CN115044553A

CN115044553A - mTOR-targeted tolerant dendritic cell and preparation method and application thereof

Info

Publication number: CN115044553A
Application number: CN202210978763.8A
Authority: CN
Inventors: 周林; 贺强; 李先亮
Original assignee: Beijing Chaoyang Hospital
Current assignee: Beijing Chaoyang Hospital
Priority date: 2022-08-16
Filing date: 2022-08-16
Publication date: 2022-09-13
Anticipated expiration: 2042-08-16
Also published as: CN115044553B

Abstract

The invention provides a mTOR-targeted tolerogenic dendritic cell and a preparation method and application thereof. The mTOR-targeted tolerogenic dendritic cell has a transcription profile of Siglec1, Spp1 and other genes which are down-regulated, PI3K/mTOR expression is absent, and a selective negative regulation effect is achieved. The mTOR-targeted tolerogenic dendritic cells are obtained by stimulating mesenchymal stem cells by using a scheme of combining low-dose rapamycin and low-concentration GM-CSF (GM-CSF), have stable negative regulation functions, molecular characteristics and special transcription profiles, and can be used for preparing various immunoregulatory cells.

Description

mTOR-targeted tolerant dendritic cell and preparation method and application thereof

Technical Field

The invention relates to a tolerogenic dendritic cell and a preparation method and application thereof, in particular to a tolerogenic dendritic cell (Rapa-tolDC) targeting mTOR and a preparation method and application thereof, belonging to the technical field of organ transplantation immune cells and application thereof.

Background

Organ transplantation is the best treatment for end-stage organ disease, but graft rejection is the major cause of the impact on the quality of life and long-term survival of recipients. The continued development of immunosuppressive agents in the last 30 years has significantly improved the short-term survival benefit of transplant recipients, but the long-term survival rate has not changed significantly. The current immunosuppressive schemes commonly used in clinic also have a plurality of defects, and chronic graft failure, metabolic diseases, graft injuries, opportunistic infections and malignant tumors caused by long-term immunosuppression seriously threaten the long-term survival of patients, and the immunosuppressive agents are required to be taken for a lifetime.

Induction of immune tolerance is the most ideal treatment option for controlling rejection and maintaining long-term survival of recipients without immune control. Successful establishment of immune tolerance can not only solve complications and side effects caused by long-term application of immunosuppressive agents, but also maintain long-term survival of transplants, and has significant medical and economic benefits. However, there is no operational transplantation immune tolerance scheme applicable to clinical application, and a novel technical scheme for safely and effectively inducing organ transplantation immune tolerance is developed, so that a receptor is promoted to establish an immune tolerance state specific to a donor, and the immune tolerance scheme is successfully applied to human beings in a transformation way, and the method has important research value, and huge clinical application prospect and social and economic benefits.

Regulatory dendritic cells (toldcs) have a negative immune regulation function, and play a role in inducing immune tolerance by inhibiting T cell proliferation, inhibiting antigen-specific T cell activation, mediating T cell apoptosis, inducing regulatory T cells, and the like. Induction of immune tolerance using adoptive reinfusion therapy of tolDC immune cells is the hot spot of current research. The small dose of immunosuppressant can improve the survival of transplanted liver, kidney and heart by combining or independently returning tolDC; the PBMC derived tolDC IS safely and effectively induced by single reinfusion of a living liver transplantation patient by using IL-10, vitamin D3 and cytokines, but IS withdrawal IS not solved, and the end of immune tolerance IS reached. Reinfusion of autologous or syngeneic toldcs, whether or not loaded with donor antigens, can improve graft survival. These all suggest that adoptive infusion of toldcs is expected to be an ideal solution for immune tolerance. However, there are few reports related to toldcs that can be used for clinical therapy, have strong inhibition and are functionally stable.

Induction of toldcs with donor-specific immunosuppression is a primary problem facing current clinical studies. Among various currently and clinically used immunosuppressive agents, Rapa can induce anti-mature tolDC with tolerance, promote differentiation, proliferation and suppressivity of Tregs, has no effect on CsA, and has inhibitory effect on hormone and MPA; rapamycin (Rapa) withdrawal has the advantage of promoting immune tolerance in long-lived liver transplants, although the sample size is small, suggesting that Rapa may have a unique advantage in inducing immune tolerance; rapa can induce the anti-maturation tolDC with stable function at the same time, promote the differentiation of Treg, but the CsA which is commonly used in clinic has no effect, and the hormone and MPA have inhibitory effect. However, no clinical studies on Rapa-induced toldcs have been reported, and studies in animal liver transplantation are rare.

How to select appropriate drugs to prepare toldcs with stable tolerability, how to determine the dose, time and frequency of cell therapy to establish a standardized treatment regimen; migration and colonization rules in vivo after tolDC treatment, whether induced immune tolerance has donor specificity, whether a proper immunosuppression scheme needs to be combined, immune memory overcoming and the like are important problems faced by tolDC immunotherapy.

