Dendritic cell body and in-vitro induction method and application thereof

Abstract

The invention relates to the technical field of dendritic cell in-vitro induction methods, and provides a dendritic cell body and an in-vitro induction method and application thereof. Thymic stromal lymphopoietin; the invention relates to a recombinant human FLT3L-Fc fusion protein. And maturing stimulation combination. On the premise of avoiding complex use of equipment, the bottlenecks of DC yield, subgroup purity and functionality are broken through.

Description

Technical Field

[0001] This invention relates to the technical field of in vitro dendritic cell induction methods, specifically, to an in vitro dendritic cell induction method. Background Technology

[0002] DC-CIK combination therapy is a relatively mature approach to tumor biotherapy. DC cells are professional antigen-presenting cells in the body and are the central link in initiating, regulating and maintaining the immune response. They have important application value in immune regulation, immune surveillance and tumor immunotherapy.

[0003] The existing standard protocol (GM-CSF+IL-4) induces a limited number of DCs (<10-fold amplification), and the proportion of the cDC1 subset is less than 10%; the mature protocol (TNF-α+IL-1β+PGE2) promotes migration, but induces a tolerance phenotype (insufficient IL-12 secretion); the application of epigenetic regulation in DC differentiation has not yet been systematically developed. Summary of the Invention

[0004] One of the objectives of this invention is to propose an in vitro method for inducing dendritic cells, which overcomes the bottlenecks in DC yield, subpopulation purity, and functionality while avoiding the use of complex equipment.

[0005] The technical solution of the present invention is as follows:

[0006] A method for in vitro induction of dendritic cells, using the following composition for induction:

[0007] EZH2 inhibitors;

[0008] Thymic stromal lymphopoietin (TSLP);

[0009] Recombinant human FLT3L-Fc fusion protein;

[0010] Mature stimulation combination.

[0011] Furthermore, the maturation stimulation combination includes CD40L, a TLR3 agonist, and a TLR7 / 8 agonist.

[0012] Furthermore, the EZH2 inhibitor is selected from GSK126 or tazetine, at a concentration of 0.5-2 μm.

[0013] Furthermore, the concentration of the thymic stromal lymphopoietin is 20-50 ng / mL;

[0014] The concentration of the recombinant human FLT3L-Fc fusion protein is 100-200 ng / mL.

[0015] Further steps include the following:

[0016] S100: Culture human monocytes or stem cells for 0-6 days in a medium containing recombinant human FLT3L-Fc fusion protein, IL-4, GM-CSF and EZH2 inhibitor;

[0017] S200: Add thymic stromal lymphopoietin during the late differentiation stage and continue culturing;

[0018] S300: Remove differentiation factors and stimulate cells with a maturation stimulation combination for 24-48 hours;

[0019] S400: Obtain dendritic cells.

[0020] Furthermore, the EZH2 inhibitor in step S100 is added only within the first 72 hours of differentiation.

[0021] This invention provides dendritic cells obtained by the above-described in vitro induction method.

[0022] This invention also provides the application of dendritic cells in the preparation of tumor prevention products.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention utilizes an EZH2 inhibitor to relieve H3K27me3 inhibition in the early stages of DC differentiation, and to synergistically replace problematic PGE2 with TLR3 / 7 / 8+CD40L three-signaling. The entire process involves 2D culture, requiring no special equipment, and uses commercially available recombinant proteins and small molecules, thus improving ease of implementation. It overcomes the bottlenecks in DC yield, subpopulation purity, and functionality while avoiding complex equipment usage. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] This embodiment provides a method for in vitro induction of dendritic cells, which involves induction using the following core composition:

[0028] EZH2 Inhibitor: As an epigenetic regulator, the EZH2 inhibitor (histone methyltransferase inhibitor) can be selected from GSK126 or tazesta. Since genes such as CD80, CCR7, and IL12B are crucial for the functional maturation and migration of dendritic cells (DCs), this embodiment uses the EZH2 inhibitor to reduce the H3K27me3 level, thereby relieving the inhibition of the above genes.

[0029] EZH2 inhibitors, at concentrations of 0.5-2 μm, can reduce H3K27me3 levels, relieve the inhibition of immune genes (CD80 / CCR7 / IL12B), and enhance DC migration and IL-12 secretion.

[0030] Thymic stromal lymphopoietin (TSLP): As a tissue microenvironment factor, its concentration is 20-50 ng / mL. By limiting the concentration range of TSLP, STAT5 activation and CCR7 expression can be maximized, and epigenetic reprogramming can be synergistically achieved to achieve high-functioning DC maturation.

[0031] Recombinant human FLT3L-Fc fusion protein: As a long-acting differentiation factor, at a concentration of 100-200 ng / mL, it can directionally induce the differentiation of cDC1 subsets (purity >70%), and enhance the antigen presentation ability and migration receptor expression of dendritic cells.

[0032] Mature stimuli combinations include:

[0033] 1) CD40L trimer, concentration of 500-2000 ng / mL;

[0034] 2) TLR3 agonist Poly(I:C), concentration 10-100 μg / mL;

[0035] 3) TLR7 / 8 agonist R848, at a concentration of 0.5-10 μg / mL.

