KR100954887B1 - Dendritic cells matured by uncarinic acid c, compositions for immunotherapy containing the dendritic cells, and methods for preparing mature dendritic cells using uncarinic acid c - Google Patents

Abstract

PURPOSE: Matured dendritic cells by uncarinic acid C are provided to enhance the differentiation of nave T cells to Th1 cells and to induce specific reaction. CONSTITUTION: A mutured dendritic cell is obtained by adding uncarinic acid C to immature dendritic cells and culturing. A composition for immunotherapy contains dendritic cells, T cells differentiated therefrom, or cytokine produced thereby. The uncarinic acid C is isolated from Uncaria rhynchophylla. A method for producing the matured dendritic cells comprises: a step of adding uncarinic acid C to immature dendritic cells by intravenous or hypodermic infection, and a step of culturing the dendritic cells.

Description

Dendritic cells matured by uncarinic acid C, compositions for immunotherapy containing the dendritic cells, and methods for preparing mature dendritic cells using uncarinic acid C}

The present invention relates to cellular immunotherapy, and more particularly, as an active ingredient, dendrictic cells matured by uncarinic acid C, an immunotherapeutic composition containing the same, And it relates to a method for producing mature dendritic cells using Uncaric acid C.

Dendritic cells (DCs) are thought to be major antigen presenting cells (APCs), whose main function is to identify microbial structures and present them to naive T cells in lymph nodes. DCs direct various immune responses by stimulating differentiation of male CD4 T cells into helper T cells such as Th1, Th2, Treg cells and Th17 cells. Cytokines secreted by DC during initial T cell stimulation play an important role in the differentiation of subsequent effector T cells. Th1 cells, via interferon gamma (IFN-γ) production, regulate antigen presentation and immunity to intracellular pathogens, and Th2 cells produce IL-4 and IL-13 to produce specific humoral responses and parasites. Mediates immunity. Th17 cells include a recently identified Th-cell lineage that plays a proinflammatory role in autoimmune and tissue inflammation. Although different DC subsets may have some inherent potential to predominantly induce Th1, Th2, Treg cells or Th17 cells, DCs also exhibit significant functional flexibility in response to signals from microorganisms and local microenvironments.

Several substances that induce DC maturation have been reported and known DC maturation stimulants include CD40 ligand (CD40-L), lipopolysaccharide (LPS) and TNF-α. It is well known that cytokines present during immune responses both in vivo and in vitro are important factors in directing responses to Th1, Th2, Treg cells or Th17 cells. Some reports have demonstrated that external factors, such as lipids produced by parasites, can modulate DC function for the purpose of avoiding host immunity.

An important goal of DC research is the development of DC-based strategies for enhancing immune responses to tumors, infectious agents or autoimmune diseases. Recently, DC has been used in many clinical immunotherapy protocols for treating tumors. To date, clinical trials using DC appear to contribute to the development of the condition in clinical immunotherapy, although not a very strong clinical effect. Therefore, it is important to identify factors that may affect the differentiation, maturation and function of DC.

We have recently reported that some terpenes induce DC from human monocytes and induce Th1, Th2 or IL-10-producing Treg cells (Takei, M. et al. (2005) ) Epicubenol and Ferruginol induce DC from human monocytes and differentiate IL-10-producing regulatory T cells in vitro.Biochem.Biophy.Res.Commun.337, 730-738; Takei, M. et al. (2006) T-cadinol and calamenene induce dendritic cells from human monoctytes and drive Th1 polarization.Eur. J. Pharmacol. 537, 190-199; Takei, M. et al. (2007) Diterpenes inhibit IL-12 production by DC and enhance Th2 cells polarization.Biochem. Res. Commun. 355, 603-610; Takei, M. et al. (2008) Diterpene, 16-phyllocladanol enhances Th1 polarization induced by LPS-primed DC, but not TNF-α-primed DC.Biochem. Res. Commun. 370, 6-10).

3β-Hydroxy-27- (E) -feruloyloxyurs-12-en-28-oic acid of Formula 1 below: 3β-hydroxy-27- (E) -feruloyloxyurse-12-ene-28-osan ), I.e., uncarinic acid C, is isolated from Uncaria rhynchophylla and is classified phytochemically as triterpenes.

