Preparation method and application of M1 type macrophage

Abstract

The invention relates to a preparation method and application of M1 type macrophages, and the method comprises the following steps: adding activated mouse lymphocytes into a mouse macrophage culture system for co-culture to obtain the M1 type macrophages. The invention explores the identification of CD3 / CD28 activated lymphocytes, the influence of the activated lymphocytes on the morphology of macrophages and the influence of the activated lymphocytes on macrophage subtypes, and provides a brand-new method for obtaining the mouse M1 type macrophages. According to the invention, the problem that the polarization of the macrophage cannot be completely induced by singly using the recombinant IFN-gamma to stimulate the macrophage is solved. The invention provides a novel in-vitro mouse M1 type macrophage model for experimental research, M1 type macrophages induced by activated lymphocytes can better simulate an in-vivo immunoreaction process, a required cell state is obtained by utilizing co-culture of two cells in vivo, and compared with dependence on exogenous addition of viruses or compounds, the in-vitro mouse M1 type macrophage model provided by the invention has the advantages that the in-vitro immunoreaction process can be better simulated by the M1 type macrophages induced by the activated lymphocytes; according to the invention, many potential risks and bad places can be effectively avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of immunotherapy, and relates to a preparation method and application of M1 macrophages, in particular to macrophages activated by activated lymphocytes, and specifically to a method and application of polarizing macrophages into M1 macrophages. Background Art

[0002] In recent years, with the in-depth understanding of the role of the immune system in the occurrence and development of cancer, cancer immunotherapy has become a new and highly effective treatment. The core idea of ​​immunotherapy is to activate or reactivate the patient's own immune system to identify and attack tumor cells.

[0003] Currently, researchers usually use three main methods to obtain macrophages required for experiments: (1) directly isolating primary macrophages from animals or humans; (2) using colony stimulating factors (CSFs) to promote the differentiation of primary monocytes into macrophages; (3) inducing differentiation of cell lines into macrophages. Although directly isolated primary macrophages and monocytes can better reflect the physiological state in vivo, there are challenges in the collection of these primary cells, such as limited sources, short survival time in vitro, high cost, and difficult to control individual differences. Therefore, inducing differentiation of cell lines into macrophages has become the main method for obtaining macrophages. However, different differentiation induction techniques may significantly affect the function and characteristics of the obtained macrophages.

[0004] As the main immune cells in the tumor microenvironment, macrophages have multiple polarization states that determine their role in tumor progression. M2 macrophages, which show alternative polarization, tend to secrete a series of cytokines that can promote the expression of extracellular matrix and tumor angiogenesis, thereby supporting tumor growth and spread. In contrast, M1 macrophages undergo pro-inflammatory polarization, and they enhance the secretion of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 12 (IL-12). These factors can promote necrosis and apoptosis of cancer cells and have an inhibitory effect on tumor cells. M1 macrophages also express higher levels of major histocompatibility complex (MHC) molecules and co-stimulatory factors, such as CD80 and CD86. The presence of these molecules enables M1 macrophages to effectively activate the adaptive immune system and launch a more targeted attack on tumor cells.

[0005] The signaling pathways involved in regulating macrophage polarization are complex, and M1 macrophages can generally be induced by LPS and IFN-γ. At present, many mammalian pathogens and pathogenic microorganisms have been used to induce M1 macrophage polarization, and these methods are being actively explored in the field of tumor treatment. For example, microorganisms such as oncolytic viruses, adenoviruses, and Listeria have been shown to activate pattern recognition receptors (PRRs) on the surface of macrophages, thereby promoting the polarization of M1 macrophages. There are also studies that use recombinant IFN-γ to stimulate macrophages to induce macrophage polarization to M1, but in this method, the use of IFN-γ may not be able to fully simulate the complex immune microenvironment in the body, and the signaling pathways of M1 polarization are very complex. The use of IFN-γ alone may not be able to fully simulate all necessary signals, thereby affecting the polarization effect, that is, the stimulation of recombinant IFN-γ in the prior art is not sufficient to completely induce macrophages to polarize to M1.

[0006] In addition, there are also in vitro studies that obtain M1 macrophages by stimulating LPS combined with IFN-γ. For example, CN113980902A discloses an in vitro culture method for efficiently inducing macrophage polarization, including the following steps: Step 1, in serum-free conditions, THP-1 monocytes are stimulated with PMA and GM-CSF to differentiate into macrophages; Step 2, 20 ng / mL IFN-γ and 100 ng / mL LPS are used to induce the macrophages differentiated in step 1 for 24 hours to obtain M1 macrophages; Step 3, 20 ng / mL IL-4 and 20 ng / mL IL-10 are used to induce the macrophages differentiated in step 1 for 24 hours to obtain M2 macrophages. This method requires first exploring the optimal concentration and induction time, the optimal combination of cytokines that induce polarization, concentration and induction time, etc., which is very complicated. CN117050943A discloses a method for preparing macrophages, comprising (a) inducing iPSCs to differentiate into EBs in a culture medium supplemented with CHIR 99021, (b) inducing the EBs formed in step a to differentiate into hematopoietic stem and progenitor cells (HSPCs); and (c) inducing the HSPCs formed in step b to differentiate into macrophages. This method is to obtain macrophages by differentiating cells from other sources (monocytes, iPSCs), and its preparation process is complicated. The differentiation process from scratch may also introduce phenotypic variations, and the preparation process is not easy to control.

[0007] Macrophages are extremely heterogeneous and can be polarized into different subtypes under different environmental stimuli. Currently, the different effects of different subtypes of macrophages on T cell activation still need further study.

