Method for inducing and activating conventional dendritic cell subset and use thereof in Anti-tumor therapy

Abstract

A method for inducing and activating a conventional dendritic cell subset, the method comprising the following steps: A. seeding human peripheral blood stem cells or umbilical cord blood stem cells in a plate, and performing expansion culture using a basal medium supplemented with cytokine 1; B. performing in vitro induced differentiation culture on the cells after the expansion culture using a basal medium supplemented with cytokine 2; C. sorting the differentiated cells to obtain pure cDC1 cells; and D. performing activation culture on the pure cDC1 cells using a basal medium supplemented with a stimulator to obtain activated cDC1 cells. A large number of human primary cDC1 are obtained by means of induction from umbilical cord blood stem cells, and the anti-tumor ability of cDC1 is then activated by means of a combination of stimulators. Moreover, the feasibility and efficacy of cDC1 for tumor therapy are verified for the first time by means of a humanized mouse tumor model.

Description

Method for inducing and activating conventional dendritic cell subsets and its application in anti-tumor therapy TECHNICAL FIELD

[0001] The present application relates to the technical field of cell therapy, tumor prevention and treatment, autoimmune disease prevention and treatment, and vaccine development, and particularly relates to a method for inducing and activating conventional dendritic cell subsets and its application in anti-tumor therapy. BACKGROUND

[0002] As a treatment method using living cells as drugs, cell therapy has the potential to cure various tumors. Although CAR-T, CAR-NK and other cell therapies have been successful in hematological tumors, their application in solid tumors has been slow. TIL and monocyte-derived dendritic cells (moDC) also lack effective breakthroughs. Therefore, developing new cell therapies is of great significance for the treatment of solid tumors.

[0003] In recent years, multi-omics research on solid tumors has revealed important immune cell subsets involved in tumor regulation. Among them, conventional type 1 dendritic cell (cDC1) is found to play a key role in tumor immunity. Unlike moDC used in current DC vaccine clinical trials, cDC1 is differentiated from DC precursors in bone marrow, and its production depends on cytokine FLT3L-induced transcription factors IRF-8 and BATF3, while the production of moDC is mainly induced by cytokines GM-CSF and IL-4. Compared with moDC, the anti-tumor advantages of cDC1 mainly include: ① It has the strongest "cross-presentation" ability. cDC1 is the main antigen-presenting cell for activating CD8+ T cells in vivo, while moDC lacks this ability. Through "cross-presentation", cDC1 plays a role without being limited to specific antigens, and by presenting different tumor antigens to CD8+ T cells, it can act on multiple tumors. ② It has the ability to home to tumor lymph nodes. The activation of CD8+ T cells in tumor-associated lymph nodes is crucial for anti-tumor immune response. cDC1 can chemotaxis to tumor tissue to uptake antigens and migrate to lymph nodes near the tumor to activate CD8+ T cells. Other antigen-presenting cells (such as tumor-associated macrophages TAM and moDC, etc.) do not have this property. ③ Activated cDC1 highly expresses cytokines such as IL-12 and IFN-λ, which can activate NK cells and induce CD8+ T cells to produce IFN-γ, promoting the proliferation and activation of NK and CD8+ T cells and playing an anti-tumor role. Given this, cDC1 is considered a new direction for future DC treatment of tumors.

[0004] Although cDC1 has unique advantages in anti-tumor, due to its low proportion in human body and no mature in vitro induction method in early stage, this cell subpopulation cannot be used for clinical treatment test. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for inducing and activating a classical dendritic cell subpopulation and its application in anti-tumor treatment.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] In a first aspect, the present application provides a method for inducing and activating a classical dendritic cell subpopulation, wherein the classical dendritic cell subpopulation is cDC1, and the method comprises the following steps:

[0008] A. Plate human peripheral blood stem cells or umbilical cord blood stem cells, and pre-expand the cells in a basic medium added with cytokine 1;

[0009] B. Induce and differentiate the pre-expanded peripheral blood stem cells or umbilical cord blood stem cells in a basic medium added with cytokine 2 in vitro;

[0010] C. Collect the differentiated cells, and sort CD141+ cells to obtain pure cDC1 cells;

[0011] D. Activate the pure cDC1 cells obtained in step C in a basic medium added with a stimulating agent to obtain activated cDC1 cells.

[0012] As a preferred solution, in step A, the peripheral blood stem cells or umbilical cord blood stem cells are CD34+ peripheral blood stem cells or CD34+ umbilical cord blood stem cells, which are obtained by magnetic bead sorting of human peripheral blood or umbilical cord blood.

[0013] As a preferred solution, the cytokine 1 added in the basic medium includes SCF, Flt3L and TPO.

