Method for inducing and activating classical dendritic cell subpopulation and application of classical dendritic cell subpopulation in anti-tumor treatment

Abstract

The invention discloses a method for inducing and activating a classic dendritic cell subset, which comprises the following steps: A, planking peripheral blood stem cells or cord blood stem cells of a human body, and performing multiplication culture by adopting a basal culture medium added with a cell factor 1; b, carrying out in-vitro induced differentiation culture on the cells subjected to multiplication culture by adopting a basic culture medium added with a cell factor 2; c, sorting the differentiated cells to obtain pure cDC1 cells; and D, carrying out activation culture on the pure cDC1 cells by adopting the basic culture medium added with the stimulant to obtain activated cDC1 cells. According to the invention, a large amount of human primary cDC1 is obtained through induction of cord blood stem cells, and the anti-tumor ability of the cDC1 is activated through a stimulant combination. And through a humanized mouse tumor model, the feasibility and effectiveness of the cDC1 in tumor treatment are verified for the first time.

Description

Technical Field

[0001] The present invention relates to the technical fields of cell therapy, tumor prevention and treatment, autoimmune disease prevention and treatment, and vaccine development, and in particular to a method for inducing and activating a classical dendritic cell subset and an application thereof in anti-tumor treatment. Background Art

[0002] Cell therapy, as a treatment method that uses living cells as drugs, has the potential to cure a variety of tumors. Although cell therapies such as CAR-T and CAR-NK have been successful in blood tumors, their application in solid tumors has been slow. TIL and dendritic cells (monocyte derived dendritic cells, moDC) prepared from monocytes in peripheral blood also lack effective breakthroughs. Therefore, the development of new cell therapies is of great significance for the treatment of solid tumors.

[0003] In recent years, multi-omics studies of solid tumors have revealed important immune cell subsets involved in tumor regulation. Among them, conventional type 1 dendritic cell subset 1 (cDC1) has been 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 the bone marrow. Its production depends on the transcription factors IRF-8 and BATF3 induced by the cytokine FLT3L, while the production of moDC is mainly induced by the cytokines GM-CSF and IL-4. Compared with moDC, the anti-tumor advantages of cDC1 are mainly reflected in: ① It has the most powerful "cross-presentation" ability. cDC1 is the main antigen-presenting cell that activates CD8+T cells in vivo, while moDC lacks this ability. Through "cross-presentation", cDC1 is not limited to specific antigens and can play a role in a variety of tumors by presenting different tumor antigens to CD8+T cells. 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 into tumor tissue to take up antigens, migrate to lymph nodes near the tumor, and activate CD8+T cells. Other antigen-presenting cells (such as tumor-associated macrophages TAM and moDC) do not have this characteristic. 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, promote the proliferation and activation of NK and CD8+T cells, and exert anti-tumor effects. In view of this, cDC1 is considered to be a new development direction for DC treatment of tumors in the future.

[0004] Although cDC1 has unique advantages in anti-tumor, its proportion in the human body is very low and there is no mature in vitro induction method in the early stage, which makes this cell subpopulation unable to be used in clinical treatment trials. Summary of the invention

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

[0006] The objective of the present invention is achieved through the following technical solutions:

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

[0008] A. Plating human peripheral blood stem cells or umbilical cord blood stem cells, and pre-expanding and culturing them in a basal medium supplemented with cytokine 1;

[0009] B. inducing differentiation and culturing the peripheral blood stem cells or umbilical cord blood stem cells pre-amplified in step A in vitro using a basal medium supplemented with cytokine 2;

[0010] C. Collect differentiated cells and sort out CD141+ cells, which are pure cDC1 cells;

[0011] D. The pure cDC1 cells obtained in step C are activated and cultured using a basic culture medium with a stimulator added thereto to obtain activated cDC1 cells.

[0012] As a preferred embodiment, 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 embodiment, in the basic culture medium supplemented with cytokine 1, cytokine 1 includes SCF, Flt3L, and TPO.

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

[0015] As a preferred embodiment, the amplification culture conditions are: culture at 37° C. and 5% CO 2 for 4 days.

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

[0017] As a preferred embodiment, in step B, the basal medium to which cytokine 2 is added includes SCF, Flt3L, GM-CSF, IL4, IFN, and TNFα. In the present invention, a large number of cDC1 cells can be induced by adding cytokine 2.

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

[0019] As a preferred embodiment, the in vitro differentiation induction culture conditions are: culture at 37° C. and 5% CO 2 for 10 days.

