KR101913353B1 - Immuno-suppressive dendritic cell-like cell and manufacturing method thereof - Google Patents
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Abstract
The present invention relates to dendritic cell-like cells having immunosuppressive ability, and more particularly to immunosuppressive dendritic cell-like cells prepared by culturing bone marrow cells (BM) together with GM-CSF for a long time, .
The dendritic cell-like cells for immunosuppression according to the present invention can be used for the treatment of various diseases or diseases which can be treated through an immunosuppressive mechanism. In addition, the dendritic cell-like cells of the present invention with improved immunotolerance capability enable effective application as an immunosuppressive agent of dendritic cells, and thus can be applied to various autoimmune disease treatments have.
Immunosuppressive function can be used without adverse effects because it is likely to be closer to the nature of the actual in vivo immunosuppressive dendritic cells because there is no pharmaceutical substance or genetic manipulation compared to conventional immunosuppressive dendritic cells.
Description
The present invention relates to dendritic cell-like cells having immunosuppressive ability, and more particularly to immunosuppressive dendritic cell-like cells prepared by culturing bone marrow cells (BM) together with GM-CSF for a long time, .
Dendritic Cells (DCs) are powerful antigen presenting cells (APCs) that induce and control primary cell-mediated immune responses. Dendritic cells are terminally differentiated cells, present in less than 1% of all immune cells in the body, but induce lymphocyte activity much more potently than monocytes or macrophages.
In addition, dendritic cells have received increasing attention as mediators of T-cell tolerance. In contrast to mature dendritic cells (mDCs), the original function of Immature Dendritic Cells (imDC) is to induce the production of T reg cells, induction of apoptosis of autoreactive working cells, or induction of immune- To provide conditions for self-tolerance. Attempts have been made to utilize immature dendritic cells that exhibit such immunosuppressive function in therapy. Unfortunately, however, there are still some obstacles such as a very limited protocol for producing immature dendritic cells, and mature stages within the host. Thus, it has been very difficult to use immunosuppressed dendritic cells for therapeutic use.
Dendritic cells initially determine whether the immune system will work against irritants to induce an inflammatory response or tolerance, and then determine which T lymphocytes inexperienced T lymphocytes will differentiate into T lymphocytes It plays a key role in determining.
Different cytokines are involved in the differentiation and growth of the resin granules. In particular, c-Kit and Flt-3 ligands support immature dendritic cells by binding to tyrosine kinase receptors. GM-CSF (Granulocyte macrophage colony-stimulating factor) or IL-3 (Interleukin-3), a product of activated T cells and other cells, also promote the differentiation of granulosa cells and TNF (tumor necrosis factor) It blocks the differentiation pathway to the bone marrow and promotes the maturation of dendritic cells.
Previous methods for immuno-suppressive dendritic cells use ex vivo / in vitro methods of exposure to cytokines, pharmacological agents, and anti-inflammatory biological or genetic engineering methods. For example, treatment with IL-10 resulted in immuno-suppressive dendritic cells that did not express other co-stimulatory molecules even when MHC II was expressed. In this way, there is a possibility that the autoimmune disease can be treated.
However, this method is not a dendritic cell made in an in vivo situation. Therefore, depending on the surrounding environment, immuno-suppressive dendritic cells may be converted into dendritic cells inducing an immune response, and worse, Lt; / RTI > That is, there is a stability problem. In addition, since DCs must be produced for each individual in the manufacturing process, there has been a limit in that the effort is increased and the cost is increased.
The present inventors confirmed long-term treatment of bone marrow cells (BM) with cytokine GM-CSF for several months to one year to become dendritic cell-like cells having immuno-suppressive ability. These immunosuppressed dendritic cell-like cells can be applied as a therapy by injecting into patients suffering from autoimmune disease. Immuno-suppressive A new way to create dendritic cells or dendritic cell-like cells, and because of the lack of pharmaceutical material or genetic manipulation, they are close to the properties of immuno-suppressive dendritic cells in the in vivo environment There is an advantage that the possibility is high. At this time, it was confirmed that the MHC-II molecule is normal but lacks co-stimulatory molecules to inhibit the immune response, and dendritic cells normally inducing T cell immune responses are also immune-suppressed by the immunocompromised dendritic cell- Confirming that the reaction can be inhibited, thereby completing the present invention.
