Preparation method and application of m1 type macrophage

Abstract

The application relates to a preparation method and application of M1 type macrophages, and the method comprises the following steps: adding activated mouse lymphocytes to a culture system of mouse macrophages for co-culture to obtain M1 type macrophages. The application explores the identification of CD3 / CD28 activated lymphocytes, the influence of activated lymphocytes on the macrophage morphology and the influence of activated lymphocytes on the macrophage subtype, and provides a brand-new method for obtaining mouse M1 type macrophages. The application solves the problem that the stimulation of macrophages by using a recombinant IFN-gamma alone is not enough to completely induce the macrophage polarization. The application provides a new in-vitro mouse M1 type macrophage model for experimental research. The M1 type macrophages induced by the activated lymphocytes can better simulate the immune response process in the body, the required cell state is obtained by using the in-vivo co-culture of two kinds of cells, and compared with the dependence on the exogenous addition of viruses or compounds, the application can effectively avoid many potential risks and disadvantages.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy technology, and to a method for preparing and using M1 macrophages, particularly macrophages activated by activated lymphocytes, specifically a method for polarizing macrophages into M1 macrophages and its application. Background Technology

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

[0003] Currently, researchers typically employ three main methods to obtain macrophages 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; and (3) inducing passaged cell lines to differentiate into macrophages. Although directly isolated primary macrophages and monocytes better reflect the physiological state in vivo, the collection of these primary cells presents challenges, such as limited sources, short in vitro survival time, high costs, and uncontrollable individual variability. Therefore, inducing passaged cell lines to differentiate 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 primary immune cells in the tumor microenvironment, macrophages exhibit multiple polarization states, which determine their role in tumor progression. M2 macrophages, exhibiting alternative polarization, tend to secrete a series of cytokines that promote extracellular matrix expression and tumor angiogenesis, thus supporting tumor growth and spread. Conversely, M1 macrophages undergo pro-inflammatory polarization, enhancing the secretion of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12). These factors promote cancer cell necrosis and apoptosis, inhibiting tumor cell activity. 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, thereby launching a more targeted attack on tumor cells.

[0005] The signaling pathways involved in regulating macrophage polarization are complex, and M1 macrophages are generally induced by LPS and IFN-γ. Currently, numerous mammalian pathogens and microorganisms have been used to induce M1 macrophage polarization, and these methods are being actively explored in the field of cancer therapy. For example, oncolytic viruses, adenoviruses, and Listeria monocytogenes have been shown to activate pattern recognition receptors (PRRs) on the surface of macrophages, thereby promoting M1 macrophage polarization. Other studies have used recombinant IFN-γ to stimulate macrophages to induce M1 polarization. However, in this approach, IFN-γ may not fully mimic the complex immune microenvironment in vivo. The signaling pathways for M1 polarization are highly complex, and using IFN-γ alone may not fully mimic all the necessary signals, thus affecting the polarization effect. In other words, current techniques using recombinant IFN-γ are insufficient to fully induce macrophage polarization to M1.

[0006] In addition, in vitro studies have also obtained M1 macrophages through stimulation with LPS combined with IFN-γ. For example, CN113980902A discloses an efficient in vitro culture method for inducing macrophage polarization, including the following steps: Step 1, under serum-free conditions, THP-1 monocytes are stimulated with PMA and GM-CSF to differentiate into macrophages; Step 2, the macrophages differentiated in Step 1 are induced for 24 h with 20 ng / mL IFN-γ and 100 ng / mL LPS to obtain M1 macrophages; Step 3, the macrophages differentiated in Step 1 are induced for 24 h with 20 ng / mL IL-4 and 20 ng / mL IL-10 to obtain M2 macrophages. This method requires first exploring the optimal concentration and induction time, the optimal combination of cytokines for inducing polarization, and the optimal concentration and induction time, making it very complex. 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 cells and progenitor cells (HSPCs); and (c) inducing the HSPCs formed in step b to differentiate into macrophages. This method obtains macrophages by differentiating cells from other sources (monocytes, iPSCs), and its preparation process is complex. The de novo differentiation process may also introduce phenotypic variations, and the preparation process is difficult to control.

[0007] Macrophages exhibit strong heterogeneity and can polarize into different subtypes under different environmental stimuli. Currently, the different roles of different subtypes of macrophages in T cell activation still require further research.

[0008] Furthermore, 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 this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0009] Studies have found that CD3 / CD28-activated T cells can produce IFN-γ, but stimulating macrophages with recombinant IFN-γ alone is insufficient to fully 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 involved in the process of activated lymphocytes inducing M1 macrophage polarization.

[0010] To address the shortcomings of existing technologies, the first aspect of this 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 comprise activated lymphocytes. The lymphocytes are preferably activated mouse lymphocytes.