[0014] As a preferred solution, the basic medium is SFEMII, and penicillin / streptomycin is added; more preferably, the added concentration of penicillin / streptomycin is 100 U / ml penicillin and 100 ug / ml streptomycin.

[0015] As a preferred solution, the expansion culture condition is 4 days of culture at 37℃ and 5% CO2.

[0016] As a preferred solution, in the cytokine 1, the concentration of each cytokine added is: 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 5-200 ng / ml TPO.

[0017] As a preferred solution, in the cytokine 2 added to the basic medium in step B, the cytokine 2 includes SCF, Flt3L, GM-CSF, IL4, IFN, TNFα. By adding the cytokine 2, the present application can induce a large number of cDC1 cells.

[0018] As a preferred solution, the basic medium is SFEMII, and penicillin / streptomycin is added; more preferably, the concentration of the added penicillin / streptomycin is 100 U / ml penicillin and 100 ug / ml streptomycin.

[0019] As a preferred solution, the condition of the in vitro induced differentiation culture is: 10 days of culture at 37°C, 5% CO2.

[0020] As a preferred solution, the IFN is selected from at least one of IFNα, IFNβ, IFNγ, with or without the addition of TNFα.

[0021] As a preferred solution, in the cytokine 2, the concentration of each cytokine added is: 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN, 2.5-200 ng / ml TNFα.

[0022] As a further preferred solution, in the cytokine 2, the concentration of each cytokine added is: 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFNγ.

[0023] As a further preferred solution, in the cytokine 2, the concentration of each cytokine added is: 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFNα or IFNβ.

[0024] As a further preferred solution, in the cytokine 2, the adding concentration of each cytokine is respectively: 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFNγ, 2.5-200 ng / ml IFNα or IFNβ.

[0025] As a further preferred solution, in the cytokine 2, the adding concentration of each cytokine is respectively: 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFNγ, 2.5-200 ng / ml IFNα or IFNβ, 2.5-200 ng / ml TNFα.

[0026] As a preferred solution, in step C, the sorting is performed by using a magnetic bead sorting kit, and the cDC1 cells are sorted by using an amount of antibody that is 3 times or more than the amount of antibody provided in the protocol (i.e., a guide experiment procedure). By increasing the amount of antibody, the sorting efficiency of the CD141+ cells (i.e., cDC1) is further improved, and more than 95% of the cDC1 cells can be sorted out, and the purity can reach more than 99.5%. If the amount of antibody provided in the conventional protocol is used, only 60% of the cDC1 cells can be sorted out.

[0027] As a preferred solution, in step D, the stimulating agent added in the basic medium includes at least two of a TLR3 stimulating agent, a TLR1 / 2 / 6 stimulating agent, a TLR7 / 8 stimulating agent, and an inflammasome stimulating agent.

[0028] As a further preferred solution, the TLR3 stimulating agent includes but is not limited to PolyIC; the TLR1 / 2 / 6 stimulating agent includes but is not limited to PAM3CSK4; the TLR7 / 8 stimulating agent includes but is not limited to R848; and the inflammasome stimulating agent includes but is not limited to ATP.

[0029] As a preferred solution, the basic medium is SFEMII.

[0030] As a preferred solution, the condition of the activation culture is that the culture is performed at 37°C under 5% CO2 for 2-24 hours.

[0031] As a preferred solution, the concentration of each of the stimulants added is: 0-10 ug / ml TLR3 stimulant, 0-10 ug / ml TLR1 / 2 / 6 stimulant, 0-10 ug / ml TLR7 / 8 stimulant, 0-20 mmol / ml inflammasome stimulant; and the concentration of at least two of the stimulants is not 0.

[0032] As a further preferred solution, the concentration of each of the stimulants added is: 0.5-10 ug / ml TLR3 stimulant, 0.5-10 ug / ml TLR1 / 2 / 6 stimulant, 1-10 ug / ml TLR7 / 8 stimulant, 1-20 mmol / ml inflammasome stimulant.

[0033] As a preferred solution, in step D, the expression amount of IL12p70 in the activated cDC1 cells is greater than or equal to 1000 pg / ml, and the expression amount of IL1β is greater than or equal to 500 pg / ml. More preferably, the expression amount of IL12p70 is greater than or equal to 2000 pg / ml, and the expression amount of IL1β is greater than or equal to 2000 pg / ml.

[0034] In a second aspect, the present application provides a use of the classical dendritic cell subpopulation obtained according to the foregoing method in the preparation of a drug for preventing and treating tumors.

[0035] As a preferred solution, the tumor includes various solid tumors, blood tumors; specifically, including but not limited to breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, Wilms tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical cancer, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, primary thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, etc.

