METHOD FOR INCREASING DENDRITIC CELL MIGRATION ABILITY, AND USE THEREOF

Abstract

A method for increasing dendritic cell migration ability, and a use thereof are disclosed. A method according to one aspect can increase the migration ability of mature dendritic cells and increase the induction, by dendritic cells, of inflammatory cytokine production, T lymphocyte proliferation and T lymphocyte polarization, and thus can be used for the prevention or treatment of immune-related diseases.

Claims

1. A method of preparing mature dendritic cells having increased migration ability, the method comprising contacting immature dendritic cells with autotaxin.

2. The method of claim 1, wherein the immature dendritic cells are immature dendritic cells, semi-mature dendritic cells, or a combination thereof.

3. The method of claim 1, wherein the immature dendritic cells are obtained by culturing bone marrow cells from which red blood cells were removed.

4. The method of claim 1, wherein the contacting is performed in an RPMI medium.

5. The method of claim 4, wherein the medium comprises fetal bovine serum (FBS), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL), and mercaptoethanol.

6. The method of claim 1, further comprising contacting the immature dendritic cells with lipopolysaccharide (LPS), keyhole limpet hemocyanin (KLH), or a combination thereof.

7. The method of claim 1, wherein the method increases induction of inflammatory cytokine production, induction of T lymphocyte proliferation, or induction of T lymphocyte polarization of dendritic cells.

8. A pharmaceutical composition for preventing or treating a disease selected from the group consisting of autoimmune diseases, cancers, infectious diseases, and inflammatory diseases, the pharmaceutical composition comprising mature dendritic cells having increased migration ability, the mature dendritic cells being prepared by the method of claim 1.

9. Mature dendritic cells having increased migration ability, which are prepared by the method of claim 1.

10. Use of mature dendritic cells having increased migration ability, the mature dendritic cells being prepared by the method of claim 1, in preparing a medicament for preventing or treating a disease selected from the group consisting of autoimmune diseases, cancers, infectious diseases, and inflammatory diseases.

11. A method of preventing or treating a disease selected from the group consisting of autoimmune diseases, cancers, infectious diseases, and inflammatory diseases, the method comprising administering an effective amount of mature dendritic cells having increased migration ability, the mature dendritic cells being prepared by the method of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0055] FIG. 1 shows a graph showing RT-PCR results of examining autotaxin gene expression in autotaxin-specific siRNA-treated mature dendritic cells and control dendritic cells (*p<0.05);

[0056] FIG. 2 shows a graph showing Western blotting results of examining autotaxin protein expression in autotaxin-specific siRNA-treated mature dendritic cells and control dendritic cells (***p<0.001);

[0057] FIG. 3A shows histograms showing expression of the surface antigens CD11c, CD14, CD40, CD54, CD80, CD86, MHCI, and MHCII in mature dendritic cells (ATX siRNA), in which autotaxin expression was suppressed by autotaxin-specific siRNA, a negative control group, and mature dendritic cells (mDCs);

[0058] FIG. 3B shows fluorescence intensity showing expression of the surface antigens CD11c, CD14, CD40, CD54, CD80, CD86, MHCI, and MHCII in mDCs (ATX siRNA), in which autotaxin expression was suppressed by autotaxin-specific siRNA, a negative control group, and mDCs;

[0059] FIG. 4 shows a graph showing ELISA results of examining inflammatory cytokine concentrations in a co-culture medium of mDCs (ATX siRNA), in which autotaxin expression was suppressed by autotaxin-specific siRNA, and mDCs with T lymphocytes (*p<0.05);

[0060] FIG. 5 shows experimental results of examining T cell proliferation ability in autotaxin gene expression-suppressed mDCs (ATX siRNA) and mDCs;

[0061] FIG. 6 shows flow cytometry results of examining IFN-/CD4 and IL-17/CD4 in autotaxin gene expression-suppressed mDCs (ATX siRNA) and normal mDCs;

[0062] FIG. 7 shows a graph showing ELISA results of examining cytokine concentrations in a co-culture medium of autotaxin gene expression-suppressed mDCs (ATX siRNA) and mDCs with CD3+ T cells;

[0063] FIG. 8 shows Western blotting results of examining Rho A protein;

[0064] FIG. 9 shows a graph showing migration assay results of examining in vitro migration ability of mDCs according to suppression of autotaxin gene expression;