[0012] Currently, methods using exogenous substances (such as LPS, IFN-γ, etc.) to stimulate and induce M1 macrophage polarization have potential applications in immunomodulation and disease treatment. However, their limitations include: First, besides acting on target cells, they may non-specifically affect other cell types, leading to non-specific immune responses. Specifically, common stimulants include bacterial components (such as lipopolysaccharide), cytokines (such as interferon-γ), and chemicals (such as rheumatoid factor). These substances can activate macrophages and induce M1 polarization, but they often also affect other immune cells. This non-specific stimulation may lead to overactivation of the immune system, causing inflammatory responses and immune-related diseases. Second, methods using exogenous substances to stimulate and induce M1 macrophage polarization have toxic side effects. For example, some cytokines at high concentrations may have cytotoxic effects on cells, leading to abnormal macrophage proliferation or apoptosis; some chemicals may cause organ toxicity or systemic toxicity with long-term use or high doses. Third, most current research relies on stimulation by a single substance, such as a single bacterial component, a single cytokine, or a single chemical substance. However, the immune system in a living organism is a highly complex network regulated by a variety of signaling molecules and cytokines. Therefore, stimulation by a single substance may not fully mimic the real immune environment, resulting in insufficient and inaccurate function and response of M1 macrophages.

[0013] Compared to traditional methods using exogenous stimuli, this invention's method of inducing M1 macrophage polarization through cell co-culture exhibits superior effects. Specifically, co-culturing activated lymphocytes with macrophages stimulates the production of various substances, including IFN-γ. IFN-γ promotes macrophage polarization towards the M1 type, thereby enhancing their inflammatory regulation and antimicrobial activity. Furthermore, this method not only provides IFN-γ but also generates signaling molecules produced by other immune cells, such as IL-1β and TNF-α, which play crucial roles in the immune response. Therefore, inducing M1 macrophage polarization through cell co-culture involves the combined effect of multiple stimuli, better mimicking the in vivo immune environment, resulting in a more comprehensive and accurate macrophage polarization effect. On the other hand, cell co-culture facilitates intercellular interactions and signal transduction. Under activated lymphocyte and macrophage co-culture conditions, macrophages not only receive stimulation from IFN-γ but also directly contact and interact with other immune cells (such as T cells and B cells). These cell-cell interactions promote the exchange and transduction of signaling molecules, thereby enhancing macrophage polarization. Furthermore, cell co-culture provides a more dynamic and precise regulatory environment. By adjusting the proportions of different cell types and adding specific cytokines or signaling molecules, the polarization process of the cells being treated can be precisely controlled. This dynamic regulation better mimics the characteristics of in vivo immune responses, making macrophage polarization more controllable and predictable. In contrast, directly inducing M1 macrophage polarization using stimulants such as IFN-γ is relatively simple and fixed, failing to provide the precision and flexibility of the dynamic regulation of this invention.

[0014] When this invention utilizes activated lymphocytes to induce M1 macrophages for tumor immunotherapy, it exhibits more significant therapeutic effects. The interactions during cell co-culture enhance the recognition ability of M1 macrophages for tumor antigens. During co-culture, M1 macrophages may activate T cells through antigen presentation, thereby establishing stronger immune memory. These M1 macrophages with enhanced immune memory can exert better effects in subsequent treatments. Even if tumor cell surface antigens change, M1 macrophages and their activated T cells can still more quickly and effectively recognize and eliminate tumor cells. Furthermore, the dynamic regulation during co-culture allows M1 macrophages to undergo functional regulation under the influence of specific cytokines, producing responses adapted to the tumor microenvironment, thereby effectively overcoming the suppression of the immune system by the tumor. In other words, the M1 macrophages induced by the method of this invention have stronger microenvironment adaptability. When the microenvironment is poorly adapted, the following may occur: macrophages may be unable to effectively perform their functions, leading to insufficient immune response and ultimately affecting treatment efficacy; macrophage survival rates may be significantly reduced due to their inability to adapt to the new microenvironment, such as undergoing apoptosis due to inhibitory signals in the microenvironment, thus directly affecting the effectiveness 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 tumor-secreted inhibitory factors), resulting in suppressed activity. This not only reduces the anti-tumor response but may also promote tumor growth; macrophages that are not adapted to the microenvironment may also cause systemic side effects. For example, an excessive immune response may lead to tissue damage or autoimmune diseases, thereby causing complications.

[0015] Furthermore, other effective substances produced during cell co-culture (such as chemokines) can bind to certain specific substances or factors in the body, resulting in more targeted therapeutic effects. For example, tumor cells may secrete specific chemokines to attract M1 macrophages, and M1 macrophages induced through co-culture are usually more sensitive to these factors. This specific binding allows 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 comprise activated T lymphocytes. Preferably, the activated lymphocytes also comprise activated B lymphocytes.