[0008] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0009] Studies have found that T cells activated by CD3 / CD28 can produce IFN-γ, but stimulating macrophages with recombinant IFN-γ alone is not sufficient to completely induce macrophage polarization. Therefore, although it can be speculated that activated lymphocytes may induce M1 macrophage polarization through IFN-γ, there must be some unknown mechanism in the process of activated lymphocytes inducing M1 macrophage polarization.

[0010] In view of the shortcomings of the prior art, the first aspect of the present invention provides a method for preparing M1 macrophages, the method comprising: adding lymphocytes to a macrophage culture system, and co-culturing to obtain M1 macrophages.

[0011] According to a preferred embodiment, the lymphocytes include activated lymphocytes. The lymphocytes are preferably mouse activated lymphocytes.

[0012] At present, the method of inducing M1 macrophage polarization by stimulating with exogenous substances (such as LPS, IFN-γ, etc.) has potential application prospects in immune regulation and disease treatment, but its defects and limitations include: First, in addition to acting on target cells, it may also non-specifically affect other types of cells and cause non-specific immune responses. Specifically, common stimuli include bacterial components (such as lipopolysaccharide), cytokines (such as interferon-γ) and chemicals (such as rheumatoid factor), which can activate macrophages and induce M1 polarization, but they usually also have an effect on other immune cells. This non-specific stimulation may lead to excessive activation of the immune system, thereby causing inflammatory responses and the occurrence of immune-related diseases. Secondly, the method of inducing M1 macrophage polarization by stimulating with exogenous substances has the problem of toxic side effects. For example, some cytokines may have cytotoxic effects on cells at high concentrations and cause abnormal proliferation or apoptosis of macrophages; some chemicals may cause organ toxicity or systemic toxicity under long-term use or high-dose conditions. Third, at present, most studies rely on the stimulation of a single substance, such as a single bacterial component, a single cytokine or a single chemical. However, the immune system in an organism is a highly complex network that is regulated by a variety of signaling molecules and cytokines. Therefore, the stimulation of a single substance may not fully simulate the real immune environment, resulting in the function and response of M1 macrophages being incomplete and inaccurate.

[0013] Compared with the traditional stimulation by exogenous substances, the method of inducing the polarization of M1 macrophages by cell co-culture of the present invention has a more prominent effect. Specifically, the method of co-culturing activated lymphocytes with macrophages can stimulate the production of a variety of substances, including IFN-γ and the like. IFN-γ can cause macrophages to polarize to the M1 type, thereby enhancing their inflammatory regulation and antimicrobial effects. The method of co-culturing activated lymphocytes with macrophages can not only provide IFN-γ, but also produce signal molecules produced by other immune cells, such as IL-1β, TNF-α, etc., which also play an important role in immune response. Therefore, the polarization of M1 macrophages induced by the cell co-culture method is the combined effect of multiple stimulating substances, which can better simulate the in vivo immune environment, so that the effect of macrophage polarization is more comprehensive and accurate. On the other hand, the cell co-culture method can provide cell-to-cell interaction and signal transduction. Under the conditions of co-culture of activated lymphocytes and macrophages, not only are macrophages affected by stimulating substances such as IFN-γ, but they also directly contact and interact with other immune cells (such as T cells, B cells, etc.). This kind of cell-to-cell interaction can promote the exchange and conduction of signal molecules, thereby enhancing the effect of macrophage polarization. In addition, the cell co-culture method can also provide a more dynamic and sophisticated regulatory environment. By adjusting the proportion of different cell types, adding specific cytokines or signal molecules, etc., it is possible to achieve fine regulation of the polarization process of the treated cells. This dynamic regulation can better simulate the characteristics of the in vivo immune response, making the polarization process of macrophages more controllable and predictable. In contrast, the direct use of IFN-γ and other stimulating substances to induce the polarization of M1 macrophages is relatively single and fixed, and cannot provide the accuracy and flexibility of the dynamic regulation of the present invention.

[0014] When the present invention utilizes activated lymphocytes to induce M1 macrophages for tumor immunotherapy, it has a more significant therapeutic effect. The interaction during the cell co-culture process can enhance the recognition ability of M1 macrophages to tumor antigens. During the co-culture process, M1 macrophages may activate T cells through antigen presentation, thereby establishing a stronger immune memory. This M1 macrophage with enhanced immune memory can play a better effect in subsequent treatments. Even if the surface antigens of tumor cells change, M1 macrophages and their activated T cells can still identify and eliminate tumor cells more quickly and effectively. Secondly, the dynamic regulation during the co-culture process enables M1 macrophages to perform functional regulation under the influence of specific cytokines, produce a response adapted to the tumor microenvironment, thereby effectively overcoming the inhibition of the tumor on the immune system, that is, the M1 macrophages induced by the method of the present invention have stronger microenvironment adaptability. When the microenvironment is poorly adaptable, the following may occur: macrophages cannot effectively perform their functions, resulting in insufficient immune response, which ultimately affects the therapeutic effect; macrophage survival rate is significantly reduced due to inability to adapt to the new microenvironment, such as undergoing apoptosis due to inhibitory signals in the microenvironment, which directly affects the effect of immunotherapy; macrophages that are not adapted to the microenvironment may be affected by inhibitory factors in the tumor microenvironment (such as immune checkpoint molecules or inhibitory factors secreted by tumors), resulting in suppressed activity. This will not only reduce anti-tumor responses, but may also promote tumor growth; macrophages that are not adapted to the microenvironment may cause systemic side effects. For example, excessive immune responses may cause tissue damage or autoimmune diseases, leading to complications.