[0065] FIG. 10 shows a graph showing qRT-PCR results of examining changes in CCR7 expression in mDCs according to suppression of autotaxin gene expression;

[0066] FIG. 11 shows photographs showing Western blotting results of examining pp38, ERK 1/2, pJNK, and NF-B protein levels in mDCs, a negative control group, mDCs in which autotaxin expression was suppressed by autotaxin-specific siRNA, HA130-treated mDCs, and PF8380-treated mDCs;

[0067] FIG. 12A shows histograms showing flow cytometry results of examining cell surface marker expression in mDCs having improved migration ability, to which a recombinant autotaxin protein has been added during culture, and in mDCs to which no recombinant autotaxin protein has been added during culture;

[0068] FIG. 12B shows a graph showing quantification of flow cytometry results of examining cell surface marker expression in mDCs having improved migration ability, to which the recombinant autotaxin protein has been added during culture, and in mDCs to which no recombinant autotaxin protein has been added during culture;

[0069] FIG. 13 shows graphs showing ELISA results of examining expression levels of inflammatory cytokines, IL-1B, IL-6, TNF-, and IL-12p70 in mDCs (+) to which autotaxin protein has been added and mDCs () to which autotaxin protein has not been added (*p<0.05, **p<0.01);

[0070] FIG. 14 shows results of examining proliferation ability of T lymphocytes in mDCs (+) to which autotaxin protein has been added and mDCs () to which autotaxin protein has not been added;

[0071] FIG. 15 shows flow cytometry results of examining IFN-/CD4 and IL-17/CD4 in mDCs (+) cultured with the addition of autotaxin protein and mDCs () cultured without addition of autotaxin protein;

[0072] FIG. 16 shows a graph showing ELISA results of examining cytokine concentrations in a co-culture medium of mDCs (+) cultured with the addition of autotaxin protein and mDCs () with CD3+ T cells;

[0073] FIG. 17 shows a photograph showing ELISA results of examining RhoA protein expression in mDCs (+) cultured with the addition of autotaxin protein and mDCs ();

[0074] FIG. 18 shows a graph showing results of a migration assay in mDCs (+) cultured with the addition of autotaxin protein and mDCs () (***p<0.001);

[0075] FIG. 19 shows a photograph showing Western blotting results of examining intracellular pp38, ERK 1/2, pJNK, and NF-B protein expression levels in mDCs (+) cultured with the addition of autotaxin protein and mDCs ();

[0076] FIG. 20 shows a photograph showing a trace of migration to popliteal lymph nodes by mDCs (+) cultured with the addition of autotaxin protein and mDCs (), over 3 days;

[0077] FIG. 21 shows a graph showing quantification of the proportion of mDCs (+) cultured with the addition of autotaxin protein and mDCs () that migrated to popliteal lymph nodes over 3 days;

[0078] FIG. 22 shows confocal microscopy results of analyzing the proportion of mDCs (+) cultured with the addition of autotaxin protein and mDCs () that migrated to popliteal lymph nodes over 3 days; and

[0079] FIG. 23 shows flow cytometry results of examining mDCs after separating lymphocytes from removed popliteal lymph nodes and staining the lymphocytes with CD11c antibody.

MODE OF DISCLOSURE

[0080] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

Example 1: Examination of Effects of Autotaxin Gene on Characteristics of Mature Dendritic Cells

[0081] Effects of autotaxin gene on characteristics of mature dendritic cells were examined by the following experiments.

[0082] 1.1 Suppression of Autotaxin Gene Expression Level in Mature Dendritic Cells by Autotaxin-Specific siRNA

[0083] Autotaxin gene expression levels in mature dendritic cells were suppressed by using autotaxin-specific siRNA. In detail, siRNA (Bioneer, Daejeon, Korea) targeting mouse Enpp2 mRNA was introduced into cells at a concentration of 100 nM using a lipofectamine 3000 (Invitrogen, CA, USA) system. On the 6.sup.th day after dendritic cell culture, all cells were collected and suspended in a basic dendritic cell medium containing 5% fetal bovine serum, and dispensed at a density of 210.sup.5 cells/well in a 6-well culture plate. 4 hr after siRNA transfection, maturation of siRNA-treated dendritic cells was induced for 24 hr by replacing the medium with a dendritic cell culture medium containing LPS, and a fresh dendritic cell culture medium was replaced for an immature dendritic cell group. Here, scramble siRNA (Bioneer) was used as a negative control group, and the experiment was repeated in triplicate. Information about siRNA targeting Enpp2 mRNA is shown in Table 1.

