Abstract
The present invention relates to a method for inducing differentiation of bone marrow cells into myeloid-derived suppressor cells (MDSCs) by treating the bone marrow cells with a toll-like receptor agonist (TLR agonist) or type I interferon, or for inducing dendritic cells from the MDSCs.
Claims
1. A method for producing myeloid-derived suppressor cells (MDSCs), comprising: a step of treating bone marrow cells with a toll-like receptor agonist (TLR agonist) or type I interferon, to induce differentiation into myeloid-derived suppressor cells, wherein the toll-like receptor agonist is at least one of toll-like receptor 7 agonist and toll-like receptor 9 agonist, wherein the myeloid-derived suppressor cells comprise MDSCs having phenotype PDCA-1+, and wherein the toll-like receptor agonist or the type I interferon is applied at the initiation time of bone marrow cell differentiation, and then is applied one or more times within 3 days after the initiation of differentiation.
2. The method according to claim 1, wherein the toll-like receptor agonist is an agonist of an intracellular toll-like receptor.
3. The method according to claim 1, wherein the bone marrow cells are further treated with interleukin 6 together with the toll-like receptor agonist.
4. The method according to claim 3, wherein the interleukin 6 is applied in an amount of 1 to 100 ng/ml.
5. The method according to claim 1, wherein the toll-like receptor agonist or the type I interferon is applied one or more times before initiation of, at the initiation time of, or during bone marrow cell differentiation.
6. The method according to claim 5, wherein the bone marrow cell differentiation is carried out by inoculating the bone marrow cells in a medium supplemented with a growth factor and performing culture for 5 to 10 days.
7. The method according to claim 6, wherein the growth factor is at least one selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor (SCF), and interleukin 3 (IL-3).
8. The method according to claim 6, wherein the growth factor is added in an amount of 10 ng/ml to 500 ng/ml.
9. The method according to claim 1, wherein the toll-like receptor agonist or the type I interferon is applied in an amount of 10 ng/ml to 1,000 ng/ml.
10. The method according to claim 1, wherein the step to induce differentiation into myeloid-derived suppressor cells is performed by culturing the bone marrow cells in a medium supplemented with a growth factor and at least one of the toll-like receptor agonist or the type I interferon.
11. The method according to claim 1, wherein the myeloid-derived suppressor cells are monocytic myeloid-derived suppressor cells (M-MDSCs).
12. The method of claim 1, wherein the type I interferon is at least one selected from the group consisting of IFN-α, IFN-β, IFN-κ, and IFN-ω.
13. A method for preventing or treating an immune disease, the method comprising treating bone marrow cells with a toll-like receptor agonist (TLR agonist) or type 1 interferon to induce differentiation into myeloid-derived suppressor cells, and administering the myeloid-derived suppressor cells to a subject in need thereof, wherein the toll-like receptor agonist is at least one of toll-like receptor 7 agonist and toll-like receptor 9 agonist, and wherein the myeloid-derived suppressor cells comprise MDSCs having phenotype PDCA-1+.
14. The method according to claim 13, wherein the immune disease is graft rejection after organ transplantation, graft rejection after hematopoietic stem cell transplantation, an autoimmune disease, or an allergic disease.
15. The method according to claim 13, wherein the bone marrow cells are further treated with interleukin 6 together with the toll-like receptor agonist.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1) FIG. 1 illustrates a schematic diagram for three methods in which bone marrow cells are treated with a TLR agonist, in Example 1 of the present invention.
(2) FIG. 2 illustrates results obtained by treatment of bone marrow cells with various TLR agonists and then analyzing, by flow cytometry, whether CD11c.sup.+ cells have been induced, in Example 1 of the present invention.
(3) FIG. 3 illustrates results obtained by treatment of bone marrow cells with various TLR agonists and then measuring a proportion of CD11c.sup.+ cells, in Example 1 of the present invention.
(4) FIG. 4 illustrates results obtained by measuring changes in proportion of CD11c.sup.+ cells for respective concentrations of TLR agonists used to treat bone marrow cells, in Example 1 of the present invention.
(5) FIG. 5 illustrates results obtained by treating respective bone marrow cells of C57BL/6 mice and C57BL/6 MyD88.sup.−/− mice with various TLR agonists and then analyzing, by flow cytometry, whether CD11c.sup.+MHC-II.sup.+ cells have been induced, in Example 2 of the present invention.