[0018] According to a preferred embodiment, the macrophages include macrophage lines RAW264.7, THP-1, U937, bone marrow-induced macrophages (BMDM), peripheral blood-derived monocyte-macrophages (PBMCs), tumor-associated macrophages (TAMs), or primary macrophages derived from mammalian tissue. Mice are preferred as mammals.

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

[0020] According to a preferred embodiment, a method for polarizing macrophages into M1-type macrophages includes the following steps:

[0021] M0 macrophages were obtained; inactive lymphocytes were obtained; the inactive lymphocytes were activated and co-cultured with M0 macrophages to induce macrophage polarization into M1 macrophages.

[0022] According to a preferred embodiment, the steps for obtaining M0 macrophages include: collecting a 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.

[0023] Preferably, the method for separating the cell suspension includes filtration and centrifugation. Those skilled in the art will understand that filtration and centrifugation are conventional techniques for cell separation, and this application does not require restrictions on these operations to complete the basic separation process.

[0024] Preferably, cells are resuspended in DMEM complete medium. More preferably, cells are resuspended in DMEM complete medium containing 5–100 ng / mL M-CSF. More preferably, cells are resuspended in DMEM complete medium containing 10–20 ng / mL M-CSF. Particularly preferably, cells are resuspended in DMEM complete medium containing 10 ng / mL M-CSF.

[0025] Preferably, the cell concentration is adjusted to 1×10⁻⁶. 4 ~1×10 7 / mL. More preferably, the cell concentration is adjusted to 1×10⁹ / mL. 5 ~1×10 7 / mL. Particularly preferably, the cell concentration is adjusted to 2×10⁹ / mL. 6 / mL.

[0026] Preferably, the cell culture conditions are 37°C and 5% CO2.

[0027] According to a preferred embodiment, the steps for obtaining inactive lymphocytes are as follows: extracting lymph nodes from mice; grinding the lymph nodes to obtain a lymphocyte suspension; separating the lymphocyte suspension; resuspending the lymphocytes; counting and adjusting the lymphocyte concentration.

[0028] Preferably, lymphocytes are resuspended in DMEM complete medium.

[0029] Preferably, the range of lymphocyte concentration is adjusted to 1×10⁻⁶. 4 ~1×10 7 / mL. More preferably, the cell concentration is adjusted to 1×10⁹ / mL. 6 ~1×10 7 / mL.

[0030] According to a preferred embodiment, the activation method of inactive lymphocytes involves adding a stimulating molecule to the inactive lymphocytes. The stimulating molecule preferably includes a CD3 antibody and / or a CD28 antibody. The concentration of the CD3 antibody used to activate the inactive lymphocytes is preferably 1-5 μg / mL; the concentration of the CD28 antibody used to activate the inactive lymphocytes is preferably 0.1-5 μg / mL. More preferably, the concentration of the CD3 antibody used to activate the inactive lymphocytes is 1-3 μg / mL; more preferably, the concentration of the CD28 antibody used to activate the inactive lymphocytes is 0.1-3 μg / mL. Particularly preferably, the concentration of the CD3 antibody used to activate the inactive lymphocytes is 1 μg / mL; particularly preferably, the concentration of the CD28 antibody used to activate the inactive lymphocytes is 0.3 μg / mL.

[0031] 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 co-culture time of activated lymphocytes and M0 macrophages is 24 to 72 hours. The co-culture time of activated lymphocytes and M0 macrophages is more preferably 24 to 48 hours. The co-culture time of activated lymphocytes and M0 macrophages is particularly preferably 24 hours.

[0032] According to a preferred embodiment, M1 macrophage polarization is promoted by increasing the expression level of iNOS or decreasing the expression level of cCD206 in macrophages. High levels of iNOS expression enable M1 macrophages to produce higher concentrations of nitric oxide (NO), which helps inhibit the growth of pathogens and tumor cells and is one of the key mechanisms by which M1 macrophages exert cytotoxicity. In disease progression, high iNOS expression in M1 macrophages helps control acute infections and fight tumors. Regulating macrophage polarization and iNOS expression may be a strategy for treating certain diseases, such as infectious diseases, tumors, and autoimmune diseases. In experimental studies, macrophage polarization and function can be assessed by detecting iNOS mRNA and protein levels. The expression level of cCD206 can reflect macrophage polarization; high expression is generally associated with M2 polarization, while low or no expression may be associated with M1 polarization. Regulating cCD206 expression or function may be a potential strategy for treating certain inflammatory diseases, autoimmune diseases, and tumors.

[0033] The second aspect of the present invention provides M1 type macrophages prepared by the method provided in the first aspect of the present invention.