TABLE-US-00001 TABLE1 siRNA Sequence Enpp2 5-GGGUCUUGGUGAAGAAAUAdTdT-3 siRNA-1 Enpp2 5-UAUUUCACCAAGACCCdTdT-3 siRNA-2

[0084] Expression of autotaxin gene and protein in autotaxin-specific siRNA-treated mature dendritic cells and the negative control group was examined by qRT-PCR and Western blotting, respectively. For qRT-PCR, a labozol reagent (Cosmo genetech, Seoul, Korea) was used to isolate RNA from dendritic cells. cDNA was synthesized from the isolated RNA using a cDNA synthesis kit (Cosmo genetech). A SensiFAST SYBR No-ROX kit (Bioline, Sydney, Australia) was used in quantitative real-time PCR of Enpp2 gene. Reaction conditions are as follows: a reaction at 95 C. for 10 min; 35 cycles of reactions at 94 C. for 20 sec, at 62 C. for 30 sec, and at 72 C. for 20 sec; and a final reaction at 72 C. for 5 min. Gene expression levels were analyzed by normalizing a threshold cycle (Ct) value of each gene to a Ct value of GAPDH, and then by comparing changes of the Ct values.

[0085] For Western blotting, dendritic cells were suspended in an RPMI 1640 medium supplemented with 2% fetal bovine serum, and then dispensed at a density of 110.sup.6 cells/well in a 6-well plate, and cultured under conditions of 37 C. and 5% CO.sub.2 for 24 hr. 24 hr later, the cell culture medium was collected and concentrated by centrifugation at 3000 g for 20 min using an Amicon Ultra-2 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore, Germany). Intracellular proteins of dendritic cells were extracted using PRO-PREP (iNtRON Biotechnology, Gyeonggi, Korea) and a phosphatase inhibitor cocktail (Calbiochem, CA, USA). Protein concentrations were determined by a Bradford (Thermo, MA, USA) assay. The quantified proteins were separated on SDS-PAGE, and the separated proteins were adsorbed onto a PVDF membrane (Biorad, CA, USA). The PVDF membrane was blocked with 5% skim milk in PBST at room temperature for 1 hr, and treated with a 1:2000 dilution of a primary antibody with 5% skim milk, and then allowed to react at 4 C. for 18 hr. Thereafter, the membrane was washed with 1PBST for 10 min three times, and then treated with a secondary antibody at 1:5000, and then allowed to react at room temperature for 2 hr. The membrane was washed again with 1PBST three times, and then exposed to an ECL solution (Thermo) to visualize protein bands using LAS-4000 (Fuji film, Tokyo, Japan). Results were analyzed using a Multi Gauge software V3.0 (Fuji film).

[0086] FIG. 1 shows a graph showing RT-PCR results of examining autotaxin gene expression in autotaxin-specific siRNA-treated mature dendritic cells and control dendritic cells (*p<0.05).

[0087] FIG. 2 shows a graph showing Western blotting results of examining autotaxin protein expression in autotaxin-specific siRNA-treated mature dendritic cells and control dendritic cells (***p<0.001).

[0088] As shown in FIGS. 1 and 2, 25% or more reduction in autotaxin (ATX) mRNA and protein expression was observed in autotaxin-specific siRNA-treated mature dendritic cells.

[0089] 1.2 Examination of Changes in Surface Phenotype Expression in Mature Dendritic Cells According to Suppression of Autotaxin Gene Expression

[0090] To examine a relationship between the autotaxin gene expression and surface phenotype expression, flow cytometry was performed as follows. 210.sup.5 cells to 510.sup.5 cells were washed with PBS (Lonza), and then stained with fluorescence-labeled monoclonal antibodies. The used monoclonal antibodies for flow cytometry are shown in Table 2 below. To examine cell viability, propidium iodide (PI) staining was performed. For staining of cytokines in T cells, cells were treated with GolgiStop (BD Bioscience, CA, USA) for 4 hr. 4 hr later, T cells were collected and CD4 was stained, and then put and fixed in a fixation/permeabilization solution (BD) at room temperature for 30 min. After washing with FACS buffer, staining was performed using IFN- and IL-17A monoclonal antibodies for 30 min. Fluorescence-labeled cells were washed with FACS buffer, and then detected using a FACS Calibur (BD). All data were analyzed using FlowJo (Tree Star, CA, USA).