Disclosure of Invention

An object of the present invention is to provide mTOR-targeted tolerogenic dendritic cells (Rapa-toldcs).

Another object of the present invention is to provide a method for preparing the mTOR-targeted tolerant dendritic cell.

Another object of the present invention is to provide the use of the mTOR-targeted tolerant dendritic cell.

In order to provide tolDC with stable tolerance, the invention adopts rapamycin modified cytokines (GM-CSF and IL-4) to induce the differentiation of dendritic cells, prepares tolDC (Rapa-tolDC) with stable tolerance function and targeting mTOR, clarifies the transcription spectrum, molecular characteristics and negative regulation effect of the Rapa-tolDC, induces different types of regulatory immune cells based on the characteristics of the Rapa-tolDC, and further establishes and prepares a novel CD8 with donor specificity ⁺ The regulatory T cells lay a foundation for solving the problem of donor specificity in immune tolerance induction.

In particular, in one aspect, the invention provides an mTOR-targeted tolerogenic dendritic cell which has a transcription profile of Siglec1, Spp1 and the like which are down-regulated, has a PI3K/mTOR expression deletion and has a selective negative regulation effect.

According to a specific embodiment of the invention, the mTOR-targeted tolerant dendritic cells of the invention, expressing the DC marker CD11c, express low surface co-stimulatory molecules CD80, CD86 and MHC-II, secrete relatively high IL-10 and low levels of INF- γ compared to mature DCs; can induce the generation of various types of regulatory immune cells, and can generate CD8 with donor specificity ⁺ Regulatory T cells.

In another aspect, the present invention also provides a method for preparing the mTOR-targeted tolerant dendritic cell, comprising:

taking bone marrow mesenchymal stem cells or peripheral blood PMBC, carrying out adherent culture by using a phenol red-free culture solution containing 1% -10% FBS to obtain precursor cells of the DC, and adding a complete culture medium containing GM-CSF and IL-4; culturing for full change on day 2, half change on day 4, supplementing cell factors GM-CSF and IL-4, maintaining concentration, and adding rapamycin when changing liquid on day 2 and day 4; the culture was continued by day 6, half-way changing of the medium, supplementing GM-CSF to maintain the concentration unchanged, and adding LPS.

According to a specific embodiment of the invention, in the preparation method of the mTOR-targeted tolerogenic dendritic cells, the concentration of GM-CSF is 1-40ng/ml, and the concentration of IL-4 is 0.5-20 ng/ml. Preferably, the ratio of the concentration of GM-CSF to IL-4 is 2: 1.

According to a specific embodiment of the invention, in the method for preparing mTOR-targeted tolerant dendritic cells, the concentration of rapamycin is 1-40 nM. Preferably, the concentration of rapamycin is 1-10 nM.

According to a specific embodiment of the invention, the method for preparing mTOR-targeted tolerogenic dendritic cells of the invention comprises the step of preparing the LPS concentration of 0.01-1 mu g/ml.

According to a specific embodiment of the invention, in the preparation method of the mTOR-targeted tolerogenic dendritic cells, the concentration ratio of GM-CSF to IL-4 is 2: 1; rapamycin was added at 10nM concentrations at day 2 and day 4 changes, respectively, and LPS was added at 0.05. mu.g/mL at day 6.

In the present invention, the concentration of each substance mentioned above means the concentration of each substance added to the culture system, unless otherwise specified.

According to a specific embodiment of the invention, in the preparation method of the mTOR-targeted tolerant dendritic cells, the immature DC (named as Rapa-imDC in the invention) targeted to the mTOR is prepared on the 6 th day of culture, and the tolerant DC (named as Rapa-tolDC in the invention) targeted to the mTOR is prepared on the 7 th day of culture. In the present invention, the tolerogenic dendritic cells include CD11c ⁺ mTOR-targeted tolerogenic DCs and different cell subsets of CD4+, CD4-, CD8+ and CD 8-among them, or dendritic cells cultured from monocytes or induced to differentiate from bone marrow stem cells, or induced to differentiate from rapamycin or everolimus.

In another aspect, the invention also provides application of the mTOR-targeted tolerogenic dendritic cells in preparation of dendritic cell products with immune tolerance functions.

According to a specific embodiment of the invention, in the application of the mTOR-targeted tolerogenic dendritic cells, the dendritic cell preparation is used for preventing and/or treating autoimmune diseases, graft-versus-host diseases and/or organ transplant rejection.

According to a specific embodiment of the present invention, the mTOR-targeted tolerant dendritic cell targeted therapy is used in transplantation including cell transplantation, organ transplantation or tissue transplantation; wherein the cell transplantation comprises stem cell transplantation, regulatory cell transplantation, islet cell transplantation or effector cell transplantation; organ transplantation includes kidney transplantation, liver transplantation, heart transplantation, small intestine transplantation, lung transplantation, pancreas transplantation, or combined organ transplantation; the tissue transplantation includes cornea transplantation, limb transplantation or face transplantation.