[0036] As a preferred solution, the dosage of the classical dendritic cell subpopulation administered is 5×10 6 -1×10 90.1 to 10 cells / kg body weight / time; the frequency of administration is once a week.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] 1) The present application obtains a large number of human primary cDC1 by inducing from umbilical cord blood stem cells, and activates the anti-tumor ability of cDC1 by stimulating agent combination. And through the tumor model of humanized mice, the feasibility and effectiveness of cDC1 for tumor treatment are first verified.

[0039] 2) The present application realizes the maximum activation of cDC1 by further optimizing the stimulating agent combination, combined use of agonists of TLR3, TLR1 / 2, TLR7 / 8 and inflammasome, greatly improves the ability of cDC1 to produce IL12p70 and IL1β, and greatly improves the level of cDC1 to produce IL12p70 and IL1β. The activated cDC1 can more efficiently activate CD8+ T cells and NK cells in vivo by secreting IL12p70, so as to produce a large number of IFNγ and GZMB and other anti-tumor factors; by secreting IL1β, it can induce the production of memory CD8+ T cells, thereby producing a persistent anti-tumor immune response.

[0040] 3) The present application establishes an efficient in vitro induction method of human cDC1, realizes the "expansion-differentiation-purification" production system of GMP standard, and compared with the existing method, the induction efficiency is improved by more than 10000 times, realizes the use of peripheral blood CD34+ stem cells or umbilical cord blood stem cells to obtain a sufficient number of autologous cDC1 and universal cDC1 cells for anti-tumor treatment, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0041] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments, made with reference to the following drawings:

[0042] Fig. 1 is a flow chart of the induction and activation of classical dendritic cell subset cDC1 and the treatment of tumors according to the present application;

[0043] Fig. 2 is the efficiency of inducing cells using different cytokine combinations in Example 1 of the present application, wherein Fig. 2A shows the efficiency of inducing cDC1 (CD141+ cells) using different cytokine combinations, and Fig. 2B shows the number of cells induced by different cytokine combinations to induce cDC1 (CD141+ cells);

[0044] Fig. 3 is the situation of cDC1 before and after magnetic bead sorting in Example 2 of the present application;

[0045] Figure 4 is the result of inducing IL12p70 cytokine and (B) IL1β cytokine by using different stimulant combinations in Example 2 of the present application; wherein Figure 4A is the expression result of IL12p70 cytokine; Figure 4B is the expression result of IL1β cytokine;

[0046] Figure 5 is the expression level of surface co-stimulatory molecule CD80 in cells after stimulation by stimulant combination 10 (polyIC + pam3csk4 + R848 + ATP) in Example 2 of the present application;

[0047] Figure 6 is the dynamic monitoring result of tumor size change of tumor model mice in each treatment group in the verification example of the present application;

[0048] Figure 7 is the activation effect of CD8+ T cells and NK cells in the tumor in each treatment group in the verification example of the present application; wherein Figure 7A is the result of flow detection of the proportion of IFN γ+GZMB+CD8+ T cells in CD8+ T cells in the tumor; Figure 7B is the result of flow detection of the proportion of IFN γ+GZMB+CD56+NK cells in NK cells in the tumor. DETAILED DESCRIPTION

[0049] The present application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0050] The existing dendritic cells for treating tumors are all peripheral blood mononuclear-derived dendritic cell subpopulations, which lack the ability to present tumor antigens, lack the ability to home to tumor-associated lymph nodes, and lack the ability to recruit and strongly activate NK cells and CD8+ T cells, thereby resulting in insufficient anti-tumor ability.

[0051] Although cDC1 has unique advantages in anti-tumor, due to its low proportion in the human body and the lack of mature in vitro induction method in the early stage, this cell subpopulation cannot be used for clinical treatment test.

[0052] Based on this, the applicant attempts to obtain a large number of human primary cDC1 from umbilical cord stem cells, and then maximizes the anti-tumor ability of cDC1 by using an optimized stimulant combination, and finally successfully establishes an efficient in vitro induction method of human cDC1, realizes the "expansion-differentiation-purification" production system of GMP standard. And through the humanized mouse tumor model, it is first verified that cDC1 is feasible and effective for tumor treatment, which removes the obstacles for the anti-tumor transformation of cDC1.