TABLE-US-00002 TABLE 2 Specificity Clone Conjugates Supplier CD11c N418 PE eBioscience CD14 Sa14-2 FITC Biolegend CD40 3/23 PE BD CD54 3E2 FITC BD CD80 16-10A1 PE eBioscience CD86 PO3 FITC Biolegend H-2Db KH95 FITC Biolegend I-A.sup.b AF6-120.1 PE BD CD4 RM4-5 APC Biolegend IFN- XMG1.2 FITC Biolegend IL-17A TC11-18H10 PE BD

[0091] FIG. 3 shows histograms (A) and fluorescence intensity (B), each showing expression of surface antigens CD11c, CD14, CD40, CD54, CD80, CD86, MHCI, and MHCII in mature dendritic cells (ATX siRNA), in which autotaxin expression was suppressed by autotaxin-specific siRNA, a negative control group, and mature dendritic cells (mDCs).

[0092] As shown in FIGS. 3A and 3B, a similar surface antigen expression was observed in each of the mDCs. In detail, it was confirmed that CD14 which is a monocyte surface antigen was rarely expressed, and CD11c which is a representative surface antigen of dendritic cells was highly expressed. Further, it was confirmed that CD40 which is a T lymphocyte stimulatory molecule, CD54 which is a T lymphocyte adhesion molecule, and CD80 and CD86 which are costimulatory molecules, were highly expressed.

[0093] Accordingly, it was confirmed that changes in the autotaxin gene expression do not affect surface phenotype expression of mDCs.

[0094] 1.3 Examination of Changes in Cytokine Expression in mDCs According to Suppression of Autotaxin Gene Expression

[0095] To examine a relationship between the autotaxin gene expression and cytokines secreted by mDCs, cytokine concentrations in culture media of mDCs were measured using ELISA as follows.

[0096] Splenocytes isolated from C57BL/6 mouse were suspended in an RPMI 1640 containing 10% fetal bovine serum, and passed through a nylon wool column to isolate CD3+T lymphocytes. 210.sup.5 mDCs and 210.sup.6 T lymphocytes were co-cultured in a 6-well pate for 3 days. Cytokine concentrations in the co-culture medium were analyzed using mouse interleukin-1 (IL-1), IL-6, tumor necrosis factor- (TNF-) (Biolegend, CA, USA), and IL-12p70 (BD Bioscience) ELISA kit. Results are expressed as meanSEM.

[0097] FIG. 4 shows a graph showing ELISA results of examining inflammatory cytokine concentrations in the co-culture medium of mDCs (ATX siRNA), in which autotaxin expression was suppressed by autotaxin-specific siRNA (ATX siRNA), and mDCs with T lymphocytes (*p<0.05).

[0098] As shown in FIG. 4, the concentrations of inflammatory cytokines in the cell culture medium of the autotaxin expression-suppressed mDCs were about 15% lower than those in the cell culture medium of the control mDCs.

[0099] Accordingly, concentrations of TNF- which is an inflammatory cytokine representing innate immunity, IL-12 which is a cytokine inducing Th1 immune response, and IL-1p and IL-6 which are representative inflammatory cytokines, were reduced, indicating that immune and inflammatory responses of autotaxin expression-suppressed mDCs were reduced.

[0100] 1.4 Examination of Changes in T Lymphocyte Proliferation Ability of mDCs According to Suppression of Autotaxin Gene Expression

[0101] To examine a relationship between the autotaxin gene expression and T lymphocyte proliferation ability of mDCs, mDCs and CD3+ T cells were co-cultured at a ratio of 1:10 for 72 hr. CD3+ cells were isolated from splenocytes of naive C57BL/6 mouse, and stained with carboxyfluorescein succinmidyl ester (CFSE) at a final concentration of 4 M. CFSE-labeled cells were washed, and counted, and then co-cultured with dendritic cells.

[0102] FIG. 5 shows experimental results of examining T cell proliferation ability in autotaxin gene expression-suppressed mDCs (ATX siRNA) and mDCs.