In another aspect, the invention also provides the application of the mTOR-targeted tolerogenic dendritic cells in preparing regulatory cells for inducing immune tolerance of a transplant recipient.

According to a specific embodiment of the invention, the regulatory cells for use in the mTOR-targeted tolerant dendritic cell of the invention comprise CD4+ CD25+ regulatory T cells, CD8 ⁺ Regulatory T cells, Foxp3 ⁺ Regulatory T cells, regulatory B cells, NON-T lymphocytes, regulatory NK cells, or regulatory macrophages.

In the present invention, Rapa-tolDC is a tolerogenic subset of DC cells with induction of donor-specific CD8 ⁺ CD45RC ^low/- Treg generation, establishing the function of transplantation immune tolerance.

In some specific embodiments of the invention, the invention induces the Rapa-tolDC (fig. 3-fig. 4) with stable tolerogenicity through an optimization scheme of combining a small dose of Rapa with a low concentration of GM-CSF + IL-4 (fig. 1), the Rapa-tolDC has a stable negative immune regulation effect (fig. 5), the transcription spectrum and the molecular characteristics of the Rapa-tolDC are defined (fig. 6), the problems of difficult selection of induced drugs, non-uniform scheme, unstable properties and the like in the existing tolDC preparation process are effectively solved, and the invention has the advantages of simple operation, short preparation period, low cost and strong repeatability.

The Rapa-tolDC prepared by the invention has stable immunoregulation function, can induce and generate different types of regulatory T cells (figure 7) and CD4 ⁺ Tregs and Breg secreting IL-10 (figure 8); induction of CD8 producing high secreting IL-10 with donor specificity ⁺ CD45RC ^low/- Tregs (fig. 9); meanwhile, the PBMC-derived Rapa-tolDC prepared by the scheme of the invention can induce the generation of Foxp3 secreting IL-10 ⁺ Regulatory T cells (fig. 10).

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings and their description illustrate exemplary embodiments of the invention and do not limit the scope of the invention unduly.

Fig. 1 is a schematic flow chart of a preparation technical scheme of mTOR-targeted tolerant DC (Rapa-tolDC) according to an embodiment of the present invention.

FIG. 2 is a graph of flow results of dendritic cells induced by different concentrations of GM-CSF regimen.

Figure 3 is a graph of the flow results of different protocols for inducing mTOR-targeted tolerant DCs.

Figure 4 is a graph of flow results of mTOR-targeted tolerant DCs versus mature dendritic cell differentiation.

FIG. 5 shows that Rapa-tolDC of the present invention promotes apoptosis of lymphocytes.

FIG. 6 is a graph showing the results of the transcription profile and molecular characteristics of the Rapa-tolDC of the present invention.

Fig. 7 is a schematic flow chart of an application of mTOR-targeted tolerant DC (Rapa-tolDC) according to an embodiment of the present invention.

FIG. 8 shows the induction of CD4 using Rapa-tolDC in the example of FIG. 7 ⁺ Treg, IL-10 secreting Breg results.

FIG. 9 shows the induction of CD8 with donor-specific high secretion of IL-10 using Rapa-tolDC in the example of FIG. 7 ⁺ CD45RC ^low/- Treg results.

FIG. 10 is a graph showing the results of mTOR-targeted tolerogenic DC induced by peripheral blood PBMCs, and shows that the generated PBMC-derived Rapa-tolDC can be prepared by the present invention, and IL-10-secreting Foxp3 can be induced ⁺ Regulatory T cells.

Detailed Description

In order to clearly understand the technical features, objects and advantages of the present invention, the technical solutions of the present invention will be described in more detail with reference to the specific embodiments and the accompanying drawings. It is obvious that the described embodiments are only examples of a part of the invention, to which the invention is not restricted. All other embodiments, which can be obtained by a person skilled in the art without making creative efforts based on the embodiments of the present invention, shall fall within the protection scope of the present invention.

It should be noted that the obtaining route of various biological reagents and materials used in the examples is only to provide a route of experimental acquisition to achieve the purpose of specific disclosure of the invention, and should not be a limitation to the source of the biological materials of the invention. In fact, any biological agent or material that can be obtained without law or ethical reasons, including isolated cells from a mammal or human such as rat, mouse, pig, or the like, or obtained from a cell bank, or purchased commercially, or prepared according to available literature, can be used instead as suggested in the examples of the present invention.

The method operations not specifically mentioned in the examples were carried out according to the conventional operations of the prior art or the operations suggested by the manufacturer's specifications.