Triterpenes have been identified as a unique class of natural products with various biological activities. Many alkaloids have been reported as antihypertensive active ingredients from the genus Uncaria. Uncarinic acid C has been shown to exhibit potent inhibitory activity against phospholipase Cγ1 (PLCγ1) and to inhibit cancer cell growth at high doses (Lee, JS, Kim, J., Kim, BY, Lrr, HS, Ahn, JS, Chang, YS (2000) Inhibition of phospholipase Cγ1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla. J. Nat. Prod. 63, 753-756.). However, the effects of uncarinic acid C on the differentiation and function of dendritic cells are not described or implied.

As mentioned above, although some terpene compounds have been reported to have pharmacological activity, there is no indication that uncarinic acid C may affect the differentiation and function of dendritic cells, a highly specialized APC for T cells. Not known.

The inventors have noted that dendritic cells may be potential targets for uncarinic acid C and have studied the ability of uncarinic acid C to human DC differentiation and function. As a result, it was confirmed that Uncarinic acid C affects the differentiation and function of dendritic cells, and came to complete the present invention.

Therefore, it is an object of the present invention to mature dendritic cells, which promote the differentiation of male T cells into Th1 cells and develop antigen specific cytotoxic T cells to induce target cell specific responses. It is to provide.

Another object of the present invention is an immunotherapeutic composition containing the dendritic cells, thereby differentiated T cells, or cytokines produced therein, in particular for immunotherapy compositions for preventing or treating tumors, infectious diseases or autoimmune diseases. It is to provide.

Still another object of the present invention is to provide a method for preparing mature dendritic cells by adding uncarinic acid C to immature dendritic cells.

The first aspect of the present invention is directed to dendritic cells, which are matured by uncarinic acid C, which promote differentiation of male T cells into Th1 cells and develop antigen specific cytotoxic T cells to induce target cell specific responses. It is about. Dendritic cells matured by uncarinic acid C can be obtained by adding uncarinic acid C to immature dendritic cells and then culturing. Uncarinic acid C is preferably added at a concentration of 0.01 to 10 μM.

The second aspect of the present invention relates to an immunotherapeutic composition containing the dendritic cells, thereby differentiated T cells or cytokines produced by the active ingredient. The composition may be for preventing or treating a tumor, infectious disease or autoimmune disease.

The third aspect of the present invention

Adding uncarinic acid C to immature dendritic cells;

Incubating the cells:

It relates to a method for producing mature dendritic cells, comprising the step. Uncarinic acid C is preferably added at a concentration of 0.01 to 10 µM.

Uncarinic acid C, together with GM-CSF and IL-4, regulates the differentiation of immature DCs from mature monocytes to mature DCs and enhances the differentiation of male T cells into Th1 type, while antigen-specific cytotoxicity. It was demonstrated that T cells were developed to induce target cell specific responses. Although the molecular phenomena that lead to the effect of Uncarinic Acid C on DC function must be identified, DC is a potential target of Uncaric Acid C. DCs matured by uncarinic acid C may be potential candidates for use as cellular immunotherapeutic agents, such as antitumor immunotherapy.

Hereinafter, the present invention will be described in more detail.

Uncaric acid C is a triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. Dendritic cells (DC) respond to immune responses by presenting antigens to male T cells, regulating Th1 / Th2 / Th3 / Treg balance, producing numerous regulatory cytokines and chemokines, and regulating survival immune effectors. It plays a pivotal role in regulation. Uncarinic acid C has been demonstrated to activate human DCs as indicated by phenotypic and functional maturation, and modified cytokine production. The expression levels of CDla, CD80, CD83, CD86, HLA-DR and CCR7, expressed by mean fluorescence intensity (MFI) against DCs matured by uncarinic acid C, increased. Production of IL-12p40 and IL-12p70 by DCs matured by Uncarinic Acid C after stimulation with J558 expressing CD40 ligand was higher than that of immature DCs or DCs matured by TNF-α. Male T cells co-cultured with allogenic uncarinic acid C-matured DC at a 1: 1 DC / T cell ratio produce large amounts of interferon-gamma (IFN-γ) depending on IL-12 secretion. Switched to Th1 cells releasing small amounts of IL-4 and IL-13. In CTL analysis, DNA fragmentation and ⁵¹ Cr release for uncarinic acid C-matured DCs were more potent compared to that of immature DCs or TNF-α-matured DCs. Uncarinic acid C-matured DCs migrated towards CCL19. These results indicate that Uncarinic Acid C regulates human DC function in a manner that favors Th1 cell polarization and that the produced DCs induce target cell specific responses of cytotoxic T cells.