[0103] As shown in FIG. 5, the autotaxin gene expression-suppressed mDCs showed about 45% lower T lymphocyte proliferation ability than the normal mDCs. Accordingly, it was confirmed that suppression of autotaxin gene expression inhibits T lymphocyte proliferation ability in mDCs.

[0104] 1.5 Examination of mDC-Mediated T Lymphocyte Polarization According to Suppression of Autotaxin Gene Expression

[0105] To examine correlation between the suppression of autotaxin gene expression and mDC-mediated T lymphocyte polarization, the following experiment was performed.

[0106] mDCs and CD3+ T cells were co-cultured at a ratio of 1:10 for 72 hr. The cells were stained with anti-CD4 and anti-IFN- antibodies, or anti-IL-17A antibody, followed by flow cytometry.

[0107] FIG. 6 shows flow cytometry results of examining IFN-/CD4 and IL-17/CD4 in autotaxin gene expression-suppressed mDCs (ATX siRNA) and normal mDCs.

[0108] As shown in FIG. 6, about 50% low differentiation of T cells into Th1 and Th17 subtype cells was observed, when co-cultured with autotaxin gene expression-suppressed mDCs, as compared with mDCs.

[0109] Further, each of mDCs and CD3+ T cells were co-cultured at a ratio of 1:10 for 72 hr, and a cell culture supernatant was harvested. Expression of cytokines (IFN-, IL-17A, IL-4, IL-10) was examined by ELISA. Data were expressed as meanSEM (n=3).

[0110] FIG. 7 shows a graph showing ELISA results of examining cytokine concentrations in a co-culture medium of autotaxin gene expression-suppressed mDCs (ATX siRNA) and mDCs with CD3+ T cells.

[0111] As shown in FIG. 7, low IFN- and IL-17 concentrations in the co-culture medium were observed in the autotaxin gene expression-suppressed mDCs.

[0112] 1.6 Examination of Change in Rho a Protein Expression According to Treatment with Autotaxin Enzyme Activity Inhibitor

[0113] The autotaxin gene expression of mDCs was suppressed by treatment with autotaxin-specific siRNA. However, since autotaxin is released and present in the cell culture medium, HA 130 (Albers, Dong et al. 2010) and PF8380 (Gierse, Thorarensen et al. 2010) which are substances capable of inhibiting autotaxin enzyme activity were added to mDCs during culture, followed by culture for 7 days.

[0114] After addition, Rho A protein which is known to be involved in cell migration in the downstream signal transduction of LPA receptor of which ligand is LPA as a product of autotaxin enzyme was examined by Western blotting, and results are shown in FIG. 8.

[0115] As shown in FIG. 8, when autotaxin-suppressed DCs (ATX siRNA) were treated with HA 130 and PF8380, Rho A protein expression was reduced by about 45% or more, as compared with mDCs.

[0116] 1.7 Examination of In Vitro Migration Ability of mDCs According to Suppression of Autotaxin Gene Expression

[0117] To examine in vitro migration ability of mDCs according to suppression of autotaxin gene expression, a migration assay was performed as follows.

[0118] A transwell plate with 8.0 m pore polycarbonate membrane (Corning, N.Y., USA) was used. mDCs cultured for 7 days were harvested, and then suspended in an RPMI 1640 medium. 510.sup.5 cells/500 L thereof was put in the upper portion of the transwell plate, and 500 L of RPMI 1640 supplemented with 2% fetal bovine serum and 100 ng/mL recombinant mouse CCL19 (R&D systems) was put in the lower portion of the transwell plate. After incubation under conditions of 37 C. and 5% CO.sub.2 for 1 hr, the culture medium in the lower portion of the transwell was transferred to a 5-mL round bottom tube, and then the number of cells was counted using a FACS Calibur for 1 min. Measurement results are shown in FIG. 9.

[0119] As shown in FIG. 9, autotaxin expression-suppressed mDCs (ATX siRNA) showed about 20% reduction in the cell migration ability, as compared with mDCs (negative control group).

[0120] 1.8 Examination of Change in CCR7 Expression According to Suppression of Autotaxin Gene Expression

[0121] CCR7 which is a homing receptor of DCs is known to be involved in migration of DCs. Therefore, to examine whether CCR7 is associated with the change in the migration ability of mDCs according to suppression of autotaxin gene expression, CCR7 expression in mDCs was examined by qRT-PCR, and results are shown in FIG. 10.