(6) FIG. 6 illustrates results obtained by treating respective bone marrow cells of C57BL/6 mice and C57BL/6 MyD88.sup.−/− mice with various TLR agonists and then measuring a proportion of CD11c.sup.+MHC-II.sup.+ cells, in Example 2 of the present invention.
(7) FIG. 7 illustrates results obtained by analyzing the phenotypes CD11c, CD11b, Gr-1, F4/80, Ly6C, CD4, CD8a, CD103, PDCA-1, B220, NK1.1, and CD49b in the cells that have been induced by differentiation after treatment of bone marrow cells with various TLR agonists, in Example 3 of the present invention.
(8) FIG. 8 illustrates results obtained by measuring proportions of CD11c.sup.+, CD11b.sup.+, Gr-1.sup.+, F4/80.sup.+, Ly6C.sup.+, CD4.sup.+, CD8α.sup.+, CD103.sup.+, PDCA-1.sup.+, B220.sup.+, NK1.1.sup.+, and CD49b.sup.+ cells in the cells that have been induced by differentiation after treatment of bone marrow cells with various TLR agonists, in Example 3 of the present invention.
(9) FIG. 9 illustrates results obtained by analyzing, by flow cytometry, an expression level of Gr-1 in the cells that have been induced by differentiation after treatment of bone marrow cells with various TLR agonists, in Example 4 of the present invention.
(10) FIG. 10 illustrates results obtained by measuring proportions of Gr-1.sup.+CD11b.sup.+CD11c.sup.−, Gr-1.sup.highCD11b.sup.+CD11c.sup.−, and Gr-1.sup.intCD11b.sup.+CD11c.sup.− cells in the cells that have been induced by differentiation after treatment of bone marrow cells with various TLR agonists, in Example 4 of the present invention.
(11) FIG. 11 illustrates results obtained by analyzing, by flow cytometry, expression levels of LyG and LyC in the cells that have been induced by differentiation after treatment of bone marrow cells with various TLR agonists, in Example 4 of the present invention.
(12) FIG. 12 illustrates results obtained by measuring proportions of CD11c.sup.− CD11b.sup.+LyG.sup.+LyC.sup.−, CD11c.sup.−CD11b.sup.+LyG.sup.+LyC.sup.int, CD11c.sup.−CD11b.sup.+LyG.sup.−LyC.sup.int, and CD11c.sup.− CD11b.sup.+LyG.sup.−LyC.sup.high cells in the cells that have been induced by differentiation after treatment of bone marrow cells with various TLR agonists, in Example 4 of the present invention.
(13) FIG. 13 illustrates results obtained by measuring proportions of CD11c.sup.− CD11b.sup.+LyG.sup.+LyC.sup.−, CD11c.sup.−CD11b.sup.+LyG.sup.+LyC.sup.int, CD11c.sup.−CD11b.sup.+LyG.sup.−LyC.sup.int, and CD11c.sup.− CD11b.sup.+LyG.sup.−LyC.sup.high cells in the cells that have been induced by differentiation after treatment of bone marrow cells, extracted from C57BL/6 mice and C57BL/6 MyD88.sup.−/− mice, with various TLR agonists, in Example 5 of the present invention.
(14) FIG. 14 graphically illustrates results obtained by measuring changes in NO.sub.2 formation level in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(15) FIG. 15 illustrates results obtained by analyzing, with Western blotting, changes in expression levels of NOS2 and arginase-1 in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(16) FIG. 16 graphically illustrates results obtained by measuring changes in expression level of IL-10 in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(17) FIG. 17 illustrates results obtained by analyzing, with FACS, changes in expression levels of arginase-1 (Arg-1) and iNOS in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(18) FIG. 18 graphically illustrates results obtained by measuring changes in expression levels of arginase-1 (Arg-1) and iNOS, and in expression level ratio of Arg-1/iNOS, in M-MDSCs which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(19) FIG. 19 illustrates results obtained by analyzing, by flow cytometry, expression levels of CD124, CD115, F4/80, and PDCA-1 in M-MDSCs which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, in Example 6 of the present invention.
(20) FIG. 20 graphically illustrates results obtained by measuring proportions of cells expressing CD124, CD115, F4/80, and PDCA-1 in M-MDSCs which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, in Example 6 of the present invention.