[0053] In the detailed description of the present application, a method for in vitro induction of classical dendritic cell subset cDC1 is provided, comprising the following steps:

[0054] A, plate human umbilical cord blood stem cells at a density of 1-20x10 5 / ml, and use a basic medium added with penicillin / streptomycin for pre-expansion culture;

[0055] B, collect the umbilical cord blood stem cells obtained after pre-expansion culture for 4 days in step A, plate at a density of 1-20x10 5 / ml, and use a basic medium added with cytokine 2 (including 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN, 2.5-200 ng / ml TNFα; the IFN is selected from IFNα, IFNβ, IFNγ) and penicillin / streptomycin for induction and differentiation culture; replace the medium every 4 days under the same conditions;

[0056] C, collect the cells obtained after induction and differentiation culture for 10 days in step B, freeze them, and then use a magnetic bead sorting kit for sorting; when sorting, use 3 times or more of the amount of antibodies provided in the protocol (i.e. the guiding experimental procedure) for addition; the CD141+ cells obtained are pure cDC1 cells;

[0057] D, add the pure cDC1 cells obtained in step C to a basic medium, and then add a stimulating agent (including 0-10 ug / ml TLR3 stimulating agent, 0-10 ug / ml TLR1 / 2 / 6 stimulating agent, 0-10 ug / ml TLR7 / 8 stimulating agent, 0-20 mmol / ml inflammasome stimulating agent; and the concentration of at least two stimulating agents is not 0) to stimulate the cDC1 cells for 4-24 hours, to obtain activated cDC1 cells, i.e. classical dendritic cell subsets.

[0058] In one specific embodiment, in step A, the umbilical cord blood stem cells are CD34+ umbilical cord blood stem cells, which are obtained by magnetic bead sorting of human umbilical cord blood. The method for obtaining the umbilical cord blood stem cells is a conventional method, which is not particularly limited in the present application.

[0059] In a specific embodiment, in step B, the cytokine 2 is any one of the following combinations:

[0060] Combination 1, 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN γ.

[0061] Combination 2, 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN α or IFN β.

[0062] Combination 3, 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN γ, 2.5-200 ng / ml IFN α or IFN β.

[0063] Combination 4, 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN γ, 2.5-200 ng / ml IFN α or IFN β, 2.5-200 ng / ml TNF α.

[0064] The concentration of the cytokines in each of the above combinations can be in the range of 5-200 ng / ml, and the cells can be induced to differentiate.

[0065] In one embodiment, in step D, the stimulants include a combination of at least two stimulants selected from 0.5-10 ug / ml TLR3 stimulants, 0.5-10 ug / ml TLR1 / 2 / 6 stimulants, 1-10 ug / ml TLR7 / 8 stimulants, and 1-20 mmol / ml inflammasome stimulants. For example, a combination of 0.5-10 ug / ml TLR3 stimulants and 0.5-10 ug / ml TLR1 / 2 / 6 stimulants, a combination of 0.5-10 ug / ml TLR3 stimulants and 1-10 ug / ml TLR7 / 8 stimulants, a combination of 0.5-10 ug / ml TLR3 stimulants and 1-20 mmol / ml inflammasome stimulants, a combination of 0.5-10 ug / ml TLR1 / 2 / 6 stimulants and 1-10 ug / ml TLR7 / 8 stimulants, a combination of 0.5-10 ug / ml TLR1 / 2 / 6 stimulants and 1-20 mmol / ml inflammasome stimulants, a combination of 1-10 ug / ml TLR7 / 8 stimulants and 1-20 mmol / ml inflammasome stimulants, a combination of 0.5-10 ug / ml TLR3 stimulants, 0.5-10 ug / ml TLR1 / 2 / 6 stimulants, 1-10 ug / ml TLR7 / 8 stimulants, and 1-20 mmol / ml inflammasome stimulants. The concentration of each stimulant is within the aforementioned range, and each can achieve the effect of activating Cdc1 cells. The specific type of each stimulant is not particularly limited, and any product that can produce the corresponding stimulation effect can be used in the present application.

[0066] In one embodiment, in steps A, B and D, the culture conditions are as follows: culture at a temperature of 37°C in a 5% CO2condition.

[0067] In one embodiment, the basal medium used in steps A, B and D is SFEMII.

[0068] In the following specific examples, each of the human cytokines (SCF, Flt3L, GM-CSF, IL4, IFNa, IFNp, IFNy) was purchased from Peprotech Company. The TLR3 stimulant PolyIC was purchased from Invivogen Company, the TLR1 / 2 / 6 stimulant PAM3CSK4 was purchased from Invivogen Company, the TLR7 / 8 stimulant R848 was purchased from Invivogen Company, and the inflammasome stimulant ATP was purchased from Invivogen Company. The A375 tumor cell line used was purchased from the National Model and Characteristic Experimental Cell Resource Bank.

[0069] Example 1 Differentiation induction of cDC1 cells

[0070] This example investigates the use of different cytokines added to the basal medium for in vitro induction and differentiation culture. The induction process is shown in Figure 1, and the specific steps are as follows:

[0071] 1. Obtaining cord blood stem cells

[0072] The cord blood was subjected to density gradient centrifugation (20°C, 300g, 20min) using Ficoll-Paque Plus (GE Healthcare) to obtain mononuclear cells, which were then separated using a CD34 positive selection kit (Miltenyi Biotec) to obtain CD34+ cord blood stem cells.