[0122] As shown in FIG. 10, it was confirmed that there was little difference in the CCR7 expression levels between the autotaxin expression-suppressed mDCs, negative control group, and mDCs.

[0123] Accordingly, it was confirmed that the change in the migration ability according to suppression of autotaxin expression is regulated independently from CCR7 expression.

[0124] 1.9 Examination of Cause of Cytokine Reduction According to Suppression of Autotaxin Gene Expression

[0125] In above exemplary embodiment, the reduced cytokine expression by suppression of autotaxin gene expression was observed. Therefore, to examine the cause of the reduced cytokine expression, intracellular signaling proteins (pp38, ERK 1/2, pJNK, and NF-B) in mDCs were examined by Western blotting.

[0126] FIG. 11 shows photographs showing Western blotting results of examining pp38, ERK 1/2, pJNK, and NF-B protein levels in mDCs, a negative control group, mDCs in which autotaxin expression was suppressed by autotaxin-specific siRNA, HA130-treated mDCs, and PF8380-treated mDCs.

[0127] As shown in FIG. 11, it was confirmed that pp38 and MAPK(ERK 1/2) expression was significantly reduced in autotaxin expression-reduced mDCs, as compared with the control group.

[0128] Therefore, it was confirmed that the change in the migration ability of mDCs by the reduced autotaxin expression is associated with the change in pp38 and MAPK expression.

[0129] Taken together, the above experimental results showed that the reduced autotaxin expression by siRNA in mDCs deteriorates overall functions of DCs, including reduction of cytokine expression, reduction of T cell proliferation-inducing ability, etc. It was also confirmed that cell migration ability is reduced according to the reduction of autotaxin expression, which is associated with the change in cytokine expression.

Example 2. Preparation of mDCs Having Improved Migration Ability

[0130] Example 1 showed that when autotaxin expression was reduced in mDCs, cell migration ability was reduced. Therefore, it was examined whether cell migration ability may be improved by adding the autotaxin protein which is an extracellular enzyme during culture of the DCs.

[0131] Animal experiments were conducted according to the Institutional Animal Care and Use Committees (IACUC) of CHA University (IRB number: IACUC170111). To isolate DCs, 6-8-week old male C57BL/6 mice were purchased for use from Orient Bio (Seongnam-si, Korea). Mouse bone marrow cells were obtained from tibia and femur of C57BL/6 mouse by washing bone marrow cavity with RPMI 1640 containing 25 mM HEPES (Lonza, MD, USA). Impurities were removed from the obtained bone marrow cells using a 70 m cell strainer, centrifugation was performed at 1600 rpm for 5 min, and then red blood cells were removed using ACK lysis buffer which is a red blood cell lysing agent. The bone marrow cells were suspended in a DC culture medium which is an RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 g/mL streptomycin, 2 mM GlutaMax, 55 nM 2-mercaptoethanol (Gibco, NY, USA), 20 ng/mL recombinant mouse (rm)GM-CSF, 20 ng/mL rmIL-4 (JW CreaGene, Gyeonggi, Korea), and 310.sup.7 cells were dispensed in a 6-well plate, followed by incubation under conditions of 37 C. and 5% CO.sub.2. On the 2.sup.nd day of culture, non-adherent cells were removed, and the DC culture medium of the same composition was added. On the 4.sup.th day of culture, half of the culture medium was collected and replaced with a fresh DC culture medium. On the 6.sup.th day of culture, all cells were collected, and a fresh DC culture medium was replaced for imDCs, and cells to be differentiated into mDCs were suspended in a DC culture medium supplemented with 1 g/mL lipopolysaccharide (LPS) and 10 g/mL KLH (Sigma Aldrich, MO, USA), and maturation was induced for 24 hr. Here, 10 g/mL of the recombinant autotaxin protein (R&D systems, MN, USA) was treated along with LPS stimulation, thereby preparing mDCs having improved migration ability by the above method.

Example 3. Characterization of mDCs Having Improved Migration Ability

[0132] 3.1 Examination of Surface Phenotype Expression

[0133] Surface marker expression was examined by flow cytometry in mDCs having improved migration ability, to which the recombinant autotaxin protein has been added during culture, and mDCs to which autotaxin protein has not been added, and shown in FIGS. 12A and 12B.