Example 1 preparation and phenotypic identification of mTOR-Targeted tolerogenic dendritic cells

1.1 preparation of BM-MSCs derived dendritic cells according to different GM-CSF + IL-4 protocols

Referring to the preparation process shown in fig. 1, bone marrow mesenchymal liver cells are obtained first, and the steps are as follows: SPF-level male Lewis rats with the weight of 200-350g are purchased from the center of Beijing Wittingle laboratory animals, killed by pulling the neck after general anesthesia, shaved and disinfected on limbs and trunk, separated by surgical instruments such as sterile scissors and tweezers in an ultra-clean bench to obtain thighbones (including upper and lower joints), and placed in cell culture solution. Removing redundant tissues such as muscle, fat and the like in a cell culture room super clean bench, and washing the surface of the femur by 1 XPBS (purchased from Solebao corporation, China); sterileSurgical scissors remove the upper and lower joints, leaving the femoral shaft. Repeatedly washing bone marrow cavity with phenol red-free RPMI-1640 (from Gibco, USA) until the color is whitish (3-5 times), filtering with 200 mesh screen, centrifuging, discarding supernatant, blowing 5ml of 1 × erythrocyte lysate (from BD, USA), mixing with marrow cell precipitate, incubating at 4 deg.C in dark for 10min, centrifuging, discarding supernatant, washing with 1 × PBS, and removing residual erythrocytes (such as erythrocyte can be removed with erythrocyte lysate); secondly, obtaining mononuclear cells by adherence, comprising the following steps: prewetting the culture dish with 1 × PBS, and centrifuging the above cells to 1.5-2 × 10 ⁶ The resulting suspension was inoculated into a flat-bottomed 6-well plate (purchased from Corning, USA) containing 2ml of FBS-free phenol red-free RPMI-1640 culture medium, and incubated at 37.5 ℃ and 5% CO ₂ Is cultured in an incubator for 2 hours. After 2h, the non-adherent cells were discarded, 2mL of phenol red-free RPMI-1640 medium containing 10% FBS (purchased from Gibco, USA) was added, and GM-CSF and IL-4 (purchased from Peprotech, USA, at a ratio of 2: 1) were added at different concentrations, respectively, at a ratio of 1ng/mL, 10ng/mL, 20ng/mL and 40ng/mL for GM-CSF, and at a ratio of 0.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL for IL-4, respectively, and the mixture was placed at 37.5 ℃ and 5% CO ₂ The cell incubator is continuously cultured for 6 days. Changing the total amount of the solution on the 2 nd day and changing the half amount of the solution on the 4 th day, wherein the solution is changed and simultaneously sufficient cytokines are supplemented to maintain the concentrations of the GM-CSF and the IL-4 unchanged; half-way fluid changes on day 6, supplemented with GM-CSF alone, maintained at constant concentration, stimulated with LPS (1. mu.g/mL) at high concentration (1. mu.g/mL), placed at 37.5 ℃ in 5% CO ₂ The cell incubator of (a) is continued for 24 hours.

1.2 flow cytometric phenotypic characterization of BM-MSCs derived dendritic cells

Collecting cells cultured to the 7 th day (i.e., continuously culturing for 24 hours after adding LPS) after stimulation by LPS, centrifuging, discarding the supernatant, washing once by 1 XPBS, centrifuging, and discarding the supernatant; thoroughly pipetting 300. mu.l of 1 XPBS and uniformly mixing to obtain a single cell suspension, equally dividing into 3 parts, respectively adding the single cell suspension into 3 flow-type sample loading tubes, marking as No. 1 standard control tubes, and adding 5. mu.l of FITC-IgG, PE-IgG and APC-IgG (purchased from BD company in USA); test sample tube No. 2, FITC-CD86, PE-CD11c and APC-CD80 (5. mu.l each from BD Co., USA) as mouse-rat monoclonal antibodies were added; in test sample tube 3, 5. mu.l each of mouse anti-rat monoclonal antibodies PE-CD11c and APC-MHC-II (purchased from BD Co., USA) was added. After mixing well, incubation for 15min at room temperature in the dark, adding 1ml of 1 × PBS, pipetting, mixing well, centrifuging, discarding the supernatant, and detecting the surface marker of the dendritic cells with a flow cytometer (BD Canto II, USA).

The results of flow cytometry detection of the expression of the induced dendritic cell surface molecules are shown in FIG. 2, and the concentrations of GM-CSF in FIG. 2, from left to right, are 1ng/mL, 10ng/mL, 20ng/mL and 40ng/mL, respectively, and the concentrations of IL-4 in FIG. 2, from left to right, are 0.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL, respectively. The results in the figure show that different concentrations of GM-CSF + IL-4 can induce BM-MSCs to differentiate into dendritic cells, and can differentiate into mature dendritic cells under the stimulation of LPS. Along with the increasing of the stimulation concentration of GM-CSF, the expression level of MHC-II on the surface of the induced and differentiated dendritic cells and the co-stimulation molecule CD86/CD80 is higher and can reach more than 90 percent (figure 2), and the molecule expression of the classical dendritic cells with antigen presentation effect is met.