(21) FIG. 21 graphically illustrates changes in expression levels of TNF-α, IL-6, IL-12p70, and IFN-β in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(22) FIG. 22 illustrates results obtained by analyzing, with FACS, changes in expression levels of PD-L1, PD-L2, Tim-3, FasL, and IDO in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(23) FIG. 23 graphically illustrates results obtained by measuring proportions of cells expressing of PD-L1, PD-L2, Tim-3, FasL, and IDO in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(24) FIG. 24 illustrates results obtained by analyzing, with FACS, changes in expression levels of PD-L1, PD-L2, Tim-3, FasL, and IDO in M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist, while applying stimulation with LPS and IFN-γ, in Example 6 of the present invention.
(25) FIG. 25 illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then identifying their T cell inhibition capacity, in Example 7 of the present invention.
(26) FIG. 26 illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR9 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then identifying their T cell inhibition capacity, in Example 7 of the present invention.
(27) FIG. 27 graphically illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then measuring changes in T cell proliferation capacity, in Example 7 of the present invention.
(28) FIG. 28 graphically illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR9 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then measuring changes in T cell proliferation capacity, in Example 7 of the present invention.
(29) FIG. 29 illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then analyzing, by flow cytometry, an expression level of Foxp3, in Example 7 of the present invention.
(30) FIG. 30 illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR9 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then analyzing, by flow cytometry, an expression level of Foxp3, in Example 7 of the present invention.
(31) FIG. 31 graphically illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR2 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then measuring a proportion of regulatory T cells, in Example 7 of the present invention.
(32) FIG. 32 graphically illustrates results obtained by subjecting M-MDSCs, which have been induced after treatment of bone marrow cells with a TLR9 agonist using the 3 method, to treatment with a co-culture of OVA-pulsed DCs and CD4 T cells, and then measuring a proportion of regulatory T cells, in Example 7 of the present invention.
(33) FIG. 33 illustrates results obtained by analyzing, by flow cytometry, expression levels of CD11c and MHC-II in the cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, in Example 8 of the present invention.
(34) FIG. 34 graphically illustrates results obtained by measuring changes in proportion of CD11c.sup.+MHC-II.sup.+ cells in the cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, while applying stimulation with LPS and IFN-γ, in Example 8 of the present invention.
(35) FIG. 35 illustrates results obtained by analyzing, by flow cytometry, expression levels of CD11b and F4/80 in the cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with M-CSF, in Example 8 of the present invention.
(36) FIG. 36 graphically illustrates results obtained by measuring changes in proportion of F4/80.sup.+CD11b.sup.+ cells in the cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with M-CSF, while applying stimulation with LPS and IFN-γ, in Example 8 of the present invention.
(37) FIG. 37 illustrates results obtained by analyzing T-cell proliferation inhibition capacity of M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, while performing culture with addition of GM-CSF, in Example 8 of the present invention.
(38) FIG. 38 graphically illustrates results obtained by measuring changes in T-cell proliferation inhibition capacity and IFN-γ secretion capacity of M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, while performing culture with addition of GM-CSF, in Example 8 of the present invention.
(39) FIG. 39 graphically illustrates results obtained by measuring a proportion of CD11c.sup.+ cells in the cells induced by culturing M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, with addition of GM-CSF, in Example 8 of the present invention.
(40) FIG. 40 illustrates results obtained by analyzing, by flow cytometry, expression levels of CD11c, PDCA-1, CD115, CCR2, MHC-II, CD64, CD11b, and Ly6C in the cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, myeloid-derived dendritic cells, or Ly6.sup.+ cell-derived dendritic cells, in Example 9 of the present invention.
(41) FIG. 41 graphically illustrates results obtained by measuring proportions of Ly6C.sup.+, MerTK.sup.+, CD11b.sup.+, CCR2.sup.+, and PDCA-1.sup.+ cells in the cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, myeloid-derived dendritic cells, or Ly6.sup.+ cell-derived dendritic cells, in Example 9 of the present invention.
(42) FIG. 42 graphically illustrates changes in expression levels of TNF-α, IL-6, IL-10, IL-12p70, and NO.sub.2 which are observed in a case where dendritic cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, myeloid-derived dendritic cells, or Ly6.sup.+ cell-derived dendritic cells are stimulated with LPS and/or IFN-γ, in Example 9 of the present invention.
(43) FIG. 43 graphically illustrates changes in expression levels of IFN-γ, IL-2, IL-10, IL-5 and IL-17A which are observed in a case where dendritic cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, myeloid-derived dendritic cells, or Ly6.sup.+ cell-derived dendritic cells are stimulated with LPS and/or IFN-γ, in Example 9 of the present invention.