[0036] Induction using the above combination of materials includes the following steps:

[0037] S100: Precursor amplification stage, cycle 0-6 days;

[0038] Human monocytes or stem cells were cultured for 0-6 days in a medium containing recombinant human FLT3L-Fc fusion protein, IL-4, GM-CSF and EZH2 inhibitor;

[0039] The culture environment was maintained at 37°C and contained 5% CO.

[0040] On the third day, half the medium was replaced to replenish the factors, providing the nutrients and factors needed for cell growth.

[0041] S200: Tissue-specific induction phase, lasting 3-6 days;

[0042] Thymic stromal lymphopoietin (TSLP) was added during the later stages of differentiation, and the culture was continued.

[0043] S300: Maturation and activation stage, cycle 6-8 days;

[0044] Remove differentiation factors and stimulate cells with a maturation stimulation combination (CD40L trimer, Poly(I:C), R848) for 24-48 hours;

[0045] S400: Obtain dendritic cells.

[0046] The EZH2 inhibitor in step S100 is added only within the first 72 hours of differentiation.

[0047] Experiment 1: DC Yield and Phenotypic Analysis

[0048] Control group: GM-CSF + IL-4 + TNF-α + IL-1β + PGE2;

[0049] This embodiment group: ERT-DC scheme;

[0050]

[0051] As can be seen from the table above, this embodiment achieves a comprehensive improvement in DC yield, maturity, and migration ability by precisely regulating the core composition (cytokine combination), maturation induction timing, and metabolic microenvironment.

[0052] Specifically, increased cell production can reduce costs and time, making it suitable for high-dose infusions or multiple reinfusions;

[0053] An increased proportion of mature CD83+ DCs can enhance immune activation and reduce the risk of immune tolerance.

[0054] Enhanced CCR7+ migration ability allows for precise targeting of lymph nodes and improved vaccine efficacy.

[0055] Experiment 2: ERT-DC induction (compared to the standard GM-CSF / IL-4 protocol)

[0056] Cell source: CD14+ monocytes from peripheral blood of healthy donors;

[0057] Factor combinations:

[0058] Amplification phase: FLT3L-Fc 100ng / mL + IL-4500U / mL + GM-CSF 800U / mL + GSK 1261μM;

[0059] Differentiation phase: TSLP 30 ng / mL (added on day 3);

[0060] Maturation stage: CD40L trimer 1000ng / mL + Poly(I:C) 30μg / mL + R8482μg / mL.

[0061] The results of DC culture detection in this embodiment are as follows:

[0062] index Traditional solution ERT-DC solution Increase Cell yield (expansion fold) 6.2 times 22.1 times 256％ cCD1 ratio 7.3％ 61.5％ 742％ IL-12p70 secretion 150pg / ml 950 pg / mL 533％ Lymph node migration rate 25％ 78％ 212％

[0063] IL-12p70 secretion capacity and lymph node migration rate are key to enhancing anti-tumor immune responses. As shown in the table above, this embodiment can reduce production costs and improve treatment stability by increasing cell yield (expansion fold), making it more suitable for large-scale clinical applications.

[0064] An increase in the cCD1 ratio can enhance anti-tumor immune responses, improve the formation of immune memory, and overcome the immunosuppressive microenvironment.

[0065] Increased IL-12p70 secretion can promote Th1 immune polarization, enhance CTL activity, and break immune tolerance.

[0066] Increased lymph node migration rate can improve antigen presentation efficiency, reduce peripheral immune tolerance, and enhance vaccine efficacy.

[0067] Example 2

[0068] Based on Example 1, this example achieves a comprehensive improvement in the yield, maturity, and migration ability of dendritic cells obtained through the above-described in vitro induction method.

[0069] Example 3

[0070] Based on Example 2, this example applies dendritic cells in the preparation of tumor prevention products to enhance the therapeutic effect.

[0071] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for in vitro induction of dendritic cells, characterized in that, Induction was performed using the following composition: EZH2 inhibitors; Thymic stromal lymphopoietin (TSLP); Recombinant human FLT3L-Fc fusion protein; Mature stimulation combination.

2. The method for in vitro induction of dendritic cells according to claim 1, characterized in that, The maturation stimulation combination includes CD40L, TLR3 agonists, and TLR7 / 8 agonists.

3. The method for in vitro induction of dendritic cells according to claim 1, characterized in that, The EZH2 inhibitor is selected from GSK126 or tazestat, with a concentration of 0.5-2 μm.

4. The method for in vitro induction of dendritic cells according to claim 1, characterized in that, The concentration of lymphopoietin in the thymic stroma is 20-50 ng / mL; The concentration of the recombinant human FLT3L-Fc fusion protein is 100-200 ng / mL.

5. The method for in vitro induction of dendritic cells according to claim 1, 2, 3, or 4, characterized in that, Includes the following steps: S100: Culture human monocytes or stem cells for 0-6 days in a medium containing recombinant human FLT3L-Fc fusion protein, IL-4, GM-CSF and EZH2 inhibitor; S200: Add thymic stromal lymphopoietin during the late differentiation stage and continue culturing; S300: Remove differentiation factors and stimulate cells with a maturation stimulation combination for 24-48 hours; S400: Obtain dendritic cells.

6. The method for in vitro induction of dendritic cells according to claim 5, characterized in that, The EZH2 inhibitor in step S100 is added only within the first 72 hours of differentiation.

7. Dendritic cells obtained by the in vitro induction method according to any one of claims 1-6.

8. The use of dendritic cells as described in claim 7 in the preparation of tumor prevention products.

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