It is an object of the present invention to provide a safe immune response-suppressing cell which is excellent in immunosuppressive ability and has no side effects such as tumor generation.
Another object of the present invention is to provide a method for producing a safe immune response-suppressing cell which is excellent in immunosuppressive ability and has no side effects such as tumorigenesis.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
In order to achieve the above object, the present invention provides immunocompromised dendritic cell-like cells prepared by treating cytokine GM-CSF with bone marrow cells for a period of 4 weeks or more.
Preferably, treating said bone marrow cells with said cytokine GM-CSF comprises culturing bone marrow cells in a medium containing cytokine GM-CSF.
Preferably, the medium containing the cytokine GM-CSF contains 10 ng / ml or more of GM-CSF.
Preferably, the immunocompromised dendritic cell-like cells express CD11c and MHC II molecules on the cell surface.
Preferably, the immunosuppressive dendritic cell-like cells have CD40, CD83 and CD86 molecules not expressed or reduced on the cell surface.
Preferably, said immunosuppressive dendritic cell-like cells have the ability to inhibit mature dendritic cells from stimulating T cells when mixed with standard mature dendritic cells.
The present invention also relates to immunosuppressive dendritic cell-like cells prepared from immune cell populations isolated from bone marrow, comprising: (1) culturing immune cells isolated from bone marrow with cytokine GM-CSF for a period of 4 weeks or more Lt; RTI ID = 0.0 > dendritic < / RTI > cell-like cells.
(2) separating cells expressing CD11c and MHC-II on the cell surface, preferably after step (1); And (3) isolating cells in which the CD40, CD83 and CD86 molecules have not been expressed or reduced on the cell surface among the cells that have undergone the step (2).
The dendritic cell-like cells for immunosuppression according to the present invention can be used for the treatment of various diseases or diseases which can be treated through an immunosuppressive mechanism. In addition, the dendritic cell-like cells of the present invention, which have improved immunotolerance ability, enable effective application as an immunosuppressant of dendritic cells, and thus can be applied to various autoimmune disease treatment and research .
Immunosuppressive function can be used without adverse effects because it is likely to be closer to the nature of the actual in vivo immunosuppressive dendritic cells because there is no pharmaceutical substance or genetic manipulation compared to conventional immunosuppressive dendritic cells.
Brief Description of the Drawings Fig. 1 is a graph showing the results of (A) a map of plasmid DNA for producing GM-CSF from CHO cells, (b) a graph showing separation of CHO / mGM-CSF cells, and (C) It is a photograph.
