctss and / or B2m.

4. The DC cell subset according to claim 1, characterized in that The genes highly expressed in the DC cell subpopulation are Cxcl9, Cxcl10, Gbp2, Gbp5, Gbp6, Ly6i, Ly6a, Ly6c2, Zbp1, Stat1, Ifi47, Isg15, Ctss and B2m; or, The genes highly expressed in the DC cell subpopulation are CXCL9, CXCL10, GBP2, GBP5, GBP6, LY6S, ZBP1, STAT1, IFI47, ISG15, CTSS and B2M; or, The genes highly expressed in the DC cell subpopulation are Cxcl9 and Ly6c2; or, The genes highly expressed in the DC cell subpopulation are CXCL9 and LY6S.

5. The DC cell subset according to any one of claims 1 to 4, characterized in that: The DC cell subsets are isolated from mammals.

6. The method for preparing a DC cell subset according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) constructing a mammal with a programmed necrosis gene defect or a mammal with a programmed necrosis gene defect in DC cells, establishing a tumor model, and collecting tumor cells at a specific time; (2) preparing the tumor cells obtained in step (1) into a single cell suspension, and using cell sorting technology to sort out the DC cell subpopulation from the single cell suspension according to specific surface markers; or, (1) collecting bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells from a subject and culturing them in vitro, adding GM-CSF and IL-4 to the culture to induce the bone marrow cells, peripheral blood mononuclear cells or umbilical cord blood mononuclear cells to differentiate into DC precursor cells, and then adding IFN-γ to the culture system to continue culturing; (2) Using cell sorting technology to sort out the DC cell subpopulation from the culture obtained in step (1) based on specific surface markers.

7. The preparation method according to claim 6, characterized in that: The subject does not express a programmed necrosis gene; Preferably, the programmed necrosis gene is RIPK3 and / or MLKL; Preferably, the tumor model is selected from breast cancer, skin melanoma, prostate cancer, cervical squamous cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, endometrial carcinoma, sarcoma and mesothelioma.

8. A drug combination, characterized in that The drug combination comprises the DC cell subset described in any one of claims 1 to 5 or the DC cell subset obtained by the preparation method described in claim 6 or 7; Preferably, the drug combination also includes a PD-1 neutralizing antibody.

9. The pharmaceutical combination according to claim 8, characterized in that The drug combination is in the form of a pharmaceutical composition; Preferably, the DC cell subset and the PD-1 neutralizing antibody are each in the form of a separate preparation; Preferably, the DC cell subset and the PD-1 neutralizing antibody are administered simultaneously or sequentially.

10. Use of the DC cell subset according to any one of claims 1 to 5, or the DC cell subset obtained by the preparation method according to claim 6 or 7, or the therapeutic composition according to claim 8 in the preparation of a drug for treating tumors; Preferably, the tumor is selected from breast cancer, skin melanoma, prostate cancer, cervical squamous cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, endometrial cancer, sarcoma and mesothelioma.

Images