Uncarinic acid C-matured dendritic cells may be injected into a patient's body according to a known method such as immunotherapy using dendritic cells, preferably by subcutaneous or intravenous injection. In addition, methods of injecting directly into or around the tumor may also be attempted.

These cell therapies extract cells from the patient's autologous cells or allogeneic cells and mass cultivate them into dendritic cells with potent immune enhancing effects, giving them the opportunity to phagocytize antigens. In this process, cancer cells or previously established allogeneic cell lines weakened by irradiation or UV irradiation may be directly incubated with dendritic cells, or cytotoxic substances such as anticancer agents or repeated freeze-thaw processes to increase the immune-enhancing capacity of the dendritic cells. After killing the cancer cells through this lysate (lysate) may be treated by dendritic cells (pulse). Alternatively, DNA, RNA, proteins or peptides of cancer cells can be used as antigens for sensitization of dendritic cells. The dendritic cells sensitized with the antigen may be injected directly into the patient, or the dendritic cells may be directly reacted with T cells extracted from the patient's blood to make cancer killing cells and then injected into the patient with activated T cells.

In particular, when the patient's own peripheral blood mononuclear cells are isolated and proliferated and differentiated in large quantities from dendritic cells having a strong immune response-inducing ability, and then injected into the body of the patient to cause an immune response, The problem can be solved. In addition, unlike treatment with cytokines, which require repeated injections, cells have the advantage of continuously producing cytokines by injecting the cells themselves. Furthermore, dendritic cells may be manipulated by minimizing immune rejection or overexpressing a growth factor gene through known genetic engineering methods.

The immunotherapeutic composition according to the present invention can induce a strong cellular immune response against diseases caused by specific antigens as well as general immune enhancing effects due to T cell proliferation inducing activity, and because it mass-produces IFN-γ anticancer and anti It can also be applied effectively to viral treatment.

In the present invention, the single dose of uncarinic acid C-matured dendritic cells is preferably 1 × 10 ⁶ to 1 × 10 ⁷ , in particular 3 × 10 ⁶ cells, but this may be due to the disease, Various related factors such as severity, route of administration, patient's weight, age and gender may be taken into consideration.

Hereinafter, the present invention will be described in detail by way of examples, but these are merely to aid the understanding of the present invention and are not intended to limit the scope of the present invention in any way.

Culture medium, reagents and monoclonal antibodies

The culture medium used in this example was RPMI 1640 (Gibco-BRL) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. Recombinant human IL-4 (IL-4), recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from R & D systems (Minnesota). For flow cytometry, monoclonal antibodies (mAbs) against the following antigens were purchased from Becton-Dickinson (California): anti-CD14-FITC (fluorescent isothiocyanate), anti-CD1a-PE ( Phycoerythrin), anti-CD40-FITC, anti-CD54-PE, anti-80-PE, anti-CD83-PE, anti-CD86-PE, anti-HLA-DR-FI, and anti-CCR-7. Endotoxin levels in all materials were below 1.0 EU / ml.

Statistical analysis

Statistical analysis of the results was performed by ANOVA. The difference was considered statistically significant when the p value was less than 0.05.

Example 1 Separation of Uncarinic Acid C from Uncaria Lincopila

Uncarinic acid C is described by Lee, JS et al. (2000) Inhibition of phospholipase Cγ1 and cancer cell proliferation by triterpene esters from Uncaria rhynchophylla. J. Nat. Prod. 63, 753-756. Uncarinic acid C had a purity of> 99%. Uncarinic acid C was dissolved in dimethylsulfoxide (DMSO). The concentration of dimethylsulfoxide in the culture medium was 0.1%, which did not affect the culture and production of cytokines under the conditions used in this example. Endotoxin of uncarinic acid C was removed using End Trap 5/1 (Profos AG, Germany). LPS concentrations were below the detection limit (<0.05 EU / mL) of the assay.