FIG. 2 shows the result of culturing bone marrow cells (BM) at various GM-CSF concentrations (A) as a standard protocol, and FIG. 2
FIG. 3 is a photograph showing the morphology of cells cultured in bone marrow cells (BM) at various GM-CSF concentrations using a standard protocol
FIG. 4 is a graph showing that the number of CD11c + MHCII + dendritic cells is increased as the concentration of GM-CSF is increased, and the graph on the lower graph shows that the amount of GM-CSF is 0.1 wt% (10 ng purified by Western blot analysis / ml) < / RTI > in the presence of GM-CSF,
FIG. 5 is a graph showing the production steps (A) and (B) of the immortalized dendritic cell-derived cell line (GJ12.3 cell line) as a CD11c + MHC II high cell obtained by repeating the flow cytometry classification method
Figure 6 shows that the immortalized DC2.4 cell line lost ability to present antigen to T cells and the early GJ12.3 cell line showed no
FIG. 7 is a photograph showing the morphology of the cells cultured in the immortalized DC2.4 cell line and the GJ12.3 cell line
Fig. 8 is a graph showing the change in the expression amount of MHC II according to the incubation time of the GJ12.3 cell line
FIG. 9 shows that the GJ12.3 cell line at the initial stage of culture can effectively stimulate allogeneic T cells in the mixed leukocyte reaction (MLR), but the long-term cultured GJ12.3 cell line stimulates allogeneic T cells Graph showing that you can not
FIG. 10 is a graph showing changes in expression of MHC-II and CD11c according to long-term culture in the GJ12.3 cell line,
FIG. 11 is a graph showing changes in expression of MHC1, CD80, CD86, CD40 and CD14 in a GJ12.3 cell line as compared with that at the initial stage of culture
FIG. 12 is a graph showing changes in expression of Ly6c, Ly6G, Gr-1, CD11b, and F4 / 80 in the GJ12.3 cell line,
FIG. 13 is a graph showing changes in expression of B220, PDCA1, 33D1, DEC205 and CD103 in the GJ12.3 cell line as compared with that at the initial stage of culture
FIG. 14 is a graph showing changes in expression of CD62L, CD117, CD135, CD4 and CD8 in a GJ12.3 cell line as compared with that at the initial stage of culture
FIG. 15 is a graph showing changes in the expression levels of CD11c and MHC II in GM-CSF-treated bone marrow cells (BM)
FIG. 16 is a graph showing changes in the expression amounts of MHC II, CD11c, MHC I and CD80 according to the culture period of GM-CSF-treated bone marrow cells (BM)
FIG. 17 is a graph showing changes in the expression amounts of CD86, CD40, CD14, CD24 and CD83 according to the culture period of GM-CSF-treated bone marrow cells (BM)
FIG. 18 is a graph showing changes in expression amounts of Ly6c, Ly6G, Gr-1, CD11b and F4 / 80 according to the culture time of GM-CSF-treated bone marrow cells (BM)
FIG. 19 is a graph showing changes in the expression amounts of B220, PDCA1, 33D1, DEC205 and CD103 according to the culture period of GM-CSF-treated bone marrow cells (BM)
FIG. 20 is a graph showing changes in the expression amounts of CD62L, CD115, CD117, CD135 and CD172 according to the culture period of GM-CSF-treated bone marrow cells (BM)
FIG. 21 is a graph showing changes in the expression amounts of CD4, CD8, CD19, CD49b and NK1.1 according to the culture period of GM-CSF-treated bone marrow cells (BM)
FIG. 22 is a graph showing changes in the amount of Ly6A / E and TER119 expressed by GM-CSF-treated bone marrow cells (BM)
23 is a graph showing the shape of a bone marrow cell (BM) treated with GM-CSF according to the incubation period (6 days, 24 weeks)
FIG. 24 shows that dendritic cell-like cells obtained by culturing bone marrow cells (BM) with GM-CSF for a prolonged period of time as compared to standard dendritic cells cultured with BM-CSF for 6 days in GM-CSF exhibit allogeneic T cells Can not be stimulated by MLR reaction
Figure 25 shows that dendritic cell-like cells prepared from bone marrow (BM) cells, which do not inhibit the MLR response of allogeneic T cells by standard dendritic cells, Graph showing effective inhibition
Hereinafter, the present invention will be described in detail.
The present invention provides immunosuppressed dendritic cell-like cells prepared by treating cytokine GM-CSF with bone marrow cells for a period of 4 weeks or more. When bone marrow cells were cultured for more than 3 weeks, the immune enhancement function, a characteristic feature of standard dendritic cells, was drastically reduced in antigen presentation and in MLR reaction, while GM- When cultured in excess of the week, the immunity enhancing function disappears and the immunosuppressive function tends to be obtained.
Also provided are immunocompromised dendritic cell-like cells prepared by treating cytokine GM-CSF with bone marrow cells for a period of greater than 16 weeks. When GM-CSF is administered to bone marrow cells for more than 16 weeks, CD11c and MHC II molecules are expressed on the cell surface as shown in the following experimental results. However, auxiliary molecules such as CD40, CD83 and CD86 molecules are not expressed in the dendritic cells Cell-like cells.