[0034] A third aspect of the present invention provides a composition comprising M1 macrophages as provided in 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 can include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. Therefore, in one aspect, the present invention provides compositions 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, including injection or infusion. Suitable formulations may comprise lymphocyte-activated macrophages in a sterile or isotonic medium (e.g., water for injection). The drug and pharmaceutical composition can be formulated into a fluid form, including a gel form. Fluid formulations can be formulated for administration to selected areas of a human or animal body via injection or infusion (e.g., via a catheter). Compositions containing lymphocyte-activated macrophages are formulated for intratumoral or intravenous administration, for example, for macrophage-targeted cancer immunotherapy.

[0035] According to a preferred embodiment, the preparation method of the composition involved in this application may include one or more steps selected from the following: isolating / purifying 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.

[0036] A fourth aspect of the present invention provides IFN-γ activated macrophages.

[0037] A fifth aspect of the present invention provides a composition comprising IFN-γ activated macrophages as provided in the fourth aspect of the present invention.

[0038] A sixth aspect of the present invention provides a method for selectively polarizing macrophages, the method comprising: treating isolated M0-type macrophages with activated lymphocytes. The present invention also provides a 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.

[0039] The seventh aspect of the present invention provides a method for preparing M2 macrophages, the method comprising treating isolated M0 macrophages with IFN-γ.

[0040] The eighth aspect of the present invention provides a method for treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of M1 macrophages provided in the second aspect of the present invention or a composition provided in the third aspect of the present invention.

[0041] Preferably, the subject has cancer, pulmonary fibrosis, liver fibrosis, liver injury, or cardiac fibrosis. More preferably, the lymphocyte-activated macrophages and the pharmaceutical composition containing lymphocyte-activated macrophages are capable of treating or preventing cancer, such as inhibiting cancer development / progression, delaying / preventing cancer onset, 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).

[0042] A ninth aspect of the present invention provides yet another method of treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of IFN-γ-activated macrophages provided in a fourth aspect of the present invention or a composition provided in a fifth aspect of the present invention. Preferably, the subject is a mammal. Preferably, the subject is a human.

[0043] The tenth aspect of the present invention provides the use of the method provided in the first aspect of the present invention, the M1 type macrophage provided in the second aspect of the present invention, the composition provided in the third aspect of the present invention, the IFN-γ activated macrophage 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 medicaments or products for macrophage-targeted cancer immunotherapy.

[0044] Technical Effects: This invention first uses CD3 / CD28 stimulation to activate lymphocytes. After naive T cells are activated by antigen stimulation, they gradually overexpress CD44 and lose CD62L, differentiating into effector cells. The results of this invention show that lymphocytes stimulated by CD3 / CD28 show increased CD44 expression and decreased CD62L expression. The method used in this invention to activate lymphocytes is successful and reliable. This invention provides a novel in vitro M1 macrophage model for research. These M1 macrophages induced by activated lymphocytes can better mimic the in vivo immune response process.

[0045] Existing technologies primarily focus on processing single cell types, such as relying solely on IFN-γ stimulation. This strategy emphasizes the role of exogenous factors, while research on intercellular interactions and synergistic effects is relatively limited. Cellular signal transduction mechanisms are complex, especially during macrophage polarization. Existing techniques are typically based on specific experimental designs, and co-culturing protocols after lymphocyte activation require significant adjustments to the experimental design. Therefore, existing technologies do not readily consider using cell co-culture to promote the generation of M1 macrophages. Compared to existing methods of directly stimulating macrophages with IFN-γ or differentiating M1 macrophages from cells of other sources, this invention utilizes activated lymphocytes to stimulate macrophages to change their polarization state, reducing operational steps and making the stimulation process easier to control and repeat. Existing macrophages possess 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 (e.g., existing techniques using monocytes or iPSCs to differentiate into macrophages may result in variations in differentiation efficiency and direction). M1 macrophages obtained through direct stimulation are easier to mass-produce and control in terms of quality.

[0046] Furthermore, while LPS and IFN-γ may activate multiple cell types (non-specifically), this invention utilizes activated lymphocytes to stimulate macrophages to polarize into M1-type macrophages. This process more closely resembles the immune response under physiological conditions (providing a similarity to naturally occurring cell-cell interactions in vivo). Activated lymphocytes can also act more specifically on macrophages through specific cell-cell contacts and signal transduction, more comprehensively mimicking the in vivo immune regulatory network. By controlling the activation state and number of lymphocytes, this invention can more precisely regulate the degree of macrophage polarization (with better experimental controllability). In terms of cost, this invention reduces costs compared to commercially available recombinant cytokines, especially when the required lymphocytes can be obtained from other parts of the experiment. Attached Figure Description

[0047] Figure 1 A schematic diagram illustrating the validation of CD3 / CD28 activated lymphocytes;

[0048] Figure 2 A schematic diagram illustrating the morphological differences of macrophages stimulated by different lymphocytes;

[0049] Figure 3 This is a schematic diagram illustrating the changes in macrophage subtypes stimulated by different lymphocytes. Detailed Implementation

[0050] The following is a detailed explanation with reference to the accompanying drawings.