[0073] 2. In vitro culture of CD34+ stem cells

[0074] The CD34+ cord blood stem cells were plated at a density of 1 x 10 5 / ml per well (24-well plate) using SFEM II as the basal medium (purchased from STEMCELL Technologies) with the addition of 100 U / ml penicillin and 100 ug / ml streptomycin (Gibco), and 100 ng / ml human SCF, 100 ng / ml human Flt3L, and 100 ng / ml human TPO. After 4 days of culture at a temperature of 37°C and 5% CO2, the cells were collected to obtain expanded cord blood stem cells.

[0075] 3. In vitro induction and differentiation of expanded cord blood stem cells

[0076] The expanded cord blood stem cells were plated at a density of 5 x 10 5 / ml per well (24-well plate) using SFEM II as the basal medium (purchased from STEMCELL Technologies) with the addition of 100 U / ml penicillin and 100 ug / ml streptomycin (Gibco), and the following combinations of human cytokines were added:

[0077] Combination 1, 100 ng / ml human SCF, 100 ng / ml human Flt3L, 2.5 ng / ml human GM-CSF, 2.5 ng / ml human IL4, and 5 ng / ml human IFNγ;

[0078] Combination 2, 100 ng / ml human SCF, 100 ng / ml human Flt3L, 2.5 ng / ml human GM-CSF, 2.5 ng / ml human IL4, and 5 ng / ml human IFNβ;

[0079] Combination 3: 100 ng / ml human SCF, 100 ng / ml human Flt3L, 2.5 ng / ml human GM-CSF, 2.5 ng / ml human IL4, 5 ng / ml human IFNβ, 5 ng / ml human IFNγ;

[0080] Combination 4: 100 ng / ml human SCF, 100 ng / ml human Flt3L, 2.5 ng / ml human GM-CSF, 2.5 ng / ml human IL4, 5 ng / ml human IFNβ, 5 ng / ml human IFNγ, and 5 ng / ml human TNFα.

[0081] The cells were cultured at 37℃ and 5% CO2, with the medium changed every 4 days under the same conditions. Cells obtained from each cytokine combination were collected on day 10 of differentiation culture and cryopreserved. The efficiency of different cytokine combinations in inducing cDC1 (CD34+ stem cells) is shown in Figure 2. As shown in Figure 2A, each cytokine combination could induce a large number of cDC1 cells, with combination 4 showing the highest induction efficiency. As shown in Figure 2B, the number of cDC1 cells induced by various cytokine combinations in this application was more than 10,000 times greater than that obtained by conventional induction methods (without pre-amplification, without IFN and TNFα).

[0082] Example 2: Sorting and Activation of cDC1 Cells

[0083] This embodiment examines cell activation using basal culture media supplemented with different stimulants. The sorting and activation process is shown in Figure 1, and the specific steps are as follows:

[0084] 1. Magnetic bead sorting of cDC1 cells

[0085] Cells collected after in vitro differentiation following combination 4 in Example 1 were resuscitated and then sorted using Miltenyi's magnetic bead sorting kit (CD141(BDCA-3) MicroBead Kit, human). cDC1 cells were sorted using three times the amount of antibody provided in the protocol. The resulting CD141+ cells were considered pure cDC1 cells with a purity of over 99.5% (see Figure 3).

[0086] 2. Activation of cDC1 cells

[0087] Pure cDC1 is 1×10 6 The cDC1 cells were added to basal culture medium (SFEMII) at a density of / ml, and each combination of stimulants was added to stimulate cDC1 cells for 4 hours. Then the cells were collected, and the levels of IL12p70 and IL1β in the supernatant and the level of the cell surface co-stimulatory molecule CD80 were detected by flow cytometry to assess the activation of cDC1 cells by each combination of stimulants.

[0088] The combinations of each stimulant used are shown in Table 1, wherein "+" means containing the stimulant, and "-" means not containing the stimulant.

[0089] Table 1: Activation of cDC1 by each combination of stimulants

[0090] The results of ELISA detection are shown in Figure 4. It can be seen from the results of induction of IL12p70 cytokine by each combination of stimulants that TLR3 stimulation (PolyIC), TLR1 / 2 / 6 stimulation (PAM3CSK4), TLR7 / 8 stimulation (R848) or inflammasome stimulation (ATP) alone cannot efficiently induce the expression of IL12p70; TLR3 stimulation (PolyIC) combined with TLR1 / 2 / 6 stimulation (PAM3CSK4), or TLR3 stimulation (PolyIC) combined with TLR7 / 8 stimulation (R848) can induce the expression of IL12p70; and the combined use of four stimulants (combination 10) can induce the highest amount of IL12p70 production (Figure 4A). It can be seen from the results of induction of IL1β cytokine by each combination of stimulants that TLR3 stimulation (PolyIC), TLR1 / 2 / 6 stimulation (PAM3CSK4), TLR7 / 8 stimulation (R848) or inflammasome stimulation (ATP) alone cannot efficiently induce the expression of IL1β; when TLR3 stimulation (PolyIC), TLR1 / 2 / 6 stimulation (PAM3CSK4) or TLR7 / 8 stimulation (R848) is combined with inflammasome (ATP) stimulation, respectively, the expression of IL1β can be induced; and the combined use of four stimulants (combination 10) can induce the highest amount of IL1β expression (Figure 4B). "***" means p<0.001.