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

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

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

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

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

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

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

[0027] As a preferred embodiment, in step D, the basal culture medium to which the stimulant is added comprises at least two of a TLR3 stimulant, a TLR1 / 2 / 6 stimulant, a TLR7 / 8 stimulant, and an inflammasome stimulant.

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

[0029] As a preferred embodiment, the basal culture medium is SFEMII.

[0030] As a preferred embodiment, the activation culture conditions are: culture at 37° C. and 5% CO 2 for 2-24 hours.

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

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

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

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

[0035] As a preferred embodiment, the tumor includes various solid tumors and 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, Wilman tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, genital urinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, ovarian cancer ... Cell carcinoma, endometrial carcinoma, adrenal cortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic granulocytic leukemia, acute myeloid leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteoblastic sarcoma, primary macroglobulinemia and retinoblastoma, etc.

[0036] As a preferred embodiment, the dosage of the classical dendritic cell subset is 5×10 6 -1×10 9cells / kg body weight / time; the dosing frequency is once a week.

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

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

[0039] 2) The present invention further optimizes the combination of stimulants and uses TLR3, TLR1 / 2, TLR7 / 8 and inflammasome agonists in combination, thereby achieving maximum activation of cDC1, greatly improving the ability of cDC1 to produce IL12p70 and IL1β, and significantly increasing the level of IL12p70 and IL1β produced by cDC1. The activated cDC1 can more efficiently activate CD8+T cells and NK cells in the body by secreting IL12p70, causing them to produce a large amount of anti-tumor factors such as IFNγ and GZMB; by secreting IL1β, it can induce the production of memory CD8+T cells, thereby producing a lasting anti-tumor immune response.

[0040] 3) The present invention establishes an efficient in vitro induction method for human cDC1, realizing a GMP-grade standard "amplification-differentiation-purification" production system. Compared with the existing methods, the induction efficiency is increased by more than 10,000 times, and it is possible to use 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0042] Figure 1 A flowchart of the invention for obtaining the induction and activation of the classical dendritic cell subset cDC1 and treating tumors;

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

[0044] Figure 3 This is the situation of cDC1 before and after magnetic bead sorting in Example 2 of the present invention;

[0045] Figure 4 The results of induction of IL12p70 cytokine and (B) IL1β cytokine by different stimulant combinations in Example 2 of the present invention; wherein, Figure 4 A is the expression result of IL12p70 cytokine; Figure 4 B is the expression result of IL1β cytokine;

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

[0047] Figure 6 The present invention verifies the dynamic monitoring results of tumor size changes of tumor model mice in each treatment group in the embodiment;

[0048] Figure 7 The present invention verifies the activation effect of each treatment group in the embodiment on CD8+T cells and NK cells in the tumor; wherein, Figure 7 A is the result of flow cytometry detection of the ratio of IFNγ+GZMB+CD8+T cells to CD8+T cells in tumors; Figure 7 B is the result of flow cytometry detection of the proportion of IFNγ+GZMB+CD56+NK cells in NK cells in the tumor. DETAILED DESCRIPTION

[0049] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0050] Existing dendritic cells used to treat tumors are all dendritic cell subpopulations derived from peripheral blood monocytes. This cell subpopulation lacks the ability to present tumor antigens, lacks the ability to home to tumor-related lymph nodes, and lacks the ability to recruit and strongly activate NK cells and CD8+T cells, resulting in insufficient anti-tumor ability.

[0051] Although cDC1 has unique advantages in anti-tumor, its proportion in the human body is very low and there is no mature in vitro induction method in the early stage, which makes this cell subpopulation unable to be used in clinical treatment trials.

[0052] Based on this, the applicant tried to induce a large number of human primary cDC1 from umbilical cord blood stem cells, and then activated the anti-tumor ability of cDC1 to the maximum extent through an optimized combination of stimulants, and finally successfully established an efficient in vitro induction method for human cDC1, realizing the "amplification-differentiation-purification" production system of GMP grade standards. And through the humanized mouse tumor model, the feasibility and effectiveness of cDC1 for tumor treatment were verified for the first time, removing obstacles for the anti-tumor transformation of cDC1.