Example 2: Generation of DCs Derived from Monocytes

All cells were isolated from human peripheral blood of normal healthy donors. Peripheral blood mononuclear cells (PBMC) were first isolated from heparinized whole blood by Ficoll-Hypaque (1.077 g / ml) (Pharmacia, Germany) density gradient centrifugation (465 g, 30 minutes, 22 ° C.). It was. PBMCs were isolated into CD14 + monocytes using a magnetic activated cell sorter (MACS) according to the manufacturer's instructions (Miltenyi Biotec, Calif.). The purity of CD14 + monocytes was always above 90%, which is clinically acceptable purity. Monocytes were incubated with GM-CSF (50 ng / ml) and IL-4 (25 ng / ml) for 6 days in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin. Cytokines were fed to the medium every 2 days. Immature DCs with RPMC 1640 containing 10% FBS and 1% penicillin-streptomycin containing GM-CSF and IL-4 for an additional 2 days with various concentrations of uncarinic acid C and TNF-α (50 ng / ml) DC was generated by stimulation. All subsequent tests were performed after 8 days of cell harvest and removal of GM-CSF, IL-4, Uncarinic Acid C or TNF-α by strong washing. To measure production of IL-6, IL-10, IL-12p40 and IL-12p70 by uncarinic acid C- or TNF-α-matured DCs, DC (2 × 10 ⁴ cells / well) was added for 24 hours. Stimulated with J558 cells (5 × 10 ⁴ cells / well) expressing CD40 ligand. Cell-free supernatants were pooled and frozen at −20 ° C. until cytokines were measured using enzyme-linked immunosorbent assay (ELISA).

Example 3: Immunophenotyping Studies

Dual-color immunofluorescence flow cytometry was performed using the following panels of monoclonal antibodies: PE-enveloped antihuman CD1a, FITC-enveloped antihuman CD14, FITC-enveloped antihuman CD40, PE-enveloped anti Human CD54, PE-antihuman CD80, PE-antihuman CD83, PE-antihuman CD86, FITC-HLA-DR and FITC-CCR-7. Negative control isotype matched with an unrelated monoclonal antibody (Becton-Dickinson). The cells were resuspended in staining medium containing PBS and 0.1% NaN ₃ and fixed with 1.0% paraformaldehyde. Isotype control was tested in parallel. Cell debris was removed by forward and side scatter gating. Samples were analyzed on a FACSCalibur (Becton-Dickinson) with CellQuest software (Becton-Dickinson). Ten thousand cells were analyzed per sample. The results are expressed as mean fluorescence intensity (MFI).

The results are shown in FIG. As shown in FIG. 1, the expression levels of CD1a, CD40, CD54, CD80, CD83, CD86 and HLA-DR represented by MFI for uncarinic acid C were increased in a dose-dependent manner. The viability of the cells at 0.001 μM and 100 μM uncarinic acid C was 90% and 55%, respectively. From these results, the concentration of uncarinic acid C was used at 0.01-10 μM for subsequent experiments. As a positive control, human monocytes were incubated with GM-CSF and IL-4 for 6 days, followed by another 2 days in the presence of TNF-α. TNF-α is a known DC maturation-promoting factor. The expression levels of CD1a, CD40, CD54, CD80, CD83, CD86 and HLA-DR represented by MFI for TNF-α (25 ng / ml) -matured DCs were higher than uncarinic acid C-matured DCs. This aspect was identical to that of the uncarinic acid C-matured DCs. On the other hand, the expression level of CCR-7, represented by MFI for uncarinic acid C-matured DCs, was increased compared to immature DCs and TNF-α-matured DCs. As a control, immature DCs (in medium) were generated by incubating human monocytes with GM-CSF and IL-4 for 8 days. At 8 days, the expression level of CD14 represented by MFI was low or undetectable.

Example 4 Effect of Uncarinic Acid C-Matured DC on Production of IL-6, IL-10, IL-12p40 and IL-12p70

DC is a professional and effective APC and their immunoregulatory and proinflammatory cytokine secretion plays a decisive role during T cell maturation, so whether cytokine production by human monocyte-derived DCs is affected by uncarinic acid C Was investigated. IL-6 is an important mediator of a wide range of biological activities that play a decisive role in the induction of pro-inflammatory and immune responses. IL-10 is a pleiotropic cytokine known to have an inhibitory effect on the adjuvant function of DC and appears to play a central role in preventing too pathological Th1 or Th2 responses in various settings. In contrast, the level of IL-12 production by bone marrow DCs during activation of male T cells is a major factor inducing the development of Th1 cells. Thus, in this Example, IL-6, IL-10, IL-12p40 and IL- in immature DCs (in medium) and DCs matured for 2 days in the presence of the factor after stimulation with J558 expressing CD40 ligand for 24 hours. 12p70 production was investigated.