(44) FIG. 44 graphically illustrates changes in T cell proliferation capacity and expression level of IFN-γ observed after subjecting CD4 T cells to treatment with dendritic cells induced by subjecting M-MDSCs, which have been obtained by treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, to treatment with GM-CSF, with myeloid-derived dendritic cells, or with Ly6.sup.+ cell-derived dendritic cells, in Example 9 of the present invention.
(45) FIG. 45 graphically illustrates results obtained by analyzing expression levels of the cytokines TNF-α, IL-6, PGE2, IFN-α, and IFN-β which are induced at early differentiation stages (8, 18, and 36 hours after TLR stimulation) after treatment of bone marrow cells with a TLR2 agonist or a TLR9 agonist using the 3 method, in Example 10 of the present invention.
(46) FIG. 46 graphically illustrates proportions of M-MDSCs and dendritic cells, obtained after treatment of bone marrow cells with TNF-α, IL-6, PGE2, IFN-α, or IFN-β, in place of a TLR agonist, using the 3 method, in Example 11 of the present invention.
(47) FIG. 47 graphically illustrates proportions of CD11c.sup.−CD11b.sup.+Ly6G.sup.−Ly6C.sup.+PDCA-1.sup.+ and CD11c.sup.−CD11b.sup.+Ly6G.sup.−Ly6C.sup.+PDCA-1.sup.− for respective treatment concentrations after treatment of bone marrow cells with TNF-α, IL-6, PGE2, IFN-α, or IFN-β, in place of a TLR agonist, using the 3 method, in Example 11 of the present invention.
(48) FIG. 48 graphically illustrates changes in proportions of MDSCs and dendritic cells in the cells harvested after treatment of bone marrow cells with recombinant IL-6 protein or an IL-6 receptor blocker together with a TLR9 agonist using the 3 method, in Example 12 of the present invention.
(49) FIG. 49 graphically illustrates a proportion of CD11c.sup.+ cells induced in a case where M-MDSCs, which have been induced after treatment of bone marrow cells with recombinant IL-6 protein or an IL-6 receptor blocker together with a TLR9 agonist using the 3 method, are subjected to treatment with GM-CSF, in Example 12 of the present invention.
(50) FIG. 50 graphically illustrates proportions of CD11c.sup.+ cells for respective treatments in a case where Ly6C.sup.+ M-MDSCs, which have been induced by treatment of bone marrow cells with a TLR2 agonist, a TLR9 agonist, or IFN-β using the 3 method, are subjected to treatment with GM-CSF, in Example 13 of the present invention.
(51) FIG. 51 graphically illustrates proportions of CD11c.sup.+ cells for respective treatments in a case where Ly6C.sup.+ M-MDSCs, which have been induced by treatment of bone marrow cells with a TLR2 agonist and further with IFN-β at 5 ng/ml or 25 ng/ml using the 3 method, are subjected to treatment with GM-CSF, in Example 13 of the present invention.
(52) FIG. 52 graphically illustrates proportions of PDCA-1.sup.+ M-MDSCs and dendritic cells after treatment of bone marrow cells, which have been harvested from wild-type mice and type I IFN receptor knockout mice, with a TLR9 agonist using the 3 method, in Example 13 of the present invention.
DETAILED DESCRIPTION OF INVENTION
(53) According to an embodiment of the present invention, there is provided a method for producing myeloid-derived suppressor cells (MDSCs), comprising a step of treating bone marrow cells with a toll-like receptor agonist (TLR agonist), to induce differentiation into myeloid-derived suppressor cells. Here, the toll-like receptor agonist used to treat the bone marrow cells is an agonist of a toll-like receptor (exogenous toll-like receptor) present in the plasma membrane and is preferably at least one of toll-like receptor 2 agonist or toll-like receptor 4 agonist for inducing differentiation into stable tolerogenic myeloid-derived suppressor cells having desired properties in the present invention. Further, in the present invention, in a case where the bone marrow cells are treated with an agonist of a toll-like receptor (intracellular toll-like receptor) present inside the cell, in particular, in the endolysosomal compartment, for example, with at least one of toll-like receptor 7 agonist or toll-like receptor 9 agonist, combined treatment with interleukin 6 may induce differentiation into stable tolerogenic myeloid-derived suppressor cells as desired in the present invention.
(54) According to another embodiment of the present invention, there is provided a method for producing myeloid-derived suppressor cells (MDSCs), comprising a step of treating bone marrow cells with type I interferon, to induce differentiation into myeloid-derived suppressor cells. Here, the type I interferon used to treat the bone marrow cells may be at least one selected from the group consisting of IFN-α, IFN-β, IFN-κ, and IFN-ω, may preferably be at least one of IFN-α or IFN-β, and may more preferably be IFN-β.