[0051] The following examples are only used to further explain the beneficial effects of polarized macrophages in this application and should not be used to limit the scope of protection of this application. Those skilled in the art can understand the specific meaning of the terms in this invention according to the specific circumstances. Unless otherwise specified, the experimental procedures described in the following examples are conventional procedures. Unless otherwise specified, the reagents used in the following examples are commercially available.

[0052] The experimental animals used in this invention were male C57BL / 6N(B6) mice, aged 8-12 weeks, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in a clean-grade standard environment. The experiments conducted in this invention were approved by the Laboratory Animal Welfare and Ethics Committee of Xuanwu Hospital, Capital Medical University (Approval No.: XW-20210531-1).

[0053] 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-amphotericidal B mixed solution (100×) (P7630, Beijing Solarbio Biotechnology Co., Ltd.); D-Hanks (calcium- and magnesium-free, phenol red-free) (H1045, Beijing Solarbio Biotechnology Co., Ltd.); Bovine serum albumin (BSA) (A7906, Sigma-Aldrich, USA); 4% tissue fixative (P1110, Beijing Solarbio Biotechnology Co., Ltd.); Wright-Giemsa staining solution-A (BA4017A, Zhuhai Beso Cell Science Technology Co., Ltd.); Wright-Giemsa staining solution-B (BA4017B, Zhuhai Beso Cell Science Technology Co., Ltd.); iNOS Rabbit pAb (A14031, Wuhan Aibotek Biotechnology Co., Ltd.); Arginase 1 (ARG1) Rabbit mAb (A4923, Wuhan Aibote Biotechnology Co., Ltd.); BD™ BD IntraSure™ Kit (641776, BD Company, USA).

[0054] Main instruments and equipment:

[0055] Flow cytometer BD FACS (BD Systems, Inc., USA); AriaTMII; Low-temperature centrifuge (Beijing Baiyang Medical Instrument Co., Ltd.); Carbon dioxide incubator (Thermo Fisher Scientific, USA); Optical microscope (Leica GmbH, Germany); 4℃ freezer (Sanyo Corporation, Japan); -80℃ freezer (Thermo Fisher Scientific, USA); Cell counter (Bio-rad Corporation, USA); TC20 cell counting plate (Bio-rad Corporation, USA); Cell culture plate (6-well TC treated) (Wuxi Nice Life Science & Technology Co., Ltd.); Cell slides (Shanghai Jing'an Biotechnology Co., Ltd.); Glass slides (Leica GmbH, Germany).

[0056] Solution preparation:

[0057] Preparation of DMEM complete medium: Add 20% FBS and 1% penicillin-streptomycin-amphotericidal solution to DMEM basal medium. Perform the entire process in a sterile operating table and store at 4°C for a short period.

[0058] Preparation of flow cytometry fixative: Dilute 4% tissue cell fixative with D-hanks buffer to 1% tissue cell fixative.

[0059] This 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 was used to analyze the significance of the two groups. P < 0.05 was considered statistically significant, and ns represented no significant difference. P values ​​are expressed in standard abbreviation format, i.e. ; ; ; .

[0060] The present invention particularly provides a method for preparing mouse M1 macrophages, the specific method of which is described in the following examples. Example 1

[0061] This embodiment provides relevant experimental methods, including extraction and culture of primary mouse bone marrow-derived macrophages (BMDM), preparation of mouse lymphocytes, co-culture of macrophages with lymphocytes in different activation states, and flow cytometry.

[0062] BMDM Extraction and Cultivation:

[0063] On day 0, B6 mice were euthanized by cervical vertebrae dislocation. The euthanized mice were sterilized by immersion in 75% alcohol for 10 min, then removed, their limbs fixed, and the hind limbs dissected and placed in pre-cooled D-Hanks buffer. The mice were then transferred to a clean bench for further processing. Excess muscle was removed from the hind limbs, and the complete tibia and femur were freed. The ends of the long bones were cut off, and the bone marrow cavity was repeatedly flushed with D-Hanks buffer using a 2 mL disposable sterile syringe. The cell suspension was transferred to a 15 mL centrifuge tube, filtered through a 70 μm cell filter, 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 cells / mL. 6 / mL, seed cells into 6-well plates, 2mL of cell suspension per well, i.e., 4×10 6 / well. Incubate in a 37°C, 5% CO2 cell culture incubator, and closely observe cell status daily under a phase-contrast microscope.