[0015] In addition, other effective substances (such as chemokines) produced during cell co-culture can combine with certain specific substances or factors in the body to form a more targeted therapeutic effect. For example, tumor cells may secrete specific chemokines to attract M1 macrophages, and M1 macrophages induced by co-culture are usually more sensitive to these factors. This specific combination enables M1 macrophages to migrate to the tumor site more effectively and exert anti-tumor effects.

[0016] According to a preferred embodiment, the lymphocytes include T lymphocytes and B lymphocytes.

[0017] According to a preferred embodiment, the activated lymphocytes include activated T lymphocytes. The activated lymphocytes preferably also include activated B lymphocytes.

[0018] According to a preferred embodiment, the macrophages include macrophages RAW264.7, macrophages THP-1, macrophages U937, bone marrow-induced macrophages BMDM, peripheral blood-derived mononuclear macrophages PBMC, tumor-associated macrophages (TAMs), or primary macrophages derived from mammalian tissues. The mammal is preferably a mouse.

[0019] According to a preferred embodiment, the macrophages comprise a bone marrow macrophage subtype.

[0020] According to a preferred embodiment, the method for polarizing macrophages into M1 macrophages comprises the following steps: Obtain M0 macrophages; obtain non-activated lymphocytes; activate the non-activated lymphocytes and co-culture them with M0 macrophages, thereby inducing the polarization of macrophages into M1 macrophages.

[0021] According to a preferred embodiment, the step of obtaining M0 macrophages includes: collecting cell suspension from the bone marrow cavity of primary mice; separating the cell suspension; resuspending the cells; counting and adjusting the cell concentration; and culturing the cells.

[0022] Preferably, the method of separating the cell suspension includes filtration and centrifugation. Those skilled in the art will appreciate that filtration and centrifugation are conventional technical means for cell separation, and these operations need not be limited in the present application, and can also complete the basic operation of separation.

[0023] Preferably, the cells are resuspended in DMEM complete medium. Preferably, the cells are resuspended in DMEM complete medium containing 5 to 100 ng / mL M-CSF. More preferably, the cells are resuspended in DMEM complete medium containing 10 to 20 ng / mL M-CSF. Particularly preferably, the cells are resuspended in DMEM complete medium containing 10 ng / mL M-CSF.

[0024] Preferably, the cell concentration is adjusted to 1×10 4 ~1×10 7 / mL. More preferably, the cell concentration is adjusted to 1×10 5 ~1×10 7 / mL. Particularly preferably, the cell concentration is adjusted to 2×10 6 / mL.

[0025] Preferably, the cells are cultured at 37°C and 5% CO2.

[0026] According to a preferred embodiment, the steps of obtaining non-activated lymphocytes are: extracting lymph nodes of mice; grinding the lymph nodes to obtain lymphocyte suspension; separating the lymphocyte suspension; resuspending the lymphocytes; counting and adjusting the lymphocyte concentration.

[0027] Preferably, DMEM complete medium is used to resuspend the lymphocytes.

[0028] Preferably, the lymphocyte concentration is adjusted to a range of 1×10 4 ~1×10 7 / mL. More preferably, the cell concentration is adjusted to 1×10 6 ~1×10 7 / mL.

[0029] According to a preferred embodiment, the method for activating non-activated lymphocytes is: adding stimulatory molecules to non-activated lymphocytes. The stimulatory molecules preferably include CD3 antibodies and / or CD28 antibodies. The concentration of CD3 antibodies used to activate non-activated lymphocytes is preferably 1-5 μg / mL; the concentration of CD28 antibodies used to activate non-activated lymphocytes is preferably 0.1-5 μg / mL. The concentration of CD3 antibodies used to activate non-activated lymphocytes is more preferably 1-3 μg / mL; the concentration of CD28 antibodies used to activate non-activated lymphocytes is more preferably 0.1-3 μg / mL. The concentration of CD3 antibodies used to activate non-activated lymphocytes is particularly preferably 1 μg / mL; the concentration of CD28 antibodies used to activate non-activated lymphocytes is particularly preferably 0.3 μg / mL.

[0030] According to a preferred embodiment, the ratio of macrophages to lymphocytes is 1:1 to 20. More preferably, the ratio of macrophages to lymphocytes is 1:10. According to a preferred embodiment, the duration of co-culture of activated lymphocytes and M0 type macrophages is 24 to 72 hours. The duration of co-culture of activated lymphocytes and M0 type macrophages is more preferably 24 to 48 hours. The duration of co-culture of activated lymphocytes and M0 type macrophages is particularly preferably 24 hours.

[0031] According to a preferred embodiment, the polarization of M1 macrophages is promoted by increasing the expression of iNOS in macrophages or reducing the expression of cCD206. High levels of iNOS expression enable M1 macrophages to produce higher concentrations of nitric oxide (NO), which helps to inhibit the growth of pathogens and tumor cells and is one of the key mechanisms for M1 macrophages to exert cytotoxicity. In the course of the disease, high iNOS expression in M1 macrophages helps to control acute infection and anti-tumor. Regulating the polarization state of macrophages and iNOS expression may become a strategy for treating certain diseases (such as infectious diseases, tumors and autoimmune diseases). In experimental studies, the polarization state and function of macrophages can be evaluated by detecting the mRNA and protein levels of iNOS. The expression level of cCD206 can reflect the polarization state of macrophages. High expression is usually associated with M2 polarization, while low expression or no expression may be associated with M1 polarization. Regulating the expression or function of cCD206 may become a potential strategy for treating certain inflammatory diseases, autoimmune diseases and tumors.

[0032] The second aspect of the present invention provides M1 macrophages prepared by the method provided by the first aspect of the present invention.