1.3 preparation scheme of mTOR-optimized tolerant DCs

According to the schematic diagram of FIG. 1, the mesenchymal stem cells were obtained by the same method as 1.1, the time points of different concentrations of GM-CSF + IL-4 intervention were unchanged, rapamycin was added at a small dose (10 nM on day 2 and 10nM on day 4) on day 2 and day 4 of culture, LPS stimulation was at a small dose (0.05. mu.g/mL) on day six and LPS stimulation was at 24h, and the cells cultured to day 7 (Rapa-tolDC) were used for flow cytometry, and staining and detection were performed in the same scheme as 1.2.

The inventor's previous protocol was also used as a control tolDC: the mesenchymal stem cells were obtained by the same method as 1.1, GM-CSF (1 ng/mL) + IL-4 (0.5 ng/mL) intervention time point, 10ng/mL rapamycin was added on the 2 nd day of culture, 0.1. mu.g/mL LPS was added on the 6 th day of culture for stimulation, and the cultured cells were harvested for 48 hours for flow cytometry, staining and detection protocol as 1.2.

Dendritic cell surface markers were flow-assayed and with increasing stimulation of GM-CSF + IL-4 (1 ng/mL, 10ng/mL, 20ng/mL for GM-CSF, respectively), on day 6 of culture (sampled before LPS stimulation was added), Rapa intervened in the stepwise upregulation of the modified DC surface co-stimulatory molecule CD86/CD80 and MHC-II expression (panel A in FIG. 3), with weaker resistance to LPS stimulation and a more mature phenotype (panel B in FIG. 3). The detection result shows that the optimized Rapa modified low-concentration scheme with a small dose can induce the generation of mTOR-targeted resistant DC with more stable phenotype.

Compared with the prior scheme, the optimized scheme has higher Rapa-tolDC generation rate and shows that the expression of CD11c is remarkably increased (87.86 +/-1.70)vs. 60.50 + -5.84, p < 0.001), and MHC-II (30.40 + -8.67)vs. 38.70 ± 3.87, p = 0.038), the expression of the surface co-stimulatory molecules CD80, CD86 was still at a lower level with no significant difference.

1.4 phenotypic differences between target mTOR-tolerant DCs and mature DCs

Immature dendritic cells differentiated by BM-MSCs induced by GM-CSF at high concentration are easily differentiated into mature DCs under stimulation of LPS, Rapa intervention can inhibit maturation of the immature dendritic cells, so that the immature dendritic cells have the effect of stably resisting stimulation of LPS, a low-concentration GM-CSF-induced mTOR-targeted tolerant DC phenotype tends to be in an immature state, the dendritic cells are prepared by respectively utilizing high-concentration and low-concentration GM-CSF schemes, the steps are the same as 1.1 and 1.3, and flow-type staining is the same as 1.2.

The flow assay results are shown in FIG. 4, where the Rapa-modified low concentration (10 ng/mL) GM-CSF + IL-4 stimulated BM-MSCs to differentiate into dendritic cells, which were cultured for 6 days and then transferred to CD11 ⁺ DC differentiation, expressed in an immature state (herein referred to as Rapa-imDC), low expression of the co-stimulatory molecules CD86/CD80 and MHC-II (panel A in FIG. 4); it was able to resist the maturation effects stimulated by LPS, still expressing lower levels of the co-stimulatory molecules CD86/CD80 and MHC-II (panel A in FIG. 4), whereas high concentration (40 ng/ml) GM-CSF + IL-4 regimen induced imDCs stimulated by LPS to Rapa were more readily differentiated towards mature DCs, both CD80/CD86 and MHC-II expression were at higher levels, which were more prone to high concentration GM-CSF induced maturationPhenotype of mature DC differentiation (panel B in FIG. 4). In addition, the phagocytic capacity of the Rapa-tolDC is reduced compared with that of the mature DC, and the uptake capacity of the dextran is reduced (63.42% vs. 95.04%).

Example 2 mTOR-Targeted tolerant DCs have Stable negative modulatory effects

In this example, the negative regulation of mTOR-targeted resistant DC (Rapa-tolDC) of the present invention was examined. A comparison was also made using the control tolDC (previous protocol).

In this and the following examples, unless otherwise noted, the tested Rapa-tolDCs of the present invention were prepared according to the protocol 1.3 in example 1, wherein GM-CSF concentration was 10ng/mL + IL-4 concentration was 5ng/mL, Rapa 10nM was added on day 2 and day 4, respectively, and LPS concentration was 0.05. mu.g/mL.

mTOR-targeted tolerogenic DC secrete elevated levels of IL-10

Reserving mTOR-targeted tolerant DC culture supernatant, and detecting the level of IL-10 secreted by the Rapa-tolDC prepared by the method in the example 1 by using an ELISA kit (operating according to the instruction); the detection result shows that the level of secreted IL-10 is remarkably increased compared with the tolDC of a control group (767.8 +/-63.02)vs. 256.9±57.87）pg/ml。

mTOR-targeted tolerogenic DC with reduced ability to stimulate lymphocyte proliferation

The preparation of Rapa-tolDC by using the method of mitomycin C inactivation example 1, mixed culture with magnetic bead sorted T cells for 48h, then adding CCK-8, incubating at 37 ℃ for 2h, detecting OD value at 450nm, and calculating stimulation index.