Also provided are immunocompromised dendritic cell-like cells prepared by treating cytokine GM-CSF with bone marrow cells for a period of more than one year. Although CD11c and MHC II molecules are expressed on the cell surface even when GM-CSF is administered to bone marrow cells for more than one year, auxiliary molecules such as CD40, CD83 and CD86 molecules are not expressed in the dendritic cells Cell-like cell characteristics are maintained.
In order to treat the cytokine GM-CSF to bone marrow cells, it is preferable to cultivate bone marrow cells in a medium containing cytokine GM-CSF. As the medium containing cytokine GM-CSF, DMC7 medium can be used. The composition of the DMC7 medium was described in Experimental Example 1 below.
The culture medium containing the cytokine GM-CSF preferably contains GM-CSF at 10 ng / ml or more. At this time, the culture medium containing GM-CSF expressing cell culture supernatant at 0.1% by weight can be used to adjust the culture medium concentration of GM-CSF to 10 ng / ml or more.
It is preferable that the immunocompromised dendritic cell-like cells have CD11c and MHC II molecules expressed on the cell surface.
It is preferable that the immunosuppressive dendritic cell-like cells have CD40, CD83 and CD86 molecules not expressed or reduced on the cell surface. At this time, the criterion for the reduction is standard dendritic cells prepared by treating GM-CSF with bone marrow cells within a standard period of 2 weeks.
Such immunosuppressive dendritic cell-like cells have the ability to inhibit mature dendritic cells from stimulating T cells when mixed with mature dendritic cells.
The present invention also relates to immunosuppressive dendritic cell-like cells prepared from immune cell populations isolated from bone marrow, comprising: (1) culturing immune cells isolated from bone marrow with cytokine GM-CSF for a period of 4 weeks or more Lt; RTI ID = 0.0 > dendritic < / RTI > cell-like cells. However, the period of more than 4 weeks may be longer than 16 weeks or more than 1 year depending on the immunosuppressive ability standard.
(2) separating the cells expressing CD11c and MHC-II on the cell surface after the step (1); And (3) isolating cells in which the CD40, CD83 and CD86 molecules have not been expressed or reduced on the cell surface among the cells that have undergone the step (2).
As used herein, 'standard' dendritic cells are used in many laboratories to cultivate dendritic cells or dendritic cell-like cells in which bone marrow (BM) cells are cultured for 5-7 days by adding GM-CSF Quot; refers to dendritic cells or dendritic cell-like cells that are produced by the method of the present invention.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Experimental Example 1. Preparation of cells and antibodies
The mice to be used for the experiment were raised at a specific germ-exclusion animal facility of the Laboratory Animal Department of Yonsei University's Yonsei University College of Medicine. OT-1 and OT-2 mice are transgenic mice (Tg) to, anti-ovalbumin T cell receptor (TCR) holds, corn transgenic CD45.1 (B6.SJL-Ptprc a Pepc b / BoyJ) C57BL / 6 mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). C57BL / 6 and BALB / c mice were purchased from Jackson Laboratory and Orient Bio (Seongnam, Korea). Animal breeding and experiments were carried out according to guidelines and protocols established by the Institutional Animal Care and Use Committee of the Yonsei University Medical College.
Cells were cultured in DMC7 medium. DMC7 medium was supplemented with 7% of L-glutamine, glucose, pyruvic acid (Catalog No. SH30243, HyClone, Logan, UT) and non-essential amino acid (HyClone) And a DMEM containing a 1X fetal calf serum (FCS: HyClone) solution. Chinese hamster ovary (CHO) cells (CHO-S, Gibco, Life Technologies, Carlsbad, Calif.) Were purchased. The DC2.4 cell line (Shen et al., 1997) was provided by permission of Dr. Kenneth Rock (of the University of Massachusetts) and by Dr. Matthew Moake (Johns Hopkins University). The J2 retroviral vector encoding the myc and raf cancer genes of the virus was provided by Dr Ulf Rapp (University of Würzburg, Germany).