[0033] The third aspect of the present invention provides a composition, the composition comprises the M1 macrophage provided by the second aspect of the present invention and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent. Lymphocyte-activated macrophages can be formulated into pharmaceutical compositions or drugs for clinical use, and may include pharmaceutically acceptable carriers, diluents, excipients or adjuvants. Therefore, on the one hand, the present invention provides a composition comprising lymphocyte-activated macrophages and pharmaceutically acceptable carriers, adjuvants, excipients or diluents. Pharmaceutically acceptable carriers, diluents, excipients or adjuvants are known in the art. The composition can be formulated for parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal administration routes, including injection or infusion. Suitable preparations may include lymphocyte-activated macrophages in sterile or isotonic media (e.g., water for injection). Drugs and pharmaceutical compositions can be formulated into fluid forms, including gel forms. The fluid formulation may be formulated for administration to a selected area of ​​the human or animal body by injection or infusion (eg, via a catheter).The composition comprising lymphocyte-activated macrophages is formulated for intratumoral or intravenous administration, eg, for macrophage-directed cancer immunotherapy.

[0034] According to a preferred embodiment, the preparation method of the composition involved in the present application may include one or more steps selected from the following: isolating / purifying the lymphocyte-activated macrophages produced by the method according to the present invention; and / or mixing the macrophages with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0035] A fourth aspect of the present invention provides IFN-γ activated macrophages.

[0036] The fifth aspect of the present invention provides a composition, comprising the IFN-γ activated macrophages provided by the fourth aspect of the present invention.

[0037] The sixth aspect of the present invention provides a method for selectively polarizing macrophages, the method comprising: treating isolated M0 macrophages with activated lymphocytes. The present invention also provides a composition, the composition comprising macrophages prepared by the method provided in the sixth aspect of the present invention and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0038] A seventh aspect of the present invention provides a method for preparing M2 macrophages, the method comprising treating isolated M0 macrophages with IFN-γ.

[0039] The eighth aspect of the present invention provides a method for treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the M1 macrophages provided by the second aspect of the present invention or the composition provided by the third aspect of the present invention.

[0040] Preferably, the subject suffers from cancer, pulmonary fibrosis, liver fibrosis, liver damage or cardiac fibrosis. More preferably, the lymphocyte-activated macrophages and the pharmaceutical composition comprising the lymphocyte-activated macrophages are capable of treating or preventing cancer, such as inhibiting the development / progression of cancer, delaying / preventing the onset of cancer, reducing / delaying / preventing tumor growth, reducing / delaying / preventing metastasis, alleviating the severity of cancer symptoms, reducing the number of cancer cells, reducing tumor size / volume, and / or increasing survival (e.g., progression-free survival).

[0041] The ninth aspect of the present invention provides another method for treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of IFN-γ activated macrophages provided by the fourth aspect of the present invention or the composition provided by the fifth aspect of the present invention. Preferably, the subject is a mammal. Preferably, the subject is a human.

[0042] The tenth aspect of the present invention provides the use of the method provided in the first aspect of the present invention, the M1 macrophages provided in the second aspect of the present invention, the composition provided in the third aspect of the present invention, the IFN-γ activated macrophages provided in the fourth aspect of the present invention, the composition provided in the fifth aspect of the present invention, the method provided in the sixth aspect of the present invention, and the method provided in the seventh aspect of the present invention in the preparation of drugs or products for macrophage-directed cancer immunotherapy.

[0043] Technical effect: The present invention first uses CD3 / CD28 stimulation to activate lymphocytes. After the initial T cells are activated by antigen stimulation, they gradually express CD44 at a high level and lose CD62L to differentiate into effector cells. The results of the present invention show that the CD44 expression level of lymphocytes stimulated by CD3 / CD28 increases and the CD62L expression level decreases. The present invention uses this method to activate lymphocytes successfully and reliably. The present invention provides a new in vitro M1 macrophage model for research. The M1 macrophages induced by activated lymphocytes can better simulate the immune response process in vivo.

[0044] The existing technology mainly focuses on the treatment of a single cell type, such as relying solely on IFN-γ stimulation. This strategy emphasizes the role of exogenous factors, while there are relatively few studies on the interaction and synergy between cells. There are complex signal transduction mechanisms between cells, especially in the process of macrophage polarization. The existing technical methods are usually based on specific experimental designs, and the scheme of co-culture after activating lymphocytes requires a large adjustment to the experimental design. Therefore, it is not easy for the existing technology to consider the idea of ​​using cell co-culture to promote the generation of M1 macrophages. Compared with the method of directly stimulating macrophages with IFN-γ and using cells from other sources to differentiate into M1 macrophages in the prior art, the present invention uses activated lymphocytes to stimulate macrophages to change the polarization state, which reduces the operation steps and makes the stimulation process easy to control and repeat. Existing macrophages have mature immune functions and phenotypes. Directly stimulating macrophage polarization can better maintain their functional characteristics, reduce the generation of other variations, and improve production efficiency (such as the prior art using monocytes or iPSCs to differentiate into macrophages may have variations in differentiation efficiency and differentiation direction). M1 macrophages obtained by direct stimulation can be more easily scaled up and quality controlled.