[0064] On day 1, change the medium to remove non-adherent cells; the adherent cells are BMDM. Discard all the old medium on top, rinse three times with D-Hanks buffer, and replenish with fresh DMEM complete medium containing 10 ng / mL M-CSF. Then, change the medium every three days with fresh DMEM complete medium containing 10 ng / mL M-CSF.

[0065] Preparation of mouse lymphocytes:

[0066] Lymph nodes were extracted from the groin, mesentery, axilla, and neck of B6 mice under aseptic conditions and placed in 1.5 mL centrifuge tubes containing pre-chilled D-Hanks buffer. Lymphocytes were extracted from the lymph nodes using a 1 mL sterile syringe core on a sterile plastic petri dish under a laminar flow hood. After filtering through a 70 μm cell filter, the cells were 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 culture medium for counting.

[0067] Co-culture of macrophages with lymphocytes in different activation states:

[0068] On day 3 of BMDM culture, different stimuli were added to the culture system of unactivated or unpolarized macrophages (M0 macrophages), specifically divided into the following four groups: 1) CD3 / CD28 control group: CD3 (final concentration 1 μg / mL) and CD28 (final concentration 0.3 μg / mL) were added; 2) Inactive lymphocyte group: Inactive B6 mouse lymphocytes were added and co-cultured at a macrophage:lymphocyte ratio of 1:10; 3) Activated lymphocyte group: Activated B6 mouse lymphocytes with CD3 (final concentration 1 μg / mL) and CD28 (final concentration 0.3 μg / mL) were added and cultured at a macrophage:lymphocyte ratio of 1:10; 4) IFN-γ group: IFN-γ (final concentration 100 ng / mL) was added.

[0069] After co-culturing for 24 h, suspension lymphocytes were isolated and macrophages were collected. Subsequently, flow cytometry was used to quantitatively analyze the immunophenotypic changes of macrophages; at the same time, Wright-Giemsa staining was used to observe the morphology of macrophages to assess their polarization status.

[0070] Flow cytometry detection of macrophage subtypes:

[0071] Discard the old culture medium for macrophages, wash three times with D-hanks buffer, add 1 mL of pre-cooled D-hanks buffer to each well, pipette the macrophages off, collect the macrophage suspension in a flow cytometer, centrifuge at 400 g for 5 min at 4°C, discard the supernatant, and resuspend the cells in 100 μL of D-hanks buffer.

[0072] Add anti-CD16 / 32 antibody to the cell suspension and incubate at 4°C in the dark for 15 min to block Fc receptors. Then add cell surface antibodies anti-CD11b-FITC and F4 / 80-PE / Dazzle™ 594 antibody, vortex to mix, and incubate at 4°C in the dark for 30 min. Add 100 μL of fixation / permeabilization reagent A and incubate at 4°C in the dark for 5 min to fix the cells. Resuspend the cells in 2 mL of D-hanks buffer, centrifuge at 400 g for 5 min at 4°C, and discard the supernatant. Add 50 μL of permeabilization reagent B and intracellular antibodies anti-iNOS-PE and anti-CD206-PE / Cyanine7 antibody, vortex to mix, and incubate at 4°C in the dark for 30 min. Wash the cells twice with D-hanks buffer, centrifuge at 400 g for 5 min at 4°C, and discard the supernatant. Resuspend the cells in 0.5 mL of D-hanks buffer and analyze bone marrow macrophage subtypes using flow cytometry.

[0073] Flow cytometry for lymphocyte phenotype detection:

[0074] Collect lymphocytes suspended in the supernatant, centrifuge at 400 g for 5 min at 4°C, discard the old culture medium, wash once with pre-chilled D-Hanks buffer, centrifuge at 400 g for 5 min at 4°C, discard the supernatant, resuspend the cells in 100 μL of D-Hanks buffer, add anti-CD4-PE / Dazzle™ 594, CD8-PE / Cyanine7, CD44-FITC, and CD62L-PE antibodies, vortex to mix, and 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, resuspend the cells in 0.5 mL of D-Hanks buffer; add 7AAD, incubate at 4°C in the dark for 5–10 min, and finally analyze the lymphocyte functional status using flow cytometry. If the sample is to be tested overnight, resuspend the cells in 1% tissue fixative and store at 4°C in the dark until analysis.