Experimental Example 2. Production of GM-CSF from Rats from CHO Cells
CDNA of mouse granulocyte / macrophage colony stimulating factor (GMCSF) was cloned by RT-PCR on all RNA samples of spleen prepared from C57BL / 6 mice. It then produces constructs consisting of GM-CSF (Park et al., 2008), internal ribosome entry site (IRES) and improved green fluorescent protein (EGFP) tagged with soluble FLAG and OLLAS. For example, SFO.GMCSF-IRES-RGFP. The GenBank accession number of the SFO.GMCSF sequence is KR029571 and the IRES-EGFP sequence is from pIRES-EGFP (Clontech, Mountain View, CA). Then, CHO cells were transformed with Lipofectamin 2000 reagent (Life Technologies). Here, as a mammalian expression vector, a plasmid DNA containing SFO.GMCSF-IRES-EGFP, a gene which is under the CMV promoter and has resistance to neomycin, and a pEGFP-N1 plasmid (Clontech) as a backbone is used 1A). Then, the CHO / GM-CSF cell line stably expressing SFO.GMCSF-IRES-EGFP was prepared by the following procedure. i) Transformed CHO cells were treated with G418 (1.5 mg / ml) for 1 week. ii) Increase the concentration of CHO cells that are highly positive for EGFP using a FACSAria II cell sorter (BD Biosciences). iii) EGFP-expressing cells classified as FACSAria II cell classifiers are cloned into CHO cell lines with limited dilution in 96-well tissue culture plates. iv) The cloned CHO cell line is selected after testing EGFP expression levels (measured by FACS, see FIG. 1B) and GM-CSF secretion levels (measured by Western blot, see FIG. 1C). Selected CHO cells (CHO / GM-CSF) expressing GM-CSF are cultured in DMC7 medium in a cell culture bag (LAMPIRE, Pipersville, PA). The medium in this state is filtered by a 0.22 μm filter (EMD Milipore, Billerica, MA) and sterilized before it is used for bone marrow cell culture.
Experimental Example 3. Bone marrow culture
A whole bone marrow cell (BM) suspension is prepared from the femur and tibia of C57BL / 6 mice as described in the paper (Inaba et al., 2009). BM cells are then counted and cultured in a 24-well tissue culture plate at a ratio of 1 × 10 6 per well containing GM-CSF at different concentrations in DMC7 medium. CHO cells cultured in conditioned media express GM-CSF as mentioned above. For short-term (up to 7 days) cultured BM cells, half of the medium is carefully removed for each well and supplemented every 2-3 days with fresh GM CSF-containing DMC7 medium. In this process, unattached or loosely adherent cells are retrieved for testing. For long-term culture of BM cells, unattached or loosely adherent cells are recovered from each well every 1-2 weeks via gentle pipetting and suction and culture medium recovery for analysis. Long-term cultured BM cells are supplemented with fresh GM-CSF-containing DMC7 medium as in the case of short-term culture.
Experimental Example 4: Allogeneic mixed lymphocyte reaction
Cell suspensions prepared by pulverizing spleen and lymph nodes in BALB / c mice with a frosted glass cell strainer (BD Biosciences) are incubated with a mixture of biotin-conjugated mAb for CD19, CD49b and MHC class II. After washing with detached buffer (containing 2% FCS and 2 mM EDTA in PBS), antibody-reactive cells are removed by biotin-conjugated magnetic beads (Dynabeads, Life Technologies). Concentrated T cells (1 × 10 7 cells / ml) were mixed with 1 μL of 0.5 mM CFSE solution, cultured at 37 ° C. for 10 minutes, and then washed with PBS by adding DMC7 medium. Four 5-10
EXPERIMENTAL EXAMPLE 5 Treatment of bone marrow cells with GM-CSF to form immortalized cell lines
BM cells are separated from C57BL / 6 mice and cultured in 24-well tissue culture plates in DMC7 medium containing 5% (v / v) CHO / GM-CSF cell culture supernatant.