[0045] In addition, LPS and IFN-γ may activate multiple cell types (non-specific), while the present invention utilizes activated lymphocytes to stimulate macrophage polarization into M1 macrophages, a process that is closer to the immune response under physiological conditions (provides a cell-to-cell interaction similar to that occurring naturally in the body), and activated lymphocytes may act more specifically on macrophages through specific cell-to-cell contact and signal transduction, more comprehensively simulating the immune regulatory network in the body. By controlling the activation state and number of lymphocytes, the present invention can more accurately regulate the polarization degree of macrophages (with better experimental controllability). In terms of cost, compared with commercially purchased recombinant cytokines, the present invention utilizes activated lymphocytes to reduce costs, especially when the required lymphocytes can be obtained from other parts of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram for validation of CD3 / CD28 activated lymphocytes; Figure 2 Schematic diagram of the morphological differences of macrophages stimulated by different lymphocytes; Figure 3 Schematic diagram of the changes in macrophage subtypes induced by different lymphocyte stimulations. DETAILED DESCRIPTION

[0047] The following is a detailed description with reference to the accompanying drawings.

[0048] The following examples are only used to further explain the beneficial effects of polarized macrophages in the present application, and should not be used to limit the scope of protection of the present application. For those of ordinary skill in the art, the specific meaning of the terms in the present invention can be understood according to the specific circumstances. The experimental procedures described in the following examples are conventional procedures unless otherwise specified. The reagents used in the following examples, etc., can be obtained through commercial channels unless otherwise specified.

[0049] The experimental animals used in the present invention were C57BL / 6N (B6) mice, male, 8-12 weeks old, provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and were all raised in a clean-grade standard environment. The experiments conducted in the present invention were approved by the Experimental Animal Welfare Ethics Committee of Xuanwu Hospital, Capital Medical University (approval number: XW-20210531-1).

[0050] Main reagents: fetal bovine serum (FBS) (AQmv09900, Beijing Aoqing Biotechnology Co., Ltd.); DMEM / high glucose version, containing L-glutamine (SH30022.01, Cytiva, USA); penicillin-streptomycin-amphotericin B mixed solution (100×) (P7630, Beijing Solebao Biotechnology Co., Ltd.); D-Hanks (without calcium and magnesium, without phenol red) (H1045, Beijing Solebao Biotechnology Co., Ltd.); bovine serum albumin (BSA) (A7906, Sigma-Aldrich, USA); 4% tissue cell fixative (P1110, Beijing Solebao Biotechnology Co., Ltd.); Wright-Giemsa staining solution-A solution (BA4017A, Zhuhai Beso Cell Science and Technology Co., Ltd.); Wright-Giemsa staining solution-B solution (BA4017B, Zhuhai Beso Cell Science and Technology Co., Ltd.); iNOS Rabbit pAb (A14031, Wuhan Abotek Biotechnology Co., Ltd.); Arginase 1 (ARG1) Rabbit mAb (A4923, Wuhan Abotec Biotechnology Co., Ltd.); BD™ BD IntraSure™ Kit (641776, BD Company, USA).

[0051] Main instruments and equipment: BD FACS flow cytometer (BD, USA); AriaTMII; low-temperature centrifuge (Beijing Baiyang Medical Instrument Co., Ltd.); CO2 incubator (Thermo Fisher, USA); optical microscope (Leica, Germany); 4°C refrigerator (Sanyo, Japan); -80°C refrigerator (Thermo Fisher, USA); cell counter (Bio-rad, USA); TC20 cell counting chamber (Bio-rad, USA); cell culture plate (6-well TC treatment) (Wuxi Nice Life Science Co., Ltd.); cell slide (Shanghai Jing'an Biotechnology Co., Ltd.); slide (Leica, Germany).

[0052] Solution preparation: Preparation of DMEM complete medium: Add 20% FBS and 1% penicillin-streptomycin-amphotericin solution to DMEM basal medium. Perform the entire process in a sterile operating table and store at 4°C for short-term storage.

[0053] Preparation of flow cytometry fixative: Dilute 4% tissue cell fixative with D-hanks buffer to 1% tissue cell fixative.

[0054] The present invention uses GraphPad Prism 8.0 software for plotting and statistical analysis. All data are expressed as mean ± standard deviation (mean ± SD). Unpaired t-test or One-way ANOVA is used to analyze the significance of two groups of data. P < 0.05 means that the difference is statistically significant, ns means that there is no significant difference, and the P value is expressed in standard abbreviation format, that is, ; ; ; .

[0055] The present invention particularly provides a method for preparing mouse M1 macrophages, and the specific method is as shown in the following examples. Example 1

[0056] This example provides relevant experimental methods, including extraction and culture of primary mouse bone marrow-derived macrophages (BMDM), preparation of mouse lymphocytes, co-culture of macrophages and lymphocytes in different activation states, and flow cytometry.

[0057] BMDM extraction and culture: On day 0, B6 mice were killed by cervical vertebral dissection. The killed mice were sterilized by soaking in 75% alcohol for 10 min and then taken out. The limbs were fixed, the lower limbs were peeled off, placed in pre-cooled D-hanks buffer, and transferred to the clean bench for operation; the excess muscles of the lower limbs were removed, the complete tibia and femur were freed, the two ends of the long bones were cut off, and the D-hanks buffer was sucked with a 2 mL disposable sterile syringe to repeatedly rinse the bone marrow cavity; the cell suspension was transferred to a 15 mL centrifuge tube, filtered with a 70 μm cell strainer, centrifuged at 400 g for 5 min at 4°C, the supernatant was discarded, and the cells were resuspended in DMEM complete medium containing 10 ng / mL M-CSF; the cells were counted and the cell concentration was adjusted to 2×10 6 / mL, the cells were inoculated in a 6-well plate, with 2 mL of cell suspension per well, i.e. 4×10 6 / well. Place in a 37°C, 5% CO2 cell culture incubator and closely observe the cell status under a phase contrast microscope every day.