[0134] As shown in FIGS. 12A and 12B, mDCs (ATX+) to which autotaxin protein has been added and mDCs (ATX-) to which autotaxin protein has not been added showed similar cell surface marker expression. Accordingly, it was confirmed that addition of autotaxin protein does not affect surface phenotype expression of DCs.

[0135] 3.2 Examination of Inflammatory Cytokine Expression

[0136] To examine the effect of autotaxin protein addition on inflammatory cytokines of DCs, mDCs having improved migration ability or mDCs were co-cultured with T lymphocytes, and concentrations of inflammatory cytokines in the co-culture medium were examined by ELISA. Data were expressed as MeanSEM (n=3).

[0137] FIG. 13 shows graphs showing ELISA results of examining expression levels of inflammatory cytokines, IL-1, IL-6, TNF-, and IL-12p70 in mDCs (+) to which autotaxin protein has been added and mDCs () to which autotaxin protein has not been added (*p<0.05, **p<0.01).

[0138] As shown in FIG. 13, it was confirmed that mDCs cultured with the addition of autotaxin protein produced high concentrations of inflammatory cytokines, as compared with mDCs to which autotaxin protein has not been added.

[0139] 3.3 Examination of T Lymphocyte Proliferation Ability

[0140] To examine the effect of autotaxin protein addition on T lymphocyte proliferation-inducing ability of DCs, mDCs having improved migration ability or mDCs were co-cultured with CD3+ T cells at a ratio of 1:10 for 72 hr. The CD3+ T cells were isolated from splenocytes of naive C57BL/6 mouse, and stained with CFSE.

[0141] FIG. 14 shows results of examining proliferation ability of T lymphocytes in mDCs (+) to which autotaxin protein has been added, and mDCs () to which autotaxin protein has not been added.

[0142] As shown in FIG. 14, it was confirmed that mDCs cultured with the addition of autotaxin protein showed about 15% high T lymphocyte proliferation ability, as compared with mDCs to which autotaxin protein has not been added.

[0143] 3.4 Examination of DC-Mediated T Cell Polarization

[0144] To examine the effect of autotaxin protein addition on DC-mediated T cell polarization, mDCs were co-cultured with CD3+ T cells at a ratio of 1:10 for 72 hr. The cells were stained with anti-CD4 and anti-IFN- antibodies or anti-IL-17A antibody, followed by flow cytometry.

[0145] FIG. 15 shows flow cytometry results of examining IFN-/CD4 and IL-17/CD4 in mDCs (+) cultured with the addition of autotaxin protein and mDCs (). IFN- is Th1 cytokine, and IL-17 is Th17 cytokine.

[0146] As shown in FIG. 15, it was confirmed that when T cells were co-cultured with mDCs having improved migration ability cultured with the addition of autotaxin protein, they were differentiated into Th1 and Th17 subtypes about 1.5 times higher than those co-cultured with mDCs to which autotaxin protein has not been added.

[0147] Further, each of mDCs was co-cultured with CD3+ T cells at a ratio of 1:10 for 72 hr, and cell culture media were harvested, and cytokine (IFN-, IL-17A, IL-4, and IL-10) expression was examined by ELISA. Data were expressed as MeanSEM (n=3).

[0148] FIG. 16 shows a graph showing ELISA results of examining cytokine concentrations in the co-culture medium of mDCs (+) cultured with the addition of autotaxin protein and mDCs () with CD3+ T cells.

[0149] As shown in FIG. 16, high IFN- and IL-17 concentrations in the co-culture medium were observed in autotaxin expression-suppressed mDCs.

[0150] 3.5 Examination of Rho a Protein and In Vitro Migration Ability

[0151] To examine the effect of autotaxin protein addition on a Rho A protein expression level in mDCs and migration ability of mDCs, experiments were performed in the same manner as in Examples 1.6 and 1.7.

[0152] FIG. 17 shows a photograph showing ELISA results of examining RhoA protein expression in mDCs (+) cultured with the addition of autotaxin protein and mDCs ().

[0153] As shown in FIG. 17, it was confirmed that RhoA protein expression was significantly increased by about 1.5 fold or more in mDCs having improved migration ability, which were cultured with the addition of autotaxin protein.

[0154] FIG. 18 shows a graph showing results of the migration assay in mDCs (+) cultured with the addition of autotaxin protein and mDCs () (***p<0.001).