The SI index of Rapa-tolDC stimulating T cell proliferation in the present invention was lower (0.93. + -. 0.05) compared to control tolDCvs. 1.22 ± 0.42, p = 0.046), indicating that the weaker the antigen presenting ability and the enhanced negative regulatory ability are in line with the requirement of inducing immune tolerance.

mTOR-targeted, tolerant DC promotes enhanced apoptotic effects in lymphocytes

Rapa-tolDCs were prepared as described in example 1, co-cultured with PBMC for 3 days at a ratio of 1:10, and flow cytometrically examined for apoptosis in effector T cells.

The results show that after 3 days of PBMC co-culture, a significant increase (33.7%) in the effect of Rapa-tolDC in promoting apoptosis of effector T cells was found (P < 0.01) compared to control tolDC (17.7%), with a significant difference (panel a-panel C in fig. 5).

All the above show that the mTOR-targeted tolerant DC prepared by the invention has a strong negative immune regulation effect and meets the requirement of inducing immune tolerance.

Example 3 transcriptome sequencing techniques to unequivocally target the molecular characteristics and transcriptional profiles of mTOR-tolerant DCs

imDC and matDC are prepared by the scheme 1.1 in example 1, Rapa-imDC and Rapa-tolDC are prepared by the scheme 1.3, centrifugation and supernatant discarding are carried out, and 1 × 10 is obtained ⁶ Extracting total RNA from the target cells by using a Trizol method, and detecting the RNA concentration by using an Agilent 2100 Bioanalyzer; removing rRNA by an RNase-H method, establishing a transcriptome library, detecting the range of an insert fragment of the library by an Agilent 2100 Bioanalyzer, and detecting the concentration of the library by using an ABI StepOneplus Real-Time PCR System (TaqMan Probe); and performing on-machine sequencing by adopting an Illumina sequencing technology SBS reagent. Sequencing results indicate that compared with matDC, the main genes of PI3K/mTOR pathway in the Rapa-tolDC are deleted or down-regulated (figure 6), and Spp1 and phlda3 are found to play key roles in regulating the differentiation of the Rapa-tolDC; this suggests that Rapa-modified mTOR-targeted tolerant DCs have specific differentiation pathways and immunogenic tolerance properties.

Example 4 mTOR-targeted tolerogenic DC induced the production of CD4 of a different phenotype ⁺ Treg

mTOR-targeted tolerant DCs were prepared using the protocol in example 1, and sorted with Anti-Rat PE CD11c magnetic beads (from America, Germany, whirlpool) to obtain CD11c with a purity > 95% or greater, as shown in FIG. 7 ⁺ The magnetic beads of Rapa-tolDC, Anti-Rat PE CD4 and CD8 (purchased from Meitian and whirlwind company, Germany) are separated to respectively obtain CD4 with the purity of more than 95 percent ⁺ T and CD8 ⁺ T cells, according to 1:4 for 7 days, collecting the mixed cells, centrifuging, discarding the supernatant, resuspending in 100. mu.l of 1 XPBS, and adding 5. mu.l of mouse anti-rat antibodyFully mixing mouse monoclonal antibodies FITC-CD4, PE-CD25, PE-CD45RC and Percp-TCR alpha beta (purchased from American BD company), incubating for 15min at room temperature in a dark place, centrifuging, discarding the supernatant, blowing and mixing uniformly with 1 XPBS 500 mu l, detecting CD4 & mu.l by using a flow cytometer ⁺ CD45RC ^low/- Treg、CD4 ⁺ CD25 ⁺ Expression level of tregs.

Flow cytometry results show that mTOR-targeted tolerant DC significantly upregulated CD4 compared to control group ⁺ CD45RC ^low/- Treg（78.58±10.23 vs. 51.30±2.26）、CD4 ⁺ CD25 ⁺ Treg（60.02±8.50 vs. 19.95 ± 4.17); the detection result shows that the mTOR-targeted tolerant DC induces the generation of different CD4 ⁺ Treg capacity (panel a in fig. 8).