(55) According to yet another embodiment of the present invention, there is provided a method for producing dendritic cells, comprising steps of: (1) treating bone marrow cells with a toll-like receptor agonist (TLR agonist), to induce differentiation into myeloid-derived suppressor cells (MDSCs); and (2) treating the differentiation-induced myeloid-derived suppressor cells with a growth factor, to induce differentiation into dendritic cells. Here, the toll-like receptor agonist used to treat the bone marrow cells is preferably an agonist of an intracellular toll-like receptor; and is more preferably at least one of toll-like receptor 7 agonist or toll-like receptor 9 agonist, among the agonists of intracellular toll-like receptors, for inducing differentiation into dendritic cells having desired properties in the present invention.
(56) Hereinafter, the present invention will be described in more detail by way of examples. These examples are merely given to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited by these examples.
EXAMPLES
[Example 1] Treatment of Bone Marrow Cells with Toll-Like Receptor Agonist
(57) Whole bone marrow cells in the femurs were harvested from C57BL/6 mice using a bone marrow harvesting needle. The harvested bone marrow cells were washed with PBS and red blood cells (RBCs) were removed using a red blood cell lysis buffer (Sigma Aldrich). The bone marrow cells were dispensed in a 6-well plate (1×10.sup.6 cells/ml; 2 ml/well) and cultured, under a condition of 5% CO.sub.2 and 37° C., in complete RPMI 1640 (c-RPMI 1640) medium supplemented with 100 U/mL of penicillin/streptomycin (Lonza, Basel, Switzerland), 10% fetal bovine serum (Lonza), 50 μM mercaptoethanol (Lonza), 0.1 mM non-essential amino acids (Lonza), and GM-CSF (20 ng/mL). Here, as illustrated in FIG. 1, each of a TLR2 agonist (Pam3CSK4), a TLR3 agonist (poly I:C), a TLR4 agonist (LPS, from Escherichia coli O111:B4), a TLR7 agonist (imiquimod, R837), and a TLR9 agonist (ODN1826) was added to the medium in an amount of 50 ng/ml at the initiation time of differentiation (1 method), on day 3 after the initiation of differentiation (2 method), or at the initiation time of differentiation and on day 3 after the initiation of differentiation (3 method). On day 3 of culture, the bone marrow cells and 1 ml of c-RPMI 1640 medium were added to each well. On day 6 of culture, the cells were collected and stained with Fluorescein-conjugated CD11c mAb to measure a proportion of CD11c.sup.+ cells. The results are illustrated in FIGS. 2 and 3. As a result, in a case where the TLR2 agonist, TLR4 agonist, TLR7 agonist, or TLR9 agonist is applied using the 3 method, capacity of inhibiting dendritic cells was strongly induced. In addition, in a case where the 3 method is used, proportions of CD11c.sup.+ cells were checked for respective treatment concentrations of each TLR agonist (TLR2 agonist (Pam3)—1, 10, and 50 ng/ml; TLR3 agonist (Poly I:C)—10, 100, and 500 ng/ml; TLR4 agonist (LPS)—1, 10, and 50 ng/ml; TLR7 agonist (imiquimod)—1, 10, and 50 ng/ml; TLR9 agonist (CPG:ODN)—1, 10, and 50 ng/ml). As a result, as illustrated in FIG. 4, it was found that differentiation into dendritic cells is inhibited in a concentration-dependent manner by the TLR agonist treated. However, in a case where the TLR3 agonist is applied, an effect of inhibiting differentiation into dendritic cells was not observed.
[Example 2] Identification of Relationship Between Inhibition of Differentiation into Dendritic Cells and MyD88
(58) Additionally, in order to identify whether an effect of inhibiting differentiation into dendritic cells is induced in a MyD88-dependent manner, C57BL/6 mice and C57BL/6 MyD88.sup.−/− mice were treated with a TLR agonist in the same manner as the 3 method in Example 1, to induce differentiation, and then a proportion of CD11c.sup.+MHC-II.sup.+ cells was measured. The results are illustrated in FIGS. 5 and 6.
(59) As illustrated in FIGS. 5 and 6, except a case where a TLR3 agonist is applied, in a case where a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, or a TLR9 agonist is applied, a proportion of CD11c.sup.+MHC-II.sup.+ cells was decreased. However, in a case where MyD88 is knocked out, a proportion of CD11c.sup.+MHC-II.sup.+ cells was maintained at 80% or higher, indicating that differentiation into dendritic cells is induced.