Immature DCs were stimulated with TNF-α (50 ng / ml) and uncarinic acid C (0.01-10 μM) to generate DCs. Cells (4 × 10 ⁴ cells / well) were stimulated for 24 hours with J558 cells expressing CD40 ligand. After 24 hours, IL-12p40, IL-12p70, IL-10 and IL-6 in culture supernatants were measured by ELISA.

The results are shown in Figures 2a to 2d (2a: IL-12p40, 2b: IL-12p70, 2c: IL-10, 2d: IL-6). Under control conditions (medium not co-cultured with J558), DCs spontaneously showed low levels of IL-6 (46 ± 9.1 pg / ml), IL-10 (3.1 ± 0.9 pg / ml), IL-12p40 (85.6 ± 6.8 pg / ml) and IL-12p70 (24.2 ± 4.8 pg / ml). Upon stimulation with J558 expressing CD40 ligand, IL-12p40 and p70 production were significantly increased at 0.1 μM of uncarinic acid C as compared to immature DCs and TNF-α-matured DCs (FIGS. 2A and B). . On the other hand, in immature DCs and TNF-α-matured DCs, production of IL-6, IL-10, IL-12p40 and IL-12p70 after stimulation with J558 expressing CD40 ligand was induced by 0.1 μM uncarinic acid C. It was lower or only detectable when compared to matured DC (FIGS. 2A, 2B, 2C and 2D).

Example 5: Allogenic Mixed Lymphocytes Reaction (Allo MLR)

CD3 + T cells were obtained from allogeneic PBMCs using magnetic cell sorting and biomolecule separation (MACS) beads (Miltenyi Biotec) for allogeneic mixed lymphocyte response analysis. Purity of the isolated cells was determined by flow cytometry using a faxcaliber CD3 + T cells> 95%. Allogeneic CD3 + T cells (2 × 10 ⁴ cells / well) were co-cultured in 96-well round bottom culture plates with graded doses (2 × 10 ² to 2 × 10 ⁴ ) of irradiated (25 Gy) DC It was. After 5 days, cells were pulsed with 1 μCi [ ³ H] -methylthymidine per well for 18 hours, then harvested and analyzed in a liquid scintillation counter.

The results are shown in FIG. The ability to induce allogeneic T cell proliferation is a functional feature of DC in vitro. When analyzing the allogeneic stimulating ability of DC in allogeneic MLR, surface marker expression changes were also reflected at the functional level. Co-culture of T cells with uncarinic acid C-matured DCs enhanced T cell proliferation. The efficiency in allogeneic MLR for uncarinic acid C-matured DCs increased in a dose-dependent manner (0.01-0.1 μM). Uncarinic acid C-matured DCs showed higher stimulation efficiency in allogeneic MLR compared to immature DCs and TNF-α-matured DCs. Co-culture of T cells with uncarinic acid C-matured DCs also increases T cell proliferation, suggesting that uncarinic acid C enhances antigen-presenting activity of DCs in allogeneic MLR.

Example 6 Effect of Uncarinic Acid C-Matured DC on Differentiation of Male T Cells into Th1 Cells

Next, the induction of Th effector cell differentiation by mature DC under different conditions was evaluated. For this purpose, Takei, M. et al. (2006) T-cadinol and calamenene induce dendritic cells from human monoctytes and drive Th1 polarization. Eur. J. Pharmacol. 537, 190-199, measurement of male T cell bias by DC was performed. Briefly, male CD4 + CD45RA + CD45RO- T cells were isolated from allogeneic PBMCs using MACS beads (Miltenyi Biotec). Purity of the isolated cells was> 95% CD4 + CD45RA + CD45RO− T cells, as measured by flow cytometry using a faxcaliber. DCs were co-cultured with male T cells (2 × 10 ⁴ cells / 200 μl) at a ratio of 1: 1 DC / T cells in 96-well U-bottom tissue culture plates. On day 5, cells were thoroughly washed and expanded with fresh medium containing 3 ng / ml rIL-2 (R & D Systems). 100 μl of culture supernatant was replaced every 3 days with the same concentration of fresh medium. On days 8-10, cells were washed, counted and T cells re-stimulated with Dynabeads CD3 / CD28 T cell expander (1 μl; Invitrogen) for 24 hours. Cell-free supernatants were pooled and frozen at −20 ° C. until cytokines were measured using ELISA. Immature DC was used as a control. IFN-γ, IL-4, IL-13 and IL-10 in the culture supernatants were measured by ELISA.