[0075] Wright-Giemsa staining:

[0076] Macrophages to be tested were cultured on cell slides, and the slides were completely covered with 4% tissue cell fixative. After standing at room temperature for 20 min, the cell slides were removed and fixed onto a glass slide. The 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 2-3 times that of solution A). The two solutions A and B were mixed by blowing with a syringe, and the staining was allowed to stand at room temperature for 7-9 min. The slides were then rinsed with running water and allowed to air dry. The morphological changes of macrophages were observed under a light microscope. Example 2

[0077] This embodiment further explores the effect of activated lymphocytes on macrophage polarization. Primary lymphocytes were extracted from the lymph nodes of B6 mice for testing. Activated lymphocytes were obtained by stimulating lymphocytes with a combination of CD3 and CD28 monoclonal antibodies (CD3 / CD28). The blank control group consisted of lymphocytes activated without CD3 / CD28. Both groups of lymphocytes were co-cultured with BMDM. After 24 h of culture, the expression of CD4, CD8, CD44, and CD62L in the blank control group and the two groups of lymphocytes activated by CD3 / CD28 was detected by flow cytometry to verify whether the CD3 / CD28-stimulated lymphocytes were successfully activated.

[0078] 1. Identification of CD3 / CD28 activated lymphocytes

[0079] Figure 1 This is a schematic diagram illustrating the validation of CD3 / CD28 activated lymphocytes, in which... Figure 1 (A) Flow cytometry detection of CD4+ in blank control lymphocytes and CD3 / CD28 activated lymphocytes. + and CD8 + Expression results of CD44 and CD62L in lymphocytes; Figure 1 (B) consists of two groups of CD4. + and CD8 + Statistical graph of average fluorescence intensity of CD44 and CD62L in lymphocytes.

[0080] CD4 in the blank control group + and CD8 + Lymphocytes are mainly CD44 - CD62L + Naïve T cells, and CD3 / CD28 activated CD4 cells + and CD8 + In addition to quiescent 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). Changes in the expression of CD44 and CD62L indicate that activation treatment causes changes in the expression levels of these markers, and activation treatment alters T cell subsets and the activated state of cells.

[0081] Mean fluorescence intensity (MFI) is a commonly used indicator to measure the expression level of cell surface markers; a higher value indicates a higher expression level of the marker. Analysis 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, indicating that CD3 / CD28 activation treatment promoted CD44 expression. + and CD8 + The expression level of CD44 in lymphocytes was significantly lower than that in the blank control group; CD3 / CD28 activated lymphocytes showed significantly lower CD62L expression levels, indicating that CD3 / CD28 activation treatment inhibited CD44 expression. + and CD8 + CD62L expression in lymphocytes ( Figure 1 B). In CD3 / CD28-activated lymphocytes, CD62L is decreased while CD44 is increased, which may indicate that T cells are shifting from a central memory phenotype to an effector memory phenotype. The altered expression of surface markers on activated lymphocytes indicates that CD3 / CD28 stimulation of lymphocyte activation was successful.

[0082] Lymphocytes and macrophages were co-cultured and divided into a blank control group and an activated lymphocyte group. The blank control group received no 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.

[0083] 2. Effects of activated lymphocytes on macrophage morphology

[0084] Different subtypes of macrophages exhibit diverse morphologies. To investigate the effect of activated lymphocytes on macrophage morphological changes, this invention cultured BMDM cells 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 (with lymphocyte stimulation as the unactivated lymphocyte group); CD3 / CD28 activated lymphocytes (with CD3 / CD28 and lymphocytes as the activated lymphocyte group); and IFN-γ (IFN-γ group). After 24 h of culture, slides were prepared, Wright-Giemsa staining was performed, and the morphological differences of the four groups of macrophages were observed under a light microscope. The results are as follows: Figure 2 As shown. Figure 2 It is a magnification of 1000.

[0085] Significantly, the macrophage morphology showed little difference among the CD3 / CD28 control group, the unactivated lymphocyte group, and the IFN-γ group, all exhibiting elongated spindle-shaped cells with synapses (pseudopodia). However, the macrophage morphology in the activated lymphocyte group underwent a significant change, specifically characterized by increased cell size, more abundant cytoplasm, and a round, "fried egg" shape. Figure 2 ).

[0086] When macrophages become round with abundant cytoplasm, it may indicate a transition to an M1 polarization state, which is associated with pro-inflammatory and bactericidal processes. This round morphology may suggest enhanced phagocytic function, crucial for clearing cellular debris and pathogens, particularly in M1-polarized macrophages. These morphological changes may also affect their antigen-presenting capacity; 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.