[0053] In a specific embodiment of the present invention, a method for inducing a classical dendritic cell subset cDC1 in vitro is provided, comprising the following steps:

[0054] A. Human umbilical cord blood stem cells were placed at a density of 1-20×10 5 / ml plating, pre-amplification culture was performed using a basal medium supplemented with cytokine 1 (including 5-200ng / ml SCF, 5-200ng / ml Flt3L, and 5-200ng / ml TPO; the concentration of each cytokine can achieve cell expansion within this range) and penicillin / streptomycin;

[0055] B. After the umbilical cord blood stem cells obtained after 4 days of pre-expansion culture in step A were collected, the density was 1-20×10 5 / ml plating, inducing differentiation culture using a basal medium supplemented with cytokines 2 (including 5-200ng / ml SCF, 5-200ng / ml Flt3L, 2.5-200ng / ml GM-CSF, 2.5-200ng / ml IL4, 2.5-200ng / ml IFN, 2.5-200ng / ml TNFα; the IFN is selected from IFNα, IFNβ, IFNγ) and penicillin / streptomycin; changing the medium every 4 days under the same conditions;

[0056] C. Collect the cells obtained after 10 days of differentiation induction culture in step B and freeze them, then use a magnetic bead sorting kit to sort them. When sorting, use 3 times or more of the antibody added in the protocol (i.e., the guiding experimental process). The CD141+ cells obtained are pure cDC1 cells;

[0057] D. Add the pure cDC1 cells obtained in step C to the basal culture medium, and then add stimulators (including 0-10ug / ml TLR3 stimulator, 0-10ug / ml TLR1 / 2 / 6 stimulator, 0-10ug / ml TLR7 / 8 stimulator, 0-20mmol / ml inflammasome stimulator; and the concentration of at least two of the stimulators 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 a specific embodiment, in step A, the cord blood stem cells are CD34+ cord blood stem cells obtained by magnetic bead sorting of human umbilical cord blood. The method for obtaining the cord blood stem cells is a conventional method and is not particularly limited in the present invention.

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

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

[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 IFNα or IFNβ.

[0062] Combination 3, 5-200ng / ml SCF, 5-200ng / ml Flt3L, 2.5-200ng / ml GM-CSF, 2.5-200ng / ml IL4, 2.5-200ng / ml IFNγ, 2.5-200ng / ml IFNα or IFNβ.

[0063] Combination 4, 5-200ng / ml SCF, 5-200ng / ml Flt3L, 2.5-200ng / ml GM-CSF, 2.5-200ng / ml IL4, 2.5-200ng / ml IFNγ, 2.5-200ng / ml IFNα or IFNβ, 2.5-200ng / ml TNFα.

[0064] When the cytokine concentrations in the above combinations are within this range, cell differentiation can be induced.

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

[0066] In a specific embodiment, the culture conditions in steps A, B and D are: culture at a temperature of 37° C. and 5% CO 2 .

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

[0068] In the following specific examples, the cytokines (SCF, Flt3L, GM-CSF, IL4, IFNα, IFNβ, IFNγ) used were purchased from Peprotech. The TLR3 stimulator PolyIC was purchased from Invivogen, the TLR1 / 2 / 6 stimulator PAM3CSK4 was purchased from Invivogen, the TLR7 / 8 stimulator R848 was purchased from Invivogen, and the inflammasome stimulator ATP was purchased from Invivogen. The A375 tumor cell line used was purchased from the National Model and Specialty Experimental Cell Resource Bank.

[0069] Example 1: Differentiation induction of cDC1 cells

[0070] This example investigates the use of basal culture medium supplemented with different cytokines for in vitro differentiation induction culture, such as Figure 1 The induction process shown in the figure has the following specific steps:

[0071] 1. Obtaining Umbilical Cord Blood Stem Cells

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

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

[0074] According to 1×10 per well 5 / ml density, CD34+ umbilical cord blood stem cells were plated (24-well plate), SFEMII was used as the basic culture medium (purchased from STEMCELL Technologies), and 100U / ml penicillin and 100ug / ml streptomycin (Gibco) were added, as well as 100ng / ml human SCF, 100ng / ml human Flt3L, and 100ng / ml human TPO. After culturing for 4 days at 37°C and 5% CO2, the cells were collected to obtain the expanded umbilical cord blood stem cells.