Meanwhile, Takei, M. et al. (2005) Epicubenol and Ferruginol induce DC from human monocytes and differentiate IL-10-producing regulatory T cells in vitro. Biochem. Biophy. Res. Commun. 337, 730-738, intracellular cytokine concentrations of harvested T cells were measured by FACS analysis. Briefly, T cells (10 ⁶ / ml) were stimulated with pobol myristate acetate (PMA) (10 ng / ml) and ionomycin (1 μg / ml) for 5 hours at 37 ° C., 5% CO ₂ . . Brefeldin A (10 μg / ml) was added during the last 2 hours of incubation to disrupt cytokine secretion. Cells were collected, fixed with 1% paraformaldehyde, permeabilized with a commercial solution (Beckman Coulter) and stained with FITC-labeled anti-IFN-γ and PE-labeled anti-IL-4 mAbs.

The results are shown in FIG. 4 (4a: IFN-γ, 4b: IL-13, 4c: IL-4, 4d: IL-10). Allogeneic uncarinic acid C- or TNF-α-matured DCs co-cultured at a 1: 1 DC / T cell ratio secrete measurable amounts of IFN-γ, while IL-4 and IL-13 secrete almost Did not do it. DCs were differentiated into co-cultured 0.1 mM uncarinic acid C and male T cells were converted to typical Th1 cells producing large amounts of IFN-γ. On the other hand, IFN-γ, IL-4 and IL-10 production by lower T cells co-cultured with immature DCs was lower.

Uncarinic acid C- and TNF-α-matured DCs induced differentiation of male T cells into Th1 responses in DC / T cells (1: 1). After expansion for 6-8 days in cytokines (IFN-γ and IL-4), concentrations were measured after 5 hours restimulation with PMA and ionomycin. The results are shown in Figure 4e. As shown in FIG. 4E, uncarinic acid C-matured DC induced more IFN-γ producing cells than IL-4.

Example 7: Cytotoxic T-lymphocytes (CTL), DNA Fragmentation and ⁵¹ Cr emission analysis

(1) CTL derivation

Takei, M. et al. (2004) Dendritic cells maturation promoted by M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, drive a potent Th1 polarization. Biochemi. Pharmacol. 68, 441-452, according to the method described in the CTL analysis. Briefly, DC (2 × 10 ⁵ ) cultured from monocytes of HLA-A0201 + donors were loaded with 20 μg / ml WT-1 peptide for 2 hours at 37 ° C. 5% CO ₂ . The WT-1 peptide may bind to HLA-A0201. In 24-well plates, autologous CD8 + T cells were cultured with 10% FBS and 1% penicillin-streptomycin containing 10 ng / ml IL-2 and 5 ng / ml IL-7 (RPMI1640: AIM-V = 1: 1 ) Was incubated with DC at 5: 1 for 10 days in 2 ml. After 10 days, CD8 + T cells were re-stimulated with irradiated (25 Gy) autologous monocytes (2: 1) loaded with WT-1 peptide and the cells were then 10 ng / ml IL-2 and 5 ng / ml IL Incubated for 10 additional days in 2 ml of 10% FBS containing -7 and 1% penicillin-streptomycin-containing medium. Using T2 cells (HLA-A0201 +) as target cells, T2 cells were loaded with 20 μg / ml WT-1 peptide or HIV-1 peptide at a concentration of 5 × 10 ⁵ cells / ml and loaded at 37 ° C. 5% CO ₂ . Incubate for 2 hours.

(2) DNA fragmentation analysis

To analyze target-induced CTL apoptosis, an 8 h [ ³ H] -methylthymidine DNA fragmentation (JAM) assay was performed. CTLs were labeled with 5 μCi [ ³ H] -methylthymidine per 10 ⁶ cells 18 hours prior to analysis in the absence of IL-2 and used as targets for tumor cells. CTLs (3 × 10 ⁴ cells / well) were harvested and co-cultured with tumor cells (T2) at 37 ° C. 5% CO ₂ for 8 hours at various tumor-to-CTL ratios and analyzed in a liquid scintillation counter (Beckman). Results are expressed as percent of DNA fragmentation based on percent reduction in cpm compared to untreated control using the following formula:

% DNA fragmentation = (control cpm-sample cpm / control cpm) × 100.