[0091] As shown in Figure 5, after cDC1 is activated by the combined use of four stimulants (combination 10, PolyIC+PAM3CSK4+R848+ATP, the curve peak on the right in the figure), compared with the control group (Ctrl, the curve peak on the left in the figure), the expression level of the surface costimulatory molecule CD80 is significantly increased.

[0092] Verification Example

[0093] In this verification example, the anti-tumor effect of cDC1 cells is verified, and the specific steps are as follows:

[0094] 1. Construction of humanized mouse tumor model

[0095] The cord blood of HLA0201 genotype was subjected to density gradient centrifugation (20℃, 300g, 20min) using Ficoll-Paque Plus (GE Healthcare) to obtain mononuclear cells, and then a part of the cells was subjected to CD34 positive selection kit (Miltenyi Biotec) to isolate CD34+ cord blood stem cells. Another part of the cells was subjected to Classical Monocyte Isolation Kit (Miltenyi Biotec) to isolate monocytes, and the monocytes were induced for 5 days using 100 ng / ml GMCSF and 20 ng / ml IL-4 to obtain moDC cells. The CD34+ cord blood stem cells were injected into the tail vein of NCG mice (Jiejue Pharmaceutical) to obtain mice with human immune system after 20 weeks. Then, 1×10 6 A375 tumor cell line was subcutaneously injected into the mice to construct a melanoma model. 2×10 6 SKOV3 tumor cell line was subcutaneously injected into the mice to construct an ovarian cancer model; 2×10 6 MDA-MB-231 cell line was subcutaneously injected into the mice to construct a breast cancer model. 2×10 6 SiHa cell line was subcutaneously injected into the mice to construct a cervical cancer model. 2×10 6 PANC-1 cell line was subcutaneously injected into the mice to construct a pancreatic cancer model. 2×10 6 HepG2 cell line was subcutaneously injected into the mice to construct a liver cancer model. 2×10 6 T98G cell line was subcutaneously injected into the mice to construct a brain glioma model. 2×10 6 HT29 cell line was subcutaneously injected into the mice to construct a colorectal cancer model.

[0096] 2. cDC1 cells for tumor model treatment

[0097] The stimulant combinations 1 (cDC1-1 group), 5 (cDC1-2 group), 6 (cDC1-3 group) and 10 (cDC1-4 group) shown in Table 1 in Example 2 were respectively used to stimulate activated cDC1 cells (consistent with the HLA typing of CD34 cells for constructing humanized mice) for intratumoral injection into melanoma model mice as cDC1 treatment groups; moDC cells (consistent with the HLA typing of CD34 cells for constructing humanized mice) were intratumorally injected into melanoma model mice as moDC treatment groups; the injection dose of each treatment group was 1×10 7 cells / kg body weight / time, once a week. And the melanoma model mice without any cell injection were used as the control group (ctrl). There were 8 mice in each group.

[0098] 3. Melanoma model detection

[0099] The changes in tumor size of mice in each treatment group and control group were dynamically monitored using a vernier caliper, and the results are shown in FIG. 6A (the six curves in the figure from top to bottom are ctrl, moDC, cDC1-1, cDC1-2, cDC1-3, cDC1-4, respectively). The results showed that activated cDC1 cells can significantly inhibit tumor growth, and the average tumor volume of cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups after 18 days was 329.79, 205.29, 188.62, and 106.55 mm 3 , respectively. While moDC cells had limited effect on inhibiting tumor growth, and the average tumor volume after 18 days was 585.86 mm 3 . The average tumor volume of the control group after 18 days was 744.37 mm 3 . Therefore, cDC1 showed advantages over moDC in anti-tumor aspect (“***” indicates p < 0.001, and “n.s.” indicates no statistical difference).

[0100] After intratumoral injection of 2 x 10 6 cDC1 cells and moDC cells, the proportion of IFN γ+GZMB+CD8+ T cells in CD8+ T cells and the proportion of IFN γ+GZMB+CD56+NK cells in NK cells in each treatment group were detected by flow cytometry, and the results are shown in FIG. 7 (“***” indicates p < 0.001, and “n.s.” indicates no statistical difference).