[0155] As shown in FIG. 18, it was confirmed that mDCs having improved migration ability, which were cultured with the addition of autotaxin protein, showed about 15% or more increased in vitro migration ability.

[0156] 3.6 Examination of Intracellular Protein Expression

[0157] To examine the effect of autotaxin protein addition on intracellular signaling proteins (pp38, ERK 1/2, pJNK, NF-B) in mDCs, expression levels of the proteins were examined by Western blotting.

[0158] FIG. 19 shows a photograph showing Western blotting results of examining intracellular pp38, ERK 1/2, pJNK, and NF-B protein expression levels in mDCs (+) cultured with the addition of autotaxin protein and mDCs ().

[0159] As shown in FIG. 19, it was confirmed that pp38, pJNK, and ERK1/2 protein expression was significantly increased in mDCs cultured with the addition of autotaxin protein.

Example 4. Evaluation of In Vivo Migration Ability of mDCs Cultured with

[0160] Addition of Recombinant Autotaxin Protein During Culture

[0161] To examine in vivo whether the migration ability of mDCs cultured with the addition of the recombinant autotaxin protein during culture was improved, migration of DCs was tracked.

[0162] In detail, DCs labeled with Near infrared dye (NIR) using a CellVue NIR815 Midi Kit for Membrane Labeling (Polysciences Inc., Warrington, UK) were suspended in PBS, and then 110.sup.5 cells/50 L were subcutaneously injected into the paw pad of C57BL/6 mouse. At 24 hour intervals immediately after injection, DC migration was tracked using Pearl Impulse (LI-COR biotechnology, NE, USA) equipment for 72 hr. Imaging was performed using a near-infrared 800-nm channel emitted at a wavelength of 778 nm and detected at 794 nm. An image on the 3.sup.rd day after injection was used as a representative image.

[0163] FIG. 20 shows a photograph showing a trace of migration to popliteal lymph nodes by mDCs (+) cultured with the addition of autotaxin protein and mDCs (), over 3 days.

[0164] As shown in FIG. 20, mDCs cultured with the addition of autotaxin protein showed 4 times higher migration to popliteal lymph node on average, as compared with mDCs cultured without the addition of autotaxin protein.

[0165] Accordingly, it was confirmed that migration ability of mDCs was improved by addition of autotaxin protein during culture.

[0166] Further, after converting the total fluorescence intensity detected in the mouse to 100%, the migration efficiency was quantified, relative to the fluorescence values of those that migrated to the popliteal lymph node, and the results are shown in FIG. 21 (*p<0.05, **p<0.01).

[0167] As shown in FIG. 21, the proportion of the DC line that migrated to the lymph node to the injected DC line was significantly increased over time.

[0168] Further, to examine the proportion of the DCs that migrated to the mouse popliteal lymph node by confocal microscopy, carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, OR, USA) fluorescence-labeled dendritic cells were injected into a mouse in the same manner as above, and 24 hr later, popliteal lymph nodes were removed. The removed popliteal lymph nodes were fixed with 4% paraformaldehyde, and frozen sections were prepared. The tissue section which was cut to a thickness of 10 m was attached to a slide and treated with a DAPI mounting solution (Immunobioscience Co., WA, USA), and then images were obtained using Zeiss LSM 510 (Carl Zeiss Co., Oberkochen, Germany). Images were analyzed using a Zeiss ZEN software (Carl Zeiss). Results are shown in FIG. 22.

[0169] As shown in FIG. 22, it was confirmed that mice injected with mDCs (+) cultured with the addition of autotaxin protein showed high migration of the cells to the popliteal lymph node, as compared with the control group injected with mDCs ().

[0170] Further, lymphocytes were isolated from the removed popliteal lymph node, and stained with anti-CD11c antibody, followed by flow cytometry. Results are shown in FIG. 23.

[0171] As shown in FIG. 23, it was confirmed that the proportion of CFSE+ and CD11c+ DCs in the popliteal lymph node of the mice injected with mDCs (+) cultured with the addition of autotaxin protein was 5 times higher than that in the control group.

[0172] Accordingly, it was confirmed that when cultured with the addition of autotaxin protein, the migration ability of mDCs is improved.

[0173] Statistical Analysis

[0174] The experimental results of this study were tested for statistical significance by Student's t-test method for at least 3 repeated experiments. Statistical significance was expressed as follows: *p<0.05, **p<0.01, ***p<0.001.