Example 5 mTOR-Targeted tolerant DCs induce Breg producing high secreted IL-10

As shown in FIG. 7, mTOR-targeted tolerant DCs were prepared using the protocol of example 1 and sorted with Anti-Rat PE CD11c magnetic beads (from Meitian and whirlpool, Germany) to obtain CD11c with a purity > 95% or greater ⁺ Magnetic beads of Rapa-tolDC, Anti-Rat PE CD45RA (purchased from America, Edison, Germany) were sorted to obtain B cells with a purity of > 95% or more, according to a 1:4, collecting the cells which are mixed and cultured for 7 days, centrifuging, discarding the supernatant, culturing by 1640 without phenol red, stimulating by monensin and ionomycin for 6 hours, centrifuging, discarding the supernatant, resuspending by 100 mul 1 XPBS, adding 5 mul of mouse anti-rat monoclonal antibody FITC-CD1d, PE-CD5 and PB-CD45RA (purchased from American BD company) respectively, mixing well, incubating for 15min at room temperature and in the dark, breaking the membrane, adding 5 mul of mouse anti-rat monoclonal antibody AF647-IL-10 (purchased from American BD company), mixing well, incubating for 60min at room temperature and in the dark, adding 2ml of membrane breaking buffer, centrifuging, discarding the supernatant, blowing and mixing well by 1 XPBS 500 mul, detecting CD45RA by a flow cytometer ⁺ CD5 ⁺ CD1d ⁺ IL-10 ⁺ Expression level of Breg.

Flow cytometry results show that mTOR-targeted tolerant DCs are capable of inducing B cell phenotype CD45RA compared with control group ⁺ CD5 ⁺ CD1d ⁺ IL-10 ⁺ Breg differentiation, IL-10 secretion levels can reach 20% -70%, with higher levels of IL-10 secretion with longer culture times. The detection result shows that the mTOR-targeted tolerant DC has CD45RA inducing high IL-10 secretion ⁺ CD5 ⁺ CD1d ⁺ Breg production capability (panel B in fig. 8).

Example 6 mTOR-targeting tolerogenic DC induces production of high IL-10 secreting CD8 with donor specific inhibitory effect ⁺ CD45RC ^low/- Treg

⁺ ^low/- mTOR-targeted tolerant DC induces the production of IL-10-highly secreted CD8CD45RCTreg

Induction of CD8 Using the protocol in example 4 ⁺ CD45RC ^low/- Treg, collecting mixed cultured cells on day 7, centrifuging, discarding supernatant, blowing 5% phenol red-free RPMI-1640500 μ l, mixing cell precipitate, adding ionomycin, PMA, and monensin, standing at 37.5 deg.C and 5% CO ₂ The cell culture box is continuously incubated for 6 hours, centrifuged, supernatant is discarded, 5 mul of monoclonal antibody FITC-CD8, PE-CD45RC and Percp-TCR alpha beta (purchased from American BD company) of mouse-rat are respectively added and fully mixed, incubation is carried out for 15min at room temperature in a dark place, 5 mul of monoclonal antibody AF647-IL-10 (purchased from American BD company) of mouse-rat is added after membrane rupture, incubation is carried out for 60min at room temperature in a dark place after full mixing, 2ml of membrane rupture buffer solution is added, centrifugation is carried out, supernatant is discarded, the mixture is blown and uniformly mixed by 1 XPBS 500 mul, and CD8 is detected by a flow cytometer ⁺ CD45RC ^low Expression levels of Treg, IL-10.

Flow cytometry results show that the normal rat spleen CD8 ⁺ CD45RC ^low/- The proportion of Treg is about 15-20%, the proportion can be increased to about 40% after separation and purification, and the Rapa-tolDC can remarkably increase the CD8 ⁺ CD45RC ^low/- Expression levels of tregs, greater than 80% (panel a in fig. 9), higher than those induced by previous protocols (72.6%); the result of nuclear factor staining showed CD8 ⁺ CD45RC ^low/- Tregs secrete high levels of IL-10, and are the primary source of IL-10 secretion (panel B in FIG. 9). The detection results show that the Rapa-tolDC has CD8 inducing high IL-10 secretion ⁺ CD45RC ^low/- The ability to generate tregs.

⁺ ^low The CD8CD45RCTreg with high IL-10 secretion can specifically inhibit the proliferation of effector T cells

Preparation of Donor-derived CD11 ⁺ Rapa-tolDC and receptor-derived CD8 ⁺ Mixing and culturing T cells at a ratio of 1:4 for 6 days, and removing CD45RC in the mixed culture system ^high Effector T cells, CFSE labeled cognate receptor, third party effector T cells, according to 1: and 4, performing mixed culture, adding a matDC from a receptor source into the control group, adding a matDC from a third party into the receptor effector T cells after performing mixed culture for 3 days, continuing to culture for 6 days, and detecting the proliferation condition of the effector T cells by using a flow cytometry.

Flow cytometry results show that the matDC and the receptor significantly stimulate CD4 ⁺ CD25 ^- CD8 induced by Rapa-tolDC compared to effector T cell proliferation ⁺ CD45RC ^low/- Tregs can remarkably inhibit rat CD4 ⁺ CD25 ^- The proliferation of effector T cells, the effect of inhibiting the same species proliferation is better than that of a xenogeneic rat, and the same species CD4 is inhibited after the third party source matDC is added ⁺ CD25 ^- The proliferative effect of effector T cells was attenuated (panel C in fig. 9). The detection result indicates that the CD8 induced by Rapa-tolDC ⁺ CD45RC ^low/- Tregs have donor-specific suppressive effects (panel D in fig. 9), laying the theoretical foundation for the induction of transplantation immune tolerance.