(60) From these results, it was found that differentiation into dendritic cells is induced in a MyD88-dependent manner.
[Example 3] Phenotypic Analysis of Cells Induced by TLR Stimulation
(61) The results obtained by analyzing phenotypes of cells harvested after treating bone marrow cells with a TLR agonist in the same manner as the 3 method in Example 1 and performing culture for 6 days are illustrated in FIGS. 7 and 8. As a result, it was found that in the cells induced by TLR stimulation, all dendritic cell phenotypes (CD11c, CD4, CD103, CD8a) decrease, whereas expression of Gr-1, Ly6C, and CD11b is induced at a high level. From these expression patterns, it was predictable that the cells induced by differentiation from the bone marrow cells are myeloid-derived suppressor cells (MDSCs).
(62) Here, it was found that in a case where among TLR agonists, especially a TLR7 or TLR9 agonist is applied, PDCA-1, a marker of plasmacytoid dendritic cells (pDCs) is highly induced; however, from the viewpoint that B220, another marker of pDCs, shows no significant difference, it can be seen that pDCs are not induced.
(63) From these results, it can be seen that different types of MDSCs are induced in a case of being treated with a TLR2 or TLR4 agonist, and in a case of being treated with a TLR7 or TLR9 agonist.
[Example 4] Identification of Phenotypes of MDSCs Induced by TLR Stimulation
(64) As illustrated in Example 3, it can be seen that the cells, which have been induced by differentiation from bone marrow cells after TLR stimulation according to the 3 method in Example 1, are MDSCs. On the other hand, subtypes of the MDSCs may be divided into monocytic-MDSCs (M-MDSCs) and granulocytic-MDSCs (G-MDSCs). Gr-1.sup.high cells mostly correspond to G-MDSCs, and Gr-1.sup.low cells may be classified as M-MDSCs.
(65) Thus, for MDSCs harvested after performing culture for 6 days with a Gr-1 antibody using the 3 method in Example 1, an expression level of Gr-1 was checked. The results are illustrated in FIGS. 9 and 10. As a result, in the cells induced by a TLR2 or TLR4 agonist, Gr-1.sup.low MDSCs were induced at a high level; on the contrary, in the cells induced by a TLR7 or TLR9 agonist, Gr-1.sup.high MDSCs were induced at a high level.
(66) Furthermore, in order to accurately classify subtypes of MDSCs, their expression patterns were identified using Ly6G and Ly6C antibodies. As a result, as illustrated in FIGS. 11 and 12, it was found that G-MDSCs (X.sub.2 cells) and M-MDSCs (X.sub.3 cells and X.sub.4 cells) are induced from bone marrow cells by TLR stimulation, and among these, a proportion of M-MDSCs is high; however, it was found that Ly6C.sup.int M-MDSCs (X.sub.3 cells) are induced in a case of being treated with a TLR2 or TLR4 agonist, and that Ly6C.sup.high M-MDSCs (X.sub.4 cells) are induced in a case of being treated with a TLR7 or TLR9 agonist.
[Example 5] Identification of Relationship Between Differentiation into MDSCs and MyD88
(67) In order to identify whether differentiation into respective MDSCs' subtypes is induced in a MyD88-dependent manner, C57BL/6 mice and C57BL/6 MyD88.sup.−/− mice were treated in the same manner as the 3 method in Example 1, and then expression patterns of Ly6G and Ly6C were identified in the same manner as in Example 4 (FIG. 13). As a result, it can be seen that Ly6C.sup.int M-MDSCs (X.sub.3 cells) are induced in a case of being treated with a TLR2 or TLR4 agonist, and that Ly6C.sup.high M-MDSCs (X.sub.4 cells) are induced in a case of being treated with a TLR7 or TLR9 agonist, indicating that differentiation into MDSCs is MyD88-dependent.
[Example 6] Molecular Parameters Induced by TLR2 or TLR9 Agonist
(68) As shown in Example 4, various factors induced in M-MDSCs were analyzed to accurately determine whether the cells induced by differentiation from bone marrow cells after TLR stimulation using the 3 method are M-MDSCs. In addition, in this analysis, in order to identify functional differences between Ly6C.sup.int M-MDSCs induced by a TLR2 or TLR4 agonist, and Ly6C.sup.high M-MDSCs induced by a TLR7 or TLR9 agonist, TLR2-M-MDSCs and TLR9-M-MDSCs were separated using a MACS system. Here, treatment with LPS and IFN-γ was performed for 24 hours to induce activity of M-MDSCs.