During the cultivation of GM-CSF and BM cells, the cells were treated with J2 retroviruses encoding the myc and raf cancer genes on
Experimental Example 6: Antigen-specific T cell response
Ovalbumin-specific OT-1 and OT-2 cells are isolated from the spleen and lymph node of OT-1 and OT-2 transgenic (Tg) mice. And labeled with CFSE as described above through mixed lymphocyte reaction. Cells cultured with GM-CSF for 5 days in syngeneic bone marrow derived cells were pulsed with ovalbumin OT-1 peptide (SIINFEKL, 1 μg / ml) for 30 minutes at 4 ° C, and the OT-2 peptide (ISQAVHAAHAEINEAGR, 10 [mu] g / ml) for 1 hour at 37 [deg.] C.
Cells exposed to varying amounts of antigen are cultured in 96-well tissue culture plates with 5 x 10 < 4 > CFSE labeled T cells. After 4-6 days, the response of CFSE labeled T cells is measured with a FACSVerse ( TM) flow cytometer (BD Biosciences).
Experimental Example 7. Western blot analysis
The purified protein or culture supernatant from CHO cell transfectants was mixed with 2X sample loading buffer (0.5 M Tris, 4.4% SDS, 20% glycerol, 4% β-mercaptoethanol, 0.01% bromophenol blue) Boil and load on a 12% SDS-polyacrylamide gel.
After electrophoresis and blotting on PVDF membranes, OLLAS-tagged proteins are detected by incubation with a hybridoma culture supernatant diluted in OLLA-2, antiOLLAS monoclonal antibody, 200: 1.
The blots were then incubated with HRP-rat IgG antibody (Southern Biotech, Birmingham, AL) complex and incubated with chemiluminescent reagent (ECL Plus, GE Healthcare Life Sciences, Pittsburgh, Pa.) And ImageQuant ™ LAS 4000 instrument ).
Multiple samples were tested and statistical comparative analyzes were performed for different groups using SigmaPlot (Systat Software, San Jose, Calif.). Statistical significance is shown in the figure as a probability value (0.05, 0.01, 0.001).
Experiment result
Experimental Example of Rat GM-CSF Production from CHO Cells. 2, in order to produce GM-CSF from rats from CHO cells, FLAG tag and OLLAS tag for soluble GMCSF cDNA and aqueous solution were arranged under the CMV promoter as shown in Fig. 1A, IRES and EGFP were allowed to express and CHO cells were transformed. (FIG. 1B), CHO cells showing high GM-CSF expression were classified as CHO / GM-CSF. (FIG. 1C), anti-OLLAS Western blot analysis indicated that the CHO / GM-CSF status medium contained approximately 10-20 μg / ml of GM-CSF.
Experimental example with bone marrow culture. 3, dendritic cells (DCs) were cultured from bone marrow cells using a standard protocol, as shown in Fig. 2 (Fig. 2). The cultures were cultured for 5 days using 0.01% to 10% different amounts of CHO / GM-CSF and no control medium. (FIG. 2A) and (FIG. 2B), the higher the concentration of GM-CSF, the greater the number of living nonadherent cells. Bone marrow (BM) cells cultured in the presence of GM-CSF produce a large number of DC expressing CD11c and MHC II (see FIG. 2A). And has a dendritic cell (DC) shape (see FIG. 3). The higher the concentration of GM-CSF, the greater the number of dendritic cells (see graph in FIG. 4). However, it can be seen in the presence of more than 0.1% of GM-CSF in the medium that some of the DC cells are still in a steady state (lower graph of FIG. 4).
Experimental example in which bone marrow cells were treated with GM-CSF to form an immortalized cell line. 5, the immortalized DC2.4 cell line lost MHC II expression ability, and although it has the shape of dendritic cells (see Fig. 7), the antigen is present in T cells (See FIG. 6). The dendritic cell-derived cell line obtained by repeated dilution of the CD11c + MHC II high cells (see FIGS. 5A and 5B) obtained by repeating the flow cytometry classification method in the present invention was named GJ12.3 cell line (FIG. 6) And (Fig. 7), the CD11c and MHC II expressions are high and have a dendritic cell (DC) shape.