Example 7 preparation of peripheral blood PBMC derived mTOR-targeted tolerogenic dendritic cells

mTOR-targeted tolerogenic dendritic cells derived from peripheral blood PBMC were prepared by replacing bone marrow mesenchymal stem cells with peripheral blood PBMC in example 1. The method comprises the following specific steps: diluting 5-10ml of peripheral blood with normal saline in equal proportion, obtaining peripheral blood PBMC by using a peripheral blood lymphocyte separating medium (Tianjin third-class male biotechnology limited) according to the ratio of the separating medium to the peripheral blood of 2:1 by using a density gradient centrifugation method, washing the PBMC once by 1 XPBS (bovine serum albumin), removing redundant red blood cells by using a red blood cell lysate, and washing the PBMC once without serum 1640; the redundant lymphocytes are removed by means of adherence,the stimulation protocol was identical to 1.3 in example 1, and on day 7 of culture, the phenotype was examined by flow cytometry, the protocol being identical to example 1. Further sorting the induced Rapa-tolDC and the magnetic beads to obtain the CD4 with the purity more than 95% ⁺ T cells, according to 1:4 for 7 days, collecting the mixed cells, centrifuging, discarding the supernatant, resuspending in 100. mu.l of 1 XPBS, and flow cytometry for CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Expression of Tregs and IL-10 secretion levels.

Flow cytometry results showed that using the protocol in example 1, phenotypically similar peripheral blood PBMC-derived mTOR-targeted tolerant DCs could be obtained, as shown by low expression of HLA-DR, CD1a, and co-stimulatory molecules CD83, CD80/CD86 (fig. 10); and can induce the production of IL-10-secreting CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Treg, direct effect is greater than indirect effect; the technical proposal provided by the invention can induce different precursor cells to differentiate into tolerogenic dendritic cells.

The above examples are merely illustrative of the preferred embodiments of the present invention and the scope of the present invention is not limited thereto. Any modification, equivalent replacement or change made on the basis of the present invention by those skilled in the art is within the protection scope of the present invention.

Claims

1. An mTOR-targeted tolerant dendritic cell, wherein the mTOR-targeted tolerant dendritic cell has a transcription profile of Siglec1 and Spp1 gene down regulation, has a PI3K/mTOR expression deletion and has a selective negative regulation effect.

2. The mTOR-targeted tolerant dendritic cell of claim 1, wherein the mTOR-targeted tolerant dendritic cell expresses the DC marker CD11c, expresses low surface co-stimulatory molecules CD80, CD86 and MHC-II, secretes relatively high IL-10 and low levels of INF- γ compared to mature DCs; can induce the generation of various types of regulatory immune cells, and can generate CD8 with donor specificity ⁺ Regulatory T cells.

3. A method of preparing mTOR-targeted tolerant dendritic cells according to claim 1 or 2 comprising:

4. The method of claim 3, wherein the concentration of GM-CSF is 1-40ng/ml, the concentration of IL-4 is 0.5-20ng/ml, the concentration of rapamycin is 1-40nM, and the concentration of LPS is 0.01-1 μ g/ml.

5. The method of claim 4, wherein the ratio of the concentration of GM-CSF to IL-4 is 2: 1; rapamycin was added at 10nM concentrations at day 2 and day 4 changes, respectively, and LPS was added at 0.05. mu.g/mL at day 6.

6. Use of the mTOR-targeted tolerant dendritic cell of claim 1 or 2 for the preparation of a dendritic cell preparation with an immune-tolerizing function.

7. The use according to claim 6, wherein the dendritic cell preparation is for the prevention and/or treatment of autoimmune diseases, graft-versus-host disease and/or organ transplant rejection.

8. Use according to claim 7, wherein the transplantation comprises cell transplantation, organ transplantation or tissue transplantation; wherein the cell transplantation comprises stem cell transplantation, regulatory cell transplantation, islet cell transplantation or effector cell transplantation; organ transplantation includes kidney transplantation, liver transplantation, heart transplantation, small intestine transplantation, lung transplantation, pancreas transplantation, or combined organ transplantation; the tissue transplantation includes cornea transplantation, limb transplantation or face transplantation.

9. Use of an mTOR-targeted tolerant dendritic cell according to claim 1 or 2 for the preparation of a regulatory cell for inducing immune tolerance in a transplant recipient.

10. The use of claim 9, wherein said regulatory cells comprise CD4+ CD25+ regulatory T cells, CD8 ⁺ Regulatory T cells, Foxp3 ⁺ Regulatory T cells, regulatory B cells, NON-T lymphocytes, regulatory NK cells, or regulatory macrophages.