[0101] 4. Dynamic detection of other tumor models

[0102] For the ovarian cancer model, the results are shown in FIG. 6B (the six curves in the figure from top to bottom are ctrl, moDC, cDC1-1, cDC1-2, cDC1-3, cDC1-4, respectively). The results showed that activated cDC1 cells can significantly inhibit tumor growth, and the average tumor volume of cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups after 18 days was 313.12, 245.62, 173.96, and 93.55 mm 3 , respectively. While moDC cells had limited effect on inhibiting tumor growth, and the average tumor volume after 18 days was 457.29 mm 3 . The average tumor volume of the control group after 18 days was 611.04 mm 3 . Therefore, cDC1 showed advantages over moDC in anti-tumor aspect (“***” indicates p < 0.001, and “n.s.” indicates no statistical difference).

[0103] For breast cancer model, the results are shown in FIG. 6C (the six curves from top to bottom in the figure are ctrl, moDC, cDC1-2, cDC1-3, cDC1-1, cDC1-4, respectively). The results show that the activated cDC1 cells can significantly inhibit the growth of tumors, and the average tumor volume of cDC1-1, cDC1-2, cDC1-3, cDC1-4 treatment groups for 18 days is 106.29, 141.11, 122.96 and 34.35 mm 3 , respectively. While the effect of moDC cells on inhibiting tumor growth is limited, the average tumor volume for 18 days is 400.15 mm 3 The average tumor volume of the control group for 18 days is 544.37 mm 3 Therefore, cDC1 shows an advantage over moDC in anti-tumor aspect (“***” represents p<0.001, “n.s.” represents no statistical difference).

[0104] For cervical cancer model, the results are shown in FIG. 6D (the six curves from top to bottom in the figure are ctrl, moDC, cDC1-2, cDC1-1, cDC1-3, cDC1-4, respectively). The results show that the activated cDC1 cells can significantly inhibit the growth of tumors, and the average tumor volume of cDC1-1, cDC1-2, cDC1-3, cDC1-4 treatment groups for 18 days is 228.79, 244.62, 204.29 and 85.55 mm 3 While the effect of moDC cells on inhibiting tumor growth is limited, the average tumor volume for 18 days is 485.86 mm 3 The average tumor volume of the control group for 18 days is 676.71 mm 3 Therefore, cDC1 shows an advantage over moDC in anti-tumor aspect (“***” represents p<0.001, “n.s.” represents no statistical difference).

[0105] For pancreatic cancer model, the results are shown in FIG. 6E (the six curves from top to bottom in the figure are ctrl, moDC, cDC1-2, cDC1-1, cDC1-3, cDC1-4, respectively). The results show that the activated cDC1 cells can significantly inhibit the growth of tumors, and the average tumor volume of cDC1-1, cDC1-2, cDC1-3, cDC1-4 treatment groups for 18 days is 178.79, 213.29, 172.96 and 67.55 mm 3 While the effect of moDC cells on inhibiting tumor growth is limited, the average tumor volume for 18 days is 485.86 mm 3 The average tumor volume of the control group for 18 days is 527.71 mm 3Therefore, cDC1s showed advantages in anti-tumor compared with moDCs (“***” represents p<0.001, and “n.s.” represents no statistical difference).

[0106] For the liver adenocarcinoma model, the results are shown in FIG. 6F (the six curves in the figure from top to bottom are ctrl, moDC, cDC1-2, cDC1-3, cDC1-1, and cDC1-4, respectively). The results show that the activated cDC1 cells can significantly inhibit the growth of tumors, and the average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups at 18 days is 196.46, 246.62, 203.29, and 107.55 mm 3 , respectively. While the effect of moDC cells on inhibiting tumor growth is limited, the average tumor volume at 18 days is 514.43 mm 3 . The average tumor volume of the control group at 18 days is 677.71 mm 3 . Therefore, cDC1s showed advantages in anti-tumor compared with moDCs (“***” represents p<0.001, and “n.s.” represents no statistical difference).

[0107] For the brain glioma model, the results are shown in FIG. 6G (the six curves in the figure from top to bottom are ctrl, moDC, cDC1-2, cDC1-3, cDC1-1, and cDC1-4, respectively). The results show that the activated cDC1 cells can significantly inhibit the growth of tumors, and the average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups at 18 days is 179.79, 229.96, 189.62, and 92.51 mm 3 , respectively. While the effect of moDC cells on inhibiting tumor growth is limited, the average tumor volume at 18 days is 571.58 mm 3 . The average tumor volume of the control group at 18 days is 611.04 mm 3 . Therefore, cDC1s showed advantages in anti-tumor compared with moDCs (“***” represents p<0.001, and “n.s.” represents no statistical difference).