(69) Specifically, a NO.sub.2 formation level was analyzed using a NO kit, expression levels of NOS2 and arginase-1 were analyzed through Western blotting, and an expression level of IL-10 was analyzed through ELISA. The results are illustrated in FIGS. 14, 15, and 16, respectively. In addition, expression levels of arginase-1 and iNOS were identified with FACS. The results are illustrated in FIGS. 17 and 18. As illustrated in FIGS. 14 to 18, it was found that expression of anti-inflammatory cytokine (IL-10) and immunosuppressive agents (Arg-1, NOS) is increased upon stimulation of M-MDSCs induced by treatment with a TLR2 or TLR9 agonist.
(70) In addition, expression levels of various markers including CD124, CD115, F4/80, and PDCA-1 were analyzed to identify phenotypes of M-MDSCs induced in a case of being treated with a TLR2 or TLR9 agonist. As a result, as illustrated in FIGS. 19 and 20, CD124 was expressed at a high level in M-MDSCs induced after TLR2 stimulation, and CD115, F4/60, and PDCA-1 were expressed at a low level in M-MDSCs induced after TLR2 stimulation.
(71) Additionally, for M-MDSCs induced in a case of being treated with a TLR2 or TLR9 agonist, expression levels of inflammatory cytokines were analyzed with ELISA, and the results are illustrated in FIG. 21. Expression levels of immunosuppressive factors were identified with FACS, and the results are illustrated in FIGS. 22 to 24. As a result, it was found that in a case of being activated, M-MDSCs induced after treatment with the TLR2 agonist inhibit expression of inflammatory cytokines at a higher level than M-MDSCs induced after treatment with the TLR9 agonist.
[Example 7] Identification of T Cell Proliferation Inhibition and Induction of Differentiation into Regulatory T Cells by M-MDSCs Induced by TLR2 or TLR9 Agonist
(72) Hereinafter, T-cell proliferation inhibition capacity and induction of differentiation into regulatory T cells, which are other characteristics of M-MDSCs, were identified. More specifically, T cells were activated through co-culture of OVA-pulsed DCs and CellTrace-labeled CD4 T cells of OT-II mice, and M-MDSCs which had been induced by differentiation using the 3 method in Example 1 were added thereto. Then, T cell inhibition capacity of M-MDSCs was identified, and the results are illustrated in FIGS. 25 to 28. An expression level of Foxp3 was checked to identify a proportion of regulatory T cells, and the results are illustrated in FIGS. 29 to 32.
(73) As illustrated in FIGS. 25 to 32, it was found that both M-MDSCs induced after TLR2 stimulation and M-MDSCs induced after TLR9 stimulation inhibit proliferation of T cells in a number-dependent manner and remarkably induce differentiation into Foxp3.sup.+CD4 T cells.
[Example 8] Evaluation of Differences, in Terms of Secondary Differentiation, Between M-MDSCs Induced by TLR2 Agonist and M-MDSCs Induced by TLR9 Agonist
(74) Respective M-MDSCs which had been induced by differentiation using the 3 method in Example 1 were dispensed in 6-well plates (1×10.sup.6 cells/ml; 2 ml/well), and then cultured for 5 days in cRPMI 1640 supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, 50 μM mercaptoethanol, 0.1 mM non-essential amino acids, and 20 ng/ml GM-CSF or M-CSF. The resulting cells were harvested. In order to identify whether differentiation into dendritic cells has been induced by the culture, staining with anti-CD11c and anti-MHC-II was performed for dendritic cells, and then analysis was performed through the flow cytometer FACSverse. The results are illustrated in FIGS. 33 and 34. In addition, in order to identify whether differentiation into macrophages has been induced by the culture, staining with anti-CD11b and anti-F40/80 was performed for macrophages, and then analysis was performed through the flow cytometer FACSverse. The results are illustrated in FIGS. 35 and 36.
(75) As illustrated in FIGS. 33 to 36, it was found that M-MDSCs induced by a TLR2 agonist have been partially induced to differentiate into macrophages and have hardly differentiated into dendritic cells, whereas M-MDSCs induced by a TLR9 agonist have been induced to differentiate into dendritic cells and macrophages.