[0058] On the first day, the medium was changed to remove non-adherent cells, and the adherent cells were BMDM; all the old culture medium on the upper layer was discarded, and the cells were rinsed three times with D-hanks buffer, and fresh DMEM complete culture medium containing 10 ng / mL M-CSF was added; thereafter, new DMEM complete culture medium containing 10 ng / mL M-CSF was replaced every three days.

[0059] Preparation of mouse lymphocytes: The lymph nodes of the inguinal, mesenteric, axillary and cervical regions of B6 mice were extracted under aseptic operation and placed in a 1.5 mL centrifuge tube filled with pre-cooled D-hanks buffer. The lymph nodes were ground into lymphocytes on a sterile plastic plate using a 1 mL sterile syringe plunger on a clean bench, filtered through a 70 μm cell strainer, and centrifuged at 400 g for 5 min at 4°C. The supernatant was discarded, and the lymphocytes were resuspended in a small amount of DMEM complete medium, and a small amount was taken for counting.

[0060] Co-culture of macrophages with lymphocytes in different activation states: On the third day of BMDM culture, different stimuli were added to the unactivated or unpolarized macrophage (M0 macrophage) culture system, and the cells were divided into the following four groups: 1) CD3 / CD28 control group: CD3 (final concentration of 1 μg / mL) and CD28 (final concentration of 0.3 μg / mL) were added; 2) non-activated lymphocyte group: unactivated B6 mouse lymphocytes were added for co-culture, and the ratio of macrophages to lymphocytes was 1:10; 3) activated lymphocyte group: B6 mouse lymphocytes activated by CD3 (final concentration of 1 μg / mL) and CD28 (final concentration of 0.3 μg / mL) were added, and the ratio of macrophages to lymphocytes was 1:10; 4) IFN-γ group: IFN-γ (final concentration of 100 ng / mL) was added.

[0061] After 24 h of co-culture, suspended lymphocytes were separated and macrophages were collected. Subsequently, flow cytometry was used to quantitatively analyze the changes in the immunophenotype of macrophages; at the same time, Wright-Giemsa staining was used to observe the morphology of macrophages to evaluate the polarization state of macrophages.

[0062] Flow cytometry detection of macrophage subtypes: The old culture medium of macrophages was discarded, and the cells were rinsed three times with D-hanks buffer. 1 mL of pre-cooled D-hanks buffer was added to each well, and the macrophages were blown off by blowing. The macrophage suspension was collected in a flow tube, and centrifuged at 400 g for 5 min at 4°C. The supernatant was discarded, and 100 μL of D-hanks buffer was added to resuspend the cells.

[0063] Add anti-CD16 / 32 antibody to the cell suspension, incubate at 4℃ in the dark for 15 min, block Fc receptors, add cell surface antibody anti-CD11b-FITC and F4 / 80-PE / Dazzle™ 594 antibody, shake and mix, incubate at 4℃ in the dark for 30 min. Add 100 μL of fixation / permeabilization reagent A solution, incubate at 4℃ in the dark for 5 min to fix the cells, add 2 mL of D-hanks buffer to resuspend the cells, centrifuge at 400 g for 5 min at 4℃, and discard the supernatant. Add 50 μL of permeabilization reagent B solution and intracellular antibody anti-iNOS-PE and anti-CD206-PE / Cyanine7 antibody, shake and mix, and incubate at 4℃ in the dark for 30 min. Wash the cells twice with D-hanks buffer, centrifuge at 400 g for 5 min at 4℃, and discard the supernatant. Add 0.5 mL of D-hanks buffer to resuspend the cells, and detect bone marrow macrophage subtypes on a flow cytometer.

[0064] Flow cytometry to detect lymphocyte phenotype: Collect the lymphocytes suspended in the supernatant, centrifuge at 400 g for 5 min at 4°C, discard the old culture medium, wash once with pre-cooled D-hanks buffer, centrifuge at 400 g for 5 min at 4°C, discard the supernatant, resuspend the cells with 100 μL of D-hanks buffer, add anti-CD4-PE / Dazzle™ 594, CD8-PE / Cyanine7, CD44-FITC, CD62L-PE antibodies, oscillate and mix, incubate at 4°C in the dark for 30 min; wash twice with 2 mL of D-hanks buffer, centrifuge at 400 g for 5 min at 4°C, discard the supernatant. Add 0.5 mL of D-hanks buffer to resuspend the cells; add 7AAD, incubate at 4°C in the dark for 5-10 min, and finally detect the functional status of lymphocytes on a flow cytometer. If the sample is tested overnight, resuspend the cells with 1% tissue fixative and store at 4°C in the dark until testing.

[0065] Wright-Giemsa staining: The macrophages to be tested were cultured on a cell slide, and the cell slide was completely covered with 4% tissue cell fixative. After standing at room temperature for 20 min, the cell slide was taken out and fixed on a glass slide. The glass slide was placed on a staining rack, and an appropriate amount of Wright-Giemsa A solution was added to completely cover the cell slide; then Wright-Giemsa B solution was added on top of solution A (the amount added was 2 to 3 times that of solution A), and the two solutions of solution A and solution B were mixed with an ear wash bulb, and the staining was allowed to stand at room temperature for 7 to 9 min, rinsed with running water, and dried naturally. Changes in the morphology of macrophages were observed under an optical microscope. Example 2

[0066] In this example, primary lymphocytes were extracted from lymph nodes of B6 mice to further explore the effect of activated lymphocytes on macrophage polarization. Lymphocytes were stimulated by CD3 monoclonal antibody combined with CD28 monoclonal antibody (CD3 / CD28) to obtain activated lymphocytes. The blank control group was lymphocytes without CD3 / CD28 activation. The two groups of lymphocytes were co-cultured with BMDM respectively. The blank control group and the two groups of lymphocytes after CD3 / CD28 activation culture for 24 h were detected by flow cytometry for the expression of CD4, CD8, CD44, and CD62L to verify whether the lymphocytes stimulated by CD3 / CD28 were successfully activated.