[0108] For the colorectal cancer model, the results are shown in FIG. 6H (the six curves in the figure from top to bottom are ctrl, moDC, cDC1-2, cDC1-1, cDC1-3, and cDC1-4, respectively). The results show that the activated cDC1 cells can significantly inhibit the growth of tumors, and the average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups at 18 days is 229.79, 279.96, 206.29, and 152.51 mm 3The moDC cells had limited effect on inhibiting tumor growth, and the average tumor volume of the moDC cells was 714.43 mm 3 after 18 days. The average tumor volume of the control group was 877.71 mm 3 after 18 days. Therefore, the cDC1 cells had an advantage over the moDC cells in terms of anti-tumor effect (“***” indicates p<0.001, and “n.s.” indicates no statistical difference).

[0109] The application has many specific application approaches, and the above description is only a preferred embodiment of the application. It should be noted that the above examples are only used to illustrate the application, and are not used to limit the protection scope of the application. For ordinary skilled persons in the art, several improvements can be made without departing from the principles of the application, and these improvements should also be considered as the protection scope of the application.

Claims

1. A method of inducing and activating a subset of classical dendritic cells, characterized in that, The method comprises the following steps: A. Plating human peripheral blood stem cells or umbilical cord blood stem cells, and pre-expanding and culturing the cells in a basic medium added with a cytokine 1; B. Inducing and differentiating the peripheral blood stem cells or umbilical cord blood stem cells pre-expanded and cultured in step A in a basic medium added with a cytokine 2; C. Collecting the differentiated cells, and sorting CD141+ cells to obtain pure cDC1 cells; D. Activating the pure cDC1 cells obtained in step C in a basic medium added with a stimulant to obtain activated cDC1 cells.

2. The method of inducing and activating a subset of classical dendritic cells according to claim 1, characterized in that, In step A, the peripheral blood stem cells or umbilical cord blood stem cells are CD34+ peripheral blood stem cells or CD34+ umbilical cord blood stem cells, which are obtained by performing magnetic bead sorting on human peripheral blood or umbilical cord blood.

3. The method of inducing and activating a subset of classical dendritic cells according to claim 1, characterized in that, The basic medium added with the cytokine 1 comprises SCF, Flt3L and TPO. The basic medium is SFEMII, and penicillin / streptomycin is added, preferably, the added concentration of the penicillin / streptomycin is 100 U / ml penicillin and 100 ug / ml streptomycin. The expansion and culture condition is 37°C, 5% CO2 for 4 days.

4. The method of inducing and activating a subset of classical dendritic cells according to claim 3, characterized in that, The added concentration of each cytokine in the cytokine 1 is 5-200 ng / ml SCF, 5-200 ng / ml Flt3L and 5-200 ng / ml TPO.

5. The method of inducing and activating a subset of classical dendritic cells according to claim 1, wherein, In step B, the basic medium added with the cytokine 2 comprises SCF, Flt3L, GM-CSF, IL4, IFN and TNFα. The basic medium is SFEMII, and penicillin / streptomycin is added, preferably, the added concentration of the penicillin / streptomycin is 100 U / ml penicillin and 100 ug / ml streptomycin. The in vitro induction and differentiation culture condition is 37°C, 5% CO2 for 10 days.

6. The method of inducing and activating a subset of classical dendritic cells according to claim 5, characterized in that, The IFN is at least one selected from IFNα, IFNβ and IFNγ, and TNFα is added or not added.

7. The method of inducing and activating a subset of classical dendritic cells according to claim 5, characterized in that, The added concentration of each cytokine in the cytokine 2 is 5-200 ng / ml SCF, 5-200 ng / ml Flt3L, 2.5-200 ng / ml GM-CSF, 2.5-200 ng / ml IL4, 2.5-200 ng / ml IFN and 2.5-200 ng / ml TNFα.

8. The method of inducing and activating a subset of classical dendritic cells according to claim 1, wherein, In step D, the basic medium added with the stimulant comprises at least two of a TLR3 stimulant, a TLR1 / 2 / 6 stimulant, a TLR7 / 8 stimulant and an inflammasome stimulant. The basic medium is SFEMII. The activation culture condition is 37°C, 5% CO2 for 2-24 hours.

9. The method of inducing and activating a subset of classical dendritic cells according to claim 8, characterized in that, The concentration of each of the stimulants added is: 0-10 ug / ml TLR3 stimulant, 0-10 ug / ml TLR1 / 2 / 6 stimulant, 0-10 ug / ml TLR7 / 8 stimulant, 0-20 mmol / ml inflammasome stimulant; and the concentration of at least two of the stimulants is not 0.

10. The use of a classical dendritic cell subpopulation obtained by the method according to any one of claims 1-9 for the preparation of a medicament for the prevention and treatment of tumors.

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