The analysis was performed three times.

(3) ⁵¹ Cr emission analysis

Nakano, M. et al. (1998) A gene encoding antigen peptides of human sequamous cell carcinoma recognized by cytotoxic T lymphocytes. J. Exp. Med. ⁵¹ Cr release assay was performed according to the method described in 187, 277-283. Briefly, effector cells were incubated with ⁵¹ Cr labeled target cells (1 × 10 ⁴ ) for 6 hours at various effector to target cell ratios, followed by harvesting the supernatant and measuring radioactivity using an automatic gamma counter. This analysis was performed three times.

(4) results

The CTL responses of autologous CD8 + T cells exhibited by DC differentiated with uncarinic acid C (0.1 μM) or TNF-α were compared. The results are shown in FIGS. 5A-5C (5a: immature DC, 5b: TNF-α-matured DC, 5c: uncarinic acid C-matured DC). In an 8 h JAM assay measuring CTL apoptosis by tumor cells, T2 cells loaded with WT-1 peptides potentiated DNA fragmentation of CTLs generated with uncarinic acid C-matured DC pulsed with WT-1 peptides. Induced. The percentage of DNA fragmentation was dependent on the increased number of CTL cells. Similar results were obtained with a ⁵¹ Cr release assay measuring lysis of target cells (FIGS. 6A-6C, 6A: immature DC, 6b: TNF-α-matured DC, 6c: uncarinic acid C-matured DC). On the other hand, DNA fragmentation percentage and ⁵¹ Cr release in JAM analysis were low or undetectable when using HIV-1 peptide (unrelated peptide) and peptide-free T2 as target cells (negative control).

Example 8: Chemotaxis

It is well reported that the expression of chemokine receptors and the ability to migrate in response to specific chemotactic stimuli are differentially regulated during activation / differentiation of DC and play an important role in the use of their function. Therefore, it was investigated whether the expression of uncarinic acid C-primed DCs correlated with their ability to migrate in response to the CCR7 ligand CCL19.

Takei, M. et al. (2006) T-cadinol and calamenene induce dendritic cells from human monoctytes and drive Th1 polarization. Eur. J. Pharmacol. 537, 190-199, DC migration was performed in a 24-well transwell cell culture chamber (5-μm pore; Costar). Briefly, 10 ⁵ cells were washed, suspended in RPMI 1640 medium containing 10% FBS and loaded into the upper chamber compartment. CCL4 (0.5 μg / ml; Peprotech), CCL19 (0.5 μg / ml; Peprotech) or chemokine free negative control was added to the lower compartment. After 4 hours incubation at 37 ° C., the cells migrated to the lower compartment through an 8 μm pore size polycarbonate filter were collected and counted.

The results are shown in Table 1.

Transfer in Response to CCL19 by Uncarinic Acid C- or TNF-α-Matured DC Without CCL19 With CCL19 Immature DC 1.8 ± 0.1 9.2 ± 2.1 TNF-α-Matured DC 1.2 ± 0.1 20.3 ± 3.5 Uncarinic Acid C-Matured DC 2.3 ± 0.6 27.8 ± 3.1

As expected, uncarinic acid C-primed DCs migrated towards CCL19 and showed a higher migration response than TNF-α-primed DCs.

1 is a diagram showing the phenotype of DC differentiated with TNF-α or uncarinic acid C (red: background signal to isotype control mAb, white: specific signal to each cell surface marker);

2A-2D are graphs showing cytokine production by DCs stimulated by J558 expressing CD40 ligands;

FIG. 3 is a graph showing allogeneic T cell stimulating ability of DCs matured with Uncarinic Acid C or TNF-α;

4A-4E show IFN-γ, IL-4, IL-13 and IL by ELISA in supernatants of male T cells stimulated by TNF-α- or uncarinic acid C (0.01-10 μM) -matured DCs. -10 is a graph showing the measurement results;

5a to 5c are graphs showing the results of DNA fragmentation analysis;

6A to 6C are graphs showing the results of ⁵¹ Cr emission analysis.

Claims (7)

delete delete delete delete delete Adding uncarinic acid C to immature dendritic cells; Incubating the cells: Comprising a step, a method for producing mature dendritic cells. The method for producing mature dendritic cells according to claim 6, wherein uncarinic acid C is added at a concentration of 0.01 to 10 µM.

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