[0067] 1. Identification of CD3 / CD28 activated lymphocytes Figure 1 Schematic diagram of CD3 / CD28 activated lymphocyte validation, wherein: Figure 1 (A) Flow cytometry analysis of blank control lymphocytes and CD4 in CD3 / CD28 activated lymphocytes. + and CD8 + The expression results of CD44 and CD62L in lymphocytes; Figure 1 (B) Two groups of CD4 + and CD8 + Statistical graph of mean fluorescence intensity of CD44 and CD62L in lymphocytes.

[0068] CD4 + and CD8 + Lymphocytes are mainly CD44 - CD62L + Initial T cells, while CD3 / CD28 activated CD4 + and CD8 + In addition to resting naive T cells, lymphocytes also include activated central memory T cells (CD44 + CD62L + ), effector memory T cells (CD44 + CD62L - ) and terminally differentiated effector memory T cells (CD44 - CD62L - )( Figure 1 A). The changes in the expression of CD44 and CD62L indicate that activation treatment causes changes in the expression levels of these markers, and activation treatment changes the activation state of T cell subsets and cells.

[0069] The mean fluorescence intensity (MFI) is a common indicator for measuring the expression level of cell surface markers. The higher the value, the higher the expression level of the marker. The analysis results of the mean fluorescence intensity of CD44 and CD62L in each group showed that the expression level of CD44 in CD3 / CD28 activated lymphocytes was significantly higher than that in the blank control group, that is, CD3 / CD28 activation treatment promoted CD4 + and CD8 + The expression of CD44 in lymphocytes; the expression level of CD62L in CD3 / CD28 activated lymphocytes was significantly lower than that in the blank control group, that is, CD3 / CD28 activation treatment inhibited the expression of CD4 + and CD8 + Expression of CD62L in lymphocytes ( Figure 1 B). In CD3 / CD28 activated lymphocytes, CD62L decreased and CD44 increased, which may indicate that T cells switch from central memory phenotype to effector memory phenotype. The expression of activated lymphocyte surface markers changes, indicating that CD3 / CD28 stimulates successful lymphocyte activation.

[0070] Lymphocytes were co-cultured with macrophages and divided into a blank control group and an activated lymphocyte group. The blank control group did not receive any stimulation, while the activated lymphocyte group received CD3 / CD28 antibody to activate lymphocytes. After 24 h of culture, the lymphocyte phenotype was detected by flow cytometry.

[0071] 2. Effects of activated lymphocytes on macrophage morphology Different subtypes of macrophages have different morphologies. In order to study the effect of activated lymphocytes on the morphological changes of macrophages, the present invention cultured BMDM on cell slides for observation. Specifically, on the third day of culture, the following were added to the M0 macrophage culture system: CD3 / CD28 (CD3 / CD28 control group), unactivated lymphocytes (lymphocyte stimulation was added as the unactivated lymphocyte group); CD3 / CD28 activated lymphocytes (CD3 / CD28 and lymphocytes were added as the activated lymphocyte group), IFN-γ (IFN-γ group). After 24 hours of culture, the slides were crawled and Wright-Giemsa stained, and the morphological differences of the four groups of macrophages were observed under an optical microscope. The results are as follows. Figure 2 shown. Figure 2 Magnification is 1000×.

[0072] Obviously, the morphology of macrophages in the CD3 / CD28 control group, the non-activated lymphocyte group, and the IFN-γ group was not much different, all showing a long spindle shape with synapses (pseudopodia). The morphology of macrophages in the activated lymphocyte group changed significantly, specifically showing an increase in cell size, rich cytoplasm, and a round "fried egg" shape ( Figure 2 ).

[0073] When macrophages become round with abundant cytoplasm, this may indicate that they are transitioning to an M1 polarization state, which is associated with pro-inflammatory, bactericidal, and other processes. The round morphology with abundant cytoplasm may indicate that the phagocytic function of macrophages is enhanced, which is important in clearing cellular debris and pathogens, especially in M1 polarized macrophages. The morphological changes of macrophages may affect their antigen presenting ability. M1 macrophages may present antigens more effectively to activate T cells, while M2 macrophages may play a role in regulating T cell responses and promoting immunosuppression.

[0074] The phagocytic ability of round macrophages with rich cytoplasm is enhanced. This morphology of macrophages has more endoplasmic reticulum and Golgi bodies, which helps them synthesize and secrete the phagocytic and digestive molecules required. The round morphology may increase the surface area of ​​the cell membrane, thereby providing more contact and phagocytic areas, allowing macrophages to more effectively encapsulate and endocytose pathogens or cell debris. Macrophages with rich cytoplasm play a key role in clearing apoptotic cells and pathogens, which is an important mechanism for maintaining tissue homeostasis and preventing the spread of infection. Macrophages of this morphology may recognize and bind to a variety of ligands, such as bacterial cell wall components, phosphatidylserine of apoptotic cells, etc. through "scavenger receptors". M2 macrophages may express lower levels of MHC-II and co-stimulatory molecules, which help them regulate T cell responses, reduce excessive immune activation, and may promote immune tolerance. In this embodiment, the morphology of macrophages in the unactivated lymphoid group and the IFN-γ group was long spindle-shaped with synapses (pseudopodia), and the macrophages in the activated lymphoid group showed that the cell volume became larger, the cytoplasm became richer, and the morphology was round "fried egg-shaped". The spindle-shaped morphology may affect the contact area between macrophages and the surrounding environment. Synapse formation may inhibit the main functions of macrophages (such as phagocytosis). In addition, the signal transmission ability of spindle-shaped macrophages, the influence of the microenvironment, and their migration and positioning abilities may affect the function of macrophages. In comparison, macrophages in the activated lymphocyte group have higher phagocytic efficiency, stronger response to cell signals, stronger migration ability, and better phagocytic function.