[0087] Round macrophages with abundant cytoplasm exhibit enhanced phagocytic capacity. This morphology of macrophages possesses more endoplasmic reticulum and Golgi apparatus, which facilitates their synthesis and secretion of necessary phagocytic and digestive molecules. The round shape may increase the surface area of ​​the cell membrane, providing more contact and phagocytic zones, allowing macrophages to more effectively encapsulate and endocytose pathogens or cellular debris. Cytoplasm-rich macrophages play a crucial role in clearing apoptotic cells and pathogens, an important mechanism for maintaining tissue homeostasis and preventing the spread of infection. This morphology of macrophages may recognize and bind to various ligands, such as bacterial cell wall components and phosphatidylserine from apoptotic cells, via "scavenger receptors." M2-type macrophages may express lower levels of MHC-II and co-stimulatory molecules, which helps them regulate T cell responses, reduce excessive immune activation, and may promote immune tolerance. In this embodiment, macrophages in the unactivated lymphocyte group and the IFN-γ group exhibited an elongated spindle shape with synapses (pseudopodia), while macrophages in the activated lymphocyte group showed increased cell volume, abundant cytoplasm, and a round, "fried egg" shape. The elongated spindle-shaped morphology may affect the contact area between macrophages and their surrounding environment. Synapse formation may inhibit major macrophage functions, such as phagocytosis. Furthermore, the signal transduction capabilities of elongated spindle-shaped macrophages, the influence of their microenvironment, and their migration and localization abilities may affect macrophage function. In comparison, macrophages in the activated lymphocyte group exhibited higher phagocytic efficiency, stronger responses to cellular signals, greater migration ability, and better phagocytic function.

[0088] M2 macrophages may exert an immunosuppressive effect in chronic infections or tumor microenvironments by suppressing T cell proliferation and activation through the secretion of anti-inflammatory factors such as IL-10. Morphological changes in 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.

[0089] 3. Effects of activated lymphocytes on macrophage subtypes

[0090] To investigate 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. Macrophage subtypes were detected by flow cytometry. iNOS was a marker for M1 macrophages, and CD206 was a marker for M2 macrophages.

[0091] In this embodiment, the streaming gate strategy is: set CD11b. + F4 / 80 + Macrophage populations were collected, and macrophage subtypes were detected by iNOS and cCD206. After co-culturing for 24 h, macrophages from four groups—CD3 / CD28 control group, unactivated lymphocyte group, activated lymphocyte group, and IFN-γ group—were collected, and the expression of CD11b, F4 / 80, iNOS, and cCD206 was detected by flow cytometry.

[0092] Figure 3 This is a schematic diagram illustrating the changes in macrophage subtypes stimulated by different lymphocytes. Figure 3 (A) Statistical results of flow cytometry analysis of macrophage subtypes in four different treatments, specifically including four CD11b groups. + F4 / 80 + Expression of iNOS and cCD206 in macrophages and iNOS - cCD206 - M0 macrophages and iNOS + cCD206 - Statistical chart of the proportion of M1 macrophages; Figure 3 (B) The expression of iNOS in macrophages under four different treatments (represented by the mean fluorescence intensity (MFI) of iNOS). Figure 3 (C) shows the expression of cCD206 in macrophages under four 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.

[0093] Figure 3 In the middle, CD11b + F4 / 80 + In macrophages, the CD3 / CD28 control group, the unactivated lymphocyte group, and the IFN-γ group showed similar performance, mainly iNOS.- cCD206 - Cellular activity showed no significant difference. Compared with the CD3 / CD28 control group, the unactivated lymphocyte group, and the IFN-γ group, the activated lymphocyte group showed higher levels of iNOS in macrophages. - cCD206 - Cells were significantly reduced, and iNOS + cCD206 - The significant increase in cells (high expression of iNOS and low expression of cCD206) indicates that the macrophages in the activated lymphocyte group have polarized from M0 type (unpolarized or nascent state) macrophages to M1 type macrophages. Figure 3 A). Among the four groups of macrophages, the iNOS expression level in macrophages from the activated lymphocyte group was significantly higher than that in the other groups. Figure 3 B), and compared with the CD3 / CD28 control group and the unactivated lymphocyte group, the expression level of cCD206 in macrophages of the activated lymphocyte group and the IFN-γ group was significantly decreased, with the lowest cCD206 expression level in the activated lymphocyte group. Figure 3 C). Therefore, this invention can effectively promote the polarization of M1 macrophages through activated lymphocytes.

[0094] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention. Equivalent substitutions of the raw materials used in this invention, the addition of auxiliary components, and the selection of specific methods are all within the scope of this invention and fall within its protection scope. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; phrases such as "preferredly" or "according to a preferred embodiment" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept.

Claims

1. A method for preparing M1 type macrophages, characterized in that, The method includes: Primary mouse bone marrow-derived macrophages were cultured for 3 days in DMEM complete medium containing M-CSF. On the third day of BMDM culture, lymphocytes were added to the culture system of unactivated or unpolarized macrophages to obtain M1 macrophages through co-culture. Lymphocytes include lymphocytes activated by stimulation with CD3 monoclonal antibody in combination with CD28 monoclonal antibody, with a ratio of macrophages to activated lymphocytes of 1:

10.

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

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

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