[0075] 3. Umbilical cord blood stem cells after induction, differentiation and expansion in vitro

[0076] The expanded cord blood stem cells were divided into 5×10 5 The cells were plated at a density of 1:100 / ml (24-well plate), SFEMII was used as the basal medium (purchased from STEMCELL Technologies), 100 U / ml penicillin and 100 ug / ml streptomycin (Gibco) were added, and the following human cytokine combinations 1-4 were added at the same time:

[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, 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, 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°C and 5% CO2, and the medium was changed every 4 days under the same conditions. The cells obtained by culturing with each cytokine combination were collected on the 10th day of differentiation culture and frozen. The efficiency of inducing cDC1 (CD34+ stem cells) with different cytokine combinations is shown in Figure 2. Figure 2 As shown. Figure 2 As shown in A, each cytokine combination can induce a large number of cDC1 cells, among which combination 4 has the highest induction efficiency. Figure 2 B It can be seen that the number of cDC1 induced by various cytokine combinations in the present application is more than 10,000 times that of the conventional induction method (without pre-amplification, without adding IFN and TNFα).

[0082] Example 2 Isolation and Activation of cDC1 Cells

[0083] This example investigates the use of basal medium supplemented with different stimulants for cell activation, such as Figure 1 The specific steps of the separation and activation process shown are as follows:

[0084] 1. Magnetic bead sorting of cDC1 cells

[0085] After the cells collected after in vitro differentiation induction in combination 4 in Example 1 were revived, they were sorted using a magnetic bead sorting kit (CD141 (BDCA-3) MicroBead Kit, human) from Miltenyi Biotec. cDC1 cells were sorted using an antibody added in an amount three times the amount provided in the protocol. The CD141+ cells sorted were pure cDC1 with a purity of more than 99.5% (see Figure 3 ).

[0086] 2. Activation of cDC1 cells

[0087] Pure cDC1 was added at 1×10 6 / ml density was added to the basal medium (SFEMII), and each stimulant combination was added to stimulate cDC1 cells for 4 hours. Then the cells were collected, and the content of IL12p70 and IL1β in the supernatant and the level of cell surface co-stimulatory molecule CD80 were detected by flow cytometry to evaluate the activation of cDC1 stimulated by each stimulant combination. The stimulant combinations used are shown in Table 1, where "+" indicates that the stimulant is contained, and "-" indicates that the stimulant is not contained.

[0088] Table 1 Activation of cDC1 by various stimulant combinations

[0089]

[0090]

[0091] ELISA test results Figure 4 As shown. The results of the induction of IL12p70 cytokines by each combination of stimulants show 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; the combination of TLR3 stimulation (PolyIC) and TLR1 / 2 / 6 stimulation (PAM3CSK4), or the combination of TLR3 stimulation (PolyIC) and TLR7 / 8 stimulation (R848) can induce the expression of IL12p70; and the combination of four stimulants (combination 10) can induce the highest amount of IL12p70 production ( Figure 4 A). The results of the induction of IL1β cytokine by each combination of stimulants show that TLR3 stimulation (PolyIC), TLR1 / 2 / 6 stimulation (PAM3CSK4), TLR7 / 8 stimulation (R848) or inflammasome stimulation (ATP) alone cannot effectively induce the expression of IL1β; when TLR3 stimulation (PolyIC), TLR1 / 2 / 6 stimulation (PAM3CSK4) or TLR7 / 8 stimulation (R848) are combined with inflammasome (ATP) stimulation, IL1β expression can be induced; and the combination of the four stimulants (combination 10) can induce the highest amount of IL1β expression ( Figure 4 B). “***” indicates p<0.001.

[0092] like Figure 5 As shown, after cDC1 was activated by a combination of four stimulants (combination 10, PolyIC+PAM3CSK4+R848+ATP, the peak of the curve on the right side of the figure), the expression level of the surface co-stimulatory molecule CD80 was significantly increased compared with the control group (Ctrl, the peak of the curve on the left side of the figure).

[0093] Verification Example

[0094] This verification example verifies the anti-tumor effect of cDC1 cells, and the specific steps are as follows:

[0095] 1. Construction of a humanized mouse tumor model

[0096] For cord blood with HLA0201 genotype, Ficoll-Paque Plus (GE Healthcare) was used to perform density gradient centrifugation (20°C, 300g, 20min) to obtain mononuclear cells, and then a portion of the cells was separated using the CD34 positive selection kit (Miltenyi Biotec) to obtain CD34+ cord blood stem cells. Another portion of the cells was separated using the Classical Monocyte Isolation Kit (Miltenyi Biotec) to obtain monocytes, and the monocytes were induced with 100ng / ml GMCSF and 20ng / ml IL-4 for 5 days to obtain moDC cells. CD34+ cord blood stem cells were injected into the tail vein of NCG mice (Jicui Yaokang), and after 20 weeks, mice with a human immune system were obtained. Next, the mice were subcutaneously injected with 1×10 6 A375 tumor cell line was used to construct a melanoma model. Mice were injected subcutaneously with 2×10 6 SKOV3 tumor cell line was used to construct an ovarian cancer model; 2×10 6 MDA-MB-231 cell line was used to construct a breast cancer model. 2×10 6 SiHa cell lines were used to construct a cervical cancer model. 6 The pancreatic cancer model was constructed by subcutaneous injection of 2×10 6 HepG2 cell lines were used to construct a liver cancer model. 2×10 6 T98G cell lines were used to construct a glioma model. 2×10 6 A HT29 cell line was used to construct a colorectal cancer model.

[0097] 2.cDC1 cells for tumor model treatment

[0098] The cDC1 cells (consistent with the HLA typing of CD34 cells constructed from humanized mice) stimulated and activated by stimulator combination 1 (cDC1-1 group), stimulator combination 5 (cDC1-2 group), stimulator combination 6 (cDC1-3 group) and stimulator combination 10 (cDC1-4 group) shown in Table 1 of Example 2 were injected intratumorally into melanoma model mice as cDC1 treatment groups; moDC cells (consistent with the HLA typing of CD34 cells constructed from humanized mice) were injected intratumorally into melanoma model mice as moDC treatment groups; the injection dose of each treatment group was 1×10 7 The mice were injected with 100 cells / kg body weight / time, once a week. The melanoma model mice without any cell injection were used as the control group (ctrl). There were 8 mice in each group.

[0099] 3. Melanoma model testing

[0100] The changes in tumor size of mice in each treatment group and control group were dynamically monitored using a vernier caliper. Figure 6 As shown in A (the six curves in the figure are ctrl, moDC, cDC1-1, cDC1-2, cDC1-3, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 329.79, 205.29, 188.62, and 106.55 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, and the average tumor volume on day 18 was 585.86 mm 3 The average tumor volume of the control group on day 18 was 744.37 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0101] After intratumoral injection of 2 × 10 6 After treatment with cDC1 cells and moDC cells, flow cytometry was used to detect the ratio of IFNγ+GZMB+CD8+T cells to CD8+T cells and the ratio of IFNγ+GZMB+CD56+NK cells to NK cells in the tumors of each treatment group. The results are as follows Figure 7 As shown ("***" indicates p<0.001, "ns" indicates no statistical difference).

[0102] 4. Dynamic detection of other tumor models

[0103] For the ovarian cancer model, the results are as follows Figure 6As shown in B (the six curves in the figure are ctrl, moDC, cDC1-1, cDC1-2, cDC1-3, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 was 313.12, 245.62, 173.96, and 93.55 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, and the average tumor volume on day 18 was 457.29 mm 3 The average tumor volume of the control group on day 18 was 611.04 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0104] For breast cancer models, the results are as follows Figure 6 C (the six curves in the figure are ctrl, moDC, cDC1-2, cDC1-3, cDC1-1, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 106.29, 141.11, 122.96, and 34.35 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, with the average tumor volume at day 18 being 400.15 mm 3 The average tumor volume of the control group on day 18 was 544.37 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0105] For the cervical cancer model, the results are as follows Figure 6 D (the six curves in the figure are ctrl, moDC, cDC1-2, cDC1-1, cDC1-3, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 228.79, 244.62, 204.29, and 85.55 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, with the average tumor volume at day 18 being 485.86 mm 3 The average tumor volume of the control group on day 18 was 676.71 mm 3Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0106] For the pancreatic cancer model, the results are as follows Figure 6 E (the six curves in the figure are ctrl, moDC, cDC1-2, cDC1-1, cDC1-3, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 178.79, 213.29, 172.96, and 67.55 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, with the average tumor volume at day 18 being 485.86 mm 3 The average tumor volume of the control group on day 18 was 527.71 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0107] For the hepatic adenocarcinoma model, the results are as follows Figure 6 As shown in F (the six curves in the figure are ctrl, moDC, cDC1-2, cDC1-3, cDC1-1, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 196.46, 246.62, 203.29, and 107.55 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, and the average tumor volume on day 18 was 514.43 mm 3 The average tumor volume of the control group on day 18 was 677.71 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0108] For the brain glioma model, the results are as follows Figure 6 G (the six curves in the figure are ctrl, moDC, cDC1-2, cDC1-3, cDC1-1, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 179.79, 229.96, 189.62, and 92.51 mm, respectively.3 However, the effect of moDC cells in inhibiting tumor growth was limited, and the average tumor volume on day 18 was 571.58 mm 3 The average tumor volume of the control group on day 18 was 611.04 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0109] For the colorectal cancer model, the results are as follows Figure 6 H (the six curves in the figure are ctrl, moDC, cDC1-2, cDC1-1, cDC1-3, cDC1-4 from top to bottom). The results showed that activated cDC1 cells can significantly inhibit tumor growth. The average tumor volume of the cDC1-1, cDC1-2, cDC1-3, and cDC1-4 treatment groups on day 18 were 229.79, 279.96, 206.29, and 152.51 mm, respectively. 3 However, the effect of moDC cells in inhibiting tumor growth was limited, and the average tumor volume on day 18 was 714.43 mm 3 The average tumor volume of the control group on day 18 was 877.71 mm 3 Therefore, cDC1 showed advantages over moDC in anti-tumor effects (“***” indicates p<0.001, and “ns” indicates no statistical difference).