[0075] M2 macrophages may inhibit the proliferation and activation of T cells by secreting anti-inflammatory factors such as IL-10, thereby exerting an immunosuppressive effect in chronic infection or tumor microenvironment. Morphological changes of macrophages may also affect their interactions with other immune cells. For example, M2 macrophages may regulate B cell differentiation and antibody production by releasing specific cytokines. In certain disease states, such as tumors, chronic infections, or autoimmune diseases, the morphology and function of macrophages may undergo specific changes to adapt to different microenvironmental conditions and immune needs.

[0076] 3. Effects of activated lymphocytes on macrophage subtypes To study the effect of activated lymphocytes on macrophage subtypes, macrophages were co-cultured with lymphocytes in different activation states for 24 h, and then macrophages were collected and detected by flow cytometry. iNOS was a marker for M1 macrophages, and CD206 was a marker for M2 macrophages.

[0077] In this embodiment, the flow gate strategy is: set CD11b + F4 / 80 + Macrophage populations were detected, and macrophage subtypes were detected by iNOS and cCD206. After 24 h of co-culture, macrophages from the CD3 / CD28 control group, non-activated lymphocyte group, activated lymphocyte group, and IFN-γ group were collected, and the expressions of CD11b, F4 / 80, iNOS, and cCD206 were detected by flow cytometry.

[0078] Figure 3 Schematic diagram of the changes in macrophage subtypes stimulated by different lymphocytes, among which: Figure 3 (A) Statistical results of flow cytometry detection of macrophage subtypes in four groups with different treatments, including four groups of CD11b + F4 / 80 + Expression of iNOS and cCD206 in macrophages and iNOS - cCD206 - M0 macrophages and iNOS + cCD206 - Statistical graph of the proportion of M1 macrophages; Figure 3 (B) The expression of iNOS in macrophages of four groups with different treatments (expressed by the mean fluorescence intensity (MFI) of iNOS); Figure 3 (C) The expression of cCD206 in macrophages of four groups with different treatments (represented by the mean fluorescent intensity (MFI) of cCD206). M1 polarization is usually associated with high expression of iNOS and low expression of cCD206; M2 polarization is usually associated with high expression of cCD206 and low expression of iNOS.

[0079] Figure 3 CD11b + F4 / 80 + In macrophages, the performance of the CD3 / CD28 control group, the non-activated lymphocyte group, and the IFN-γ group was similar, mainly iNOS - cCD206 -Compared with the CD3 / CD28 control group, the non-activated lymphocyte group, and the IFN-γ group, the expression of iNOS in macrophages in the activated lymphocyte group was significantly higher than that in the non-activated lymphocyte group. - cCD206 - The number of cells decreased significantly, and iNOS + cCD206 - The number of cells increased significantly (high expression of iNOS and low expression of cCD206), indicating that the macrophages in the activated lymphocyte group polarized from M0 (unpolarized or initial state) macrophages to M1 macrophages ( Figure 3 A). Among the four groups of macrophages, the iNOS expression level in the macrophages of the activated lymphocyte group was significantly higher than that of the other groups ( Figure 3 B), compared with the CD3 / CD28 control group and the non-activated lymphocyte group, the expression of cCD206 in macrophages in the activated lymphocyte group and the IFN-γ group was significantly decreased, among which the expression of cCD206 in the activated lymphocyte group was the lowest ( Figure 3 C). Therefore, the present invention can effectively promote the polarization of M1 macrophages through activated lymphocytes.

[0080] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention. The equivalent replacement of the raw materials selected by the present invention, the addition of auxiliary components, the selection of specific methods, etc., also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the description of the present invention and its drawings are illustrative and do not constitute limitations on the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The description of the present invention contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", which means that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept.

Claims

1. A method for preparing M1 macrophages, characterized in that: The method comprises: adding lymphocytes to a culture system of macrophages, and co-culturing to obtain M1 macrophages.

2. The method according to claim 1, characterized in that The lymphocytes include activated lymphocytes.

3. The method according to claim 1 or 2, characterized in that: The macrophages include a subtype of bone marrow macrophages.

4. The method according to claim 1, characterized in that: The method promotes the polarization of M1 macrophages by increasing the expression level of iNOS in macrophages or decreasing the expression level of cCD206.

5. An M1 macrophage, characterized in that: The method is prepared by any one of claims 1 to 4.

6. A composition, characterized in that The method comprises the M1 macrophage according to claim 5 and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

7. A method for preparing a composition, characterized in that: The method comprises one or more steps selected from the following: isolating / purifying the M1 macrophages produced according to the method of any one of claims 1 to 4; and / or mixing the M1 macrophages with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

8. A method for selectively polarizing macrophages, characterized in that: The method comprises: treating isolated M0 macrophages with activated lymphocytes.

9. A composition, characterized in that The invention comprises macrophages prepared by the method according to claim 8 and a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

10. Use of the method according to any one of claims 1 to 4, the M1 macrophage according to claim 5, the composition according to claim 6, the preparation method according to claim 7, the method according to claim 8 or the composition according to claim 9 in the preparation of a drug or product for macrophage-directed cancer immunotherapy.

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