Experimental example of the characteristics of the GJ12.3 cell line prepared in the present invention and whether it can elicit T cell response. 6, the DC2.4 cell line showed low MHC II expression, and the GJ12.3 cell line generated in the present invention showed that the expression of MHC II was continuously lowered for 10 weeks, The expression of MHC I is also maintained after 10 weeks (see FIG. 8). GJ12.3 cell lines, which were only recently produced, were able to stimulate allogeneic T cells effectively in mixed leukocyte reaction (MLR) as well as BM cells cultured with GM-CSF (see Figure 9) . However, it was confirmed that the ability of T cells to elicit an immune response to the ovalbumin OT-1 peptide and the OT-2 peptide decreased after 11 weeks (see FIG. 10). In addition, cell surface molecules other than CD11c and MHC II were compared over various periods of culture, with the result that there was no significant change in expression of molecules other than MHC II (see FIGS. 11, 12, 13 and 14).
The long-term culture of BM cells grown in GM-CSF-supplemented medium with high CD11c and MHC II expression levels was not homogeneous in the first month, but after 6 months, the CD11c + MHC II + (See Fig. 15), and most other cell surface molecules such as MHC I and CD80 also change more homogeneously over time (Figs. 16, 17, 18, 19, 20 , Fig. 21 and Fig. 22), it was confirmed that the shape of the dendritic cells did not change even when cultured for a long period of time (see Fig. 23).
Eventually, when long-term bone marrow (BM) cells are co-cultured with GM-CSF, high-level MHC II surface-expressed cells can be obtained (see FIG. 15), but these cells stimulate allogeneic T cells with MLR (Fig. 24). ≪ tb > < TABLE > The ability to rapidly stimulate CD4 + T cells and CD8 + T cells was reduced. This appears to be due to the reduced expression of T-cell stimulatory molecules (CD40, CD83, CD86), despite the presence of MHC II molecules. Furthermore, in order to investigate whether supernatant of BM cells cultured with GM-CSF for a long period of time has a supressive effect on T cell responses, we examined whether MLR responses of allogeneic T cells were normal 25). ≪ / RTI >
In conclusion, dendritic cells produced by standard methods of GM-CSF-mediated bone marrow (BM) cell culture for 5-7 days are able to stimulate T cells, Non-homogeneous bone marrow-derived cells of dendritic cells. On the other hand, when bone marrow cells were cultured with GM-CSF for more than 4 weeks and over a period of more than 1 year, cells expressing homogeneously high levels of MHC II and CD11c molecules were produced but lost ability to stimulate T cells Cells. It was confirmed that mixing the cells prepared by culturing the bone marrow with GM-CSF for a long period of time with the dendritic cells capable of stimulating T cells prepared by standard methods can suppress the immune response Thereby completing the invention.
Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
Treatment of the bone marrow cells with the cytokine GM-CSF,
A dendritic cell-like cell for immunosuppression characterized by culturing bone marrow cells in a medium containing cytokine GM-CSF.
The medium containing the cytokine GM-CSF,
GM-CSF in an amount of 10 ng / ml or more.
The immunosuppressive dendritic cell-
Wherein the CD11c and MHC II molecules are expressed on the cell surface.
The immunosuppressive dendritic cell-
Wherein the CD40, CD83 and CD86 molecules are not expressed or reduced on the cell surface.
(1) culturing immune cells isolated from bone marrow by treating cytokine GM-CSF for a period of more than 16 weeks,
Production of dendritic cell-like cells for immunosuppression, inhibiting the antigen presenting ability of mature dendritic cells when mixed with mature dendritic cells, inhibiting mature dendritic cells from stimulating T cells Way.
After the step (1)
(2) separating cells expressing CD11c and MHC-II on the cell surface; And
(3) separating the cells in which the CD40, CD83 and CD86 molecules have not been expressed or reduced in the cells subjected to the step (2), to prepare a dendritic cell-like cell for immunosuppression Way.
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