[0110] The present invention has many specific application paths, and the above is only a preferred embodiment of the present invention. It should be pointed out that the above embodiments are only used to illustrate the present invention, and are not used to limit the protection scope of the present invention. For those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A method for inducing and activating classical dendritic cell subsets, characterized in that: The following steps are involved: A. Plating human peripheral blood stem cells or umbilical cord blood stem cells, and pre-expanding and culturing them in a basal medium supplemented with cytokine 1; B. inducing differentiation and culturing the peripheral blood stem cells or umbilical cord blood stem cells pre-amplified in step A in vitro using a basal medium supplemented with cytokine 2; C. Collect differentiated cells and sort out CD141+ cells, which are pure cDC1 cells; D. Activate and culture the pure cDC1 cells obtained in step C using a basal culture medium with added stimulants to obtain activated cDC1 cells.

2. The method for inducing and activating classical dendritic cell subsets 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 magnetic bead sorting of human peripheral blood or umbilical cord blood.

3. The method for inducing and activating classical dendritic cell subsets according to claim 1, characterized in that: The basal medium added with cytokine 1 includes SCF, Flt3L, and TPO; The basal medium is SFEMII, and penicillin / streptomycin is added. Preferably, the concentration of penicillin / streptomycin added is 100U / ml penicillin and 100ug / ml streptomycin; The amplification culture conditions are: culture at 37° C. and 5% CO 2 for 4 days.

4. The method for inducing and activating classical dendritic cell subsets according to claim 3, characterized in that: In the cytokine 1, the added concentrations of the cytokines are: 5-200ng / ml SCF, 5-200ng / ml Flt3L, 5-200ng / ml TPO.

5. The method for inducing and activating classical dendritic cell subsets according to claim 1, characterized in that: In step B, the basal medium is supplemented with cytokine 2, wherein cytokine 2 includes SCF, Flt3L, GM-CSF, IL4, IFN, and TNFα; The basal medium is SFEMII, and penicillin / streptomycin is added. Preferably, the concentration of penicillin / streptomycin added is 100U / ml penicillin and 100ug / ml streptomycin; The in vitro differentiation induction culture conditions are: culture at 37° C. and 5% CO 2 for 10 days.

6. The method for inducing and activating classical dendritic cell subsets according to claim 5, characterized in that: The IFN is selected from at least one of IFNα, IFNβ and IFNγ, with or without TNFα.

7. The method for inducing and activating classical dendritic cell subsets according to claim 5, characterized in that: In the cytokine 2, the added concentrations of the cytokines are: 5-200ng / ml SCF, 5-200ng / ml Flt3L, 2.5-200ng / ml GM-CSF, 2.5-200ng / ml IL4, 2.5-200ng / ml IFN, and 2.5-200ng / ml TNFα.

8. The method for inducing and activating classical dendritic cell subsets according to claim 1, characterized in that: In step D, the basal medium to which the stimulant is added comprises at least two of a TLR3 stimulant, a TLR1 / 2 / 6 stimulant, a TLR7 / 8 stimulant, and an inflammasome stimulant; The basal medium is SFEMII; The activation culture conditions are: culture at 37° C. and 5% CO 2 for 2-24 hours.

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

10. Use of a classical dendritic cell subset obtained according to the method according to any one of claims 1 to 9 in preparing drugs for preventing and treating tumors.

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