(76) Additionally, T cell proliferation inhibition capacity and degree of secondary differentiation into dendritic cells were evaluated while culturing respective M-MDSCs, which had been induced by differentiation using the 3 method in Example 1, with addition of GM-CSF in the presence of T cells. As a result, as illustrated in FIGS. 37 and 38, it was found that M-MDSCs induced by TLR2 stimulation do not show such changes and show consistently T cell proliferation inhibition capacity and IFN-γ inhibition capacity, whereas M-MDSCs induced by TLR9 stimulation show remarkably decreased T cell proliferation inhibition capacity and IFN-γ inhibition capacity in the presence of GM-CSF.
(77) In addition, degree of secondary differentiation into CD11c was identified under these conditions. As a result, as illustrated in FIG. 39, it was found that M-MDSCs induced by TLR2 stimulation stably maintain cellular characteristics, whereas M-MDSCs induced by TLR9 stimulation have been induced to differentiate into dendritic cells.
(78) From these results, it can be seen that M-MDSCs induced by differentiation using a TLR2 agonist have not been induced to differentiate into dendritic cells and stably maintain characteristics of M-MDSCs even in a case of being treated with a growth factor, whereas M-MDSCs induced by differentiation using a TLR9 agonist have been secondarily induced to differentiate into dendritic cells in a case of being further treated with a growth factor.
[Example 9] Functional Analysis for Dendritic Cells and Macrophages which had been Secondarily Differentiated from M-MDSCs Induced by TLR9 Agonist
(79) Hereinafter, characteristics of the dendritic cells, which had been secondarily differentiated from M-MDSCs induced by differentiation using the TLR9 agonist in the present invention, were analyzed by comparison with those of dendritic cells obtained by differentiation using other methods.
(80) In vitro methods of inducing differentiation into dendritic cells include a method of inducing differentiation using bone marrow cells and a method of inducing differentiation via Ly6C.sup.+ cells. Here, using the method of inducing differentiation using bone marrow cells, cells isolated from the mouse femoral bone marrow were cultured for 5 days in the same manner as the method of inducing differentiation into dendritic cells in Example 8 (bone marrow-derived dendritic cells, BMDCs). In addition, Ly6C.sup.+ cells were isolated from the mouse femoral bone marrow through Ly6C MicroBeads and then cultured as described above (Ly6C.sup.+-derived DCs). After 5 days, these cells were subjected to CD11c MicroBeads, to reisolate only dendritic cells (>90%).
(81) The dendritic cells isolated by the above methods and the dendritic cells obtained by secondary differentiation in Example 8 were analyzed in terms of phenotypes, and the results are illustrated in FIGS. 40 and 41.
(82) In addition, the respective three dendritic cells were dispensed in cRPMI 1640 medium in 48-well plates (1×10.sup.5 cells/ml), and then treated with LPS and IFN-γ as stimulants for the dendritic cells. Expression levels of cytokines induced by the respective dendritic cells were analyzed with ELISA, and their NO.sub.2 formation levels were analyzed with a NO kit. The results are illustrated in FIGS. 42 and 43. As a result, it was found that the dendritic cells, which have been secondarily differentiated from M-MDSCs induced by differentiation using a TLR9 agonist induce IL-12p70 and IL-10, which are important for differentiation into Th1- and Th2-type cells, at higher levels than the dendritic cells induced by other methods.
(83) Additionally, in order to identify an effect of the dendritic cells according to the present invention on T cells, the three dendritic cells as obtained above were stimulated with OVA323-339 peptide for 1 hour and then co-cultured, at a ratio of 1:5 (DCs: T cells), with CD4 T cells isolated from OT-II mice for 3 days. Subsequently, T cell proliferation capacity and IFN-γ expression levels were analyzed. As illustrated in FIG. 44, it was found that the dendritic cells, which have been secondarily differentiated from M-MDSCs induced by differentiation using the TLR9 agonist, exhibit remarkably superior T cell proliferation capacity as compared with Ly6C.sup.+-derived dendritic cells; and it was found that the dendritic cells, which have been secondarily differentiated from M-MDSCs induced by differentiation using the TLR9 agonist, allow Th1 cell-induced IFN-γ to be induced at a very high level as compared with the dendritic cells obtained by differentiation using other methods.
(84) From these results, it can be seen that the dendritic cells, which have been secondarily differentiated from M-MDSCs induced by differentiation using the TLR9 agonist, can effectively induce naive T cells into Th1 cells.
[Example 10] Effect of Cytokine on M-MDSCs Induced by TLR2 or TLR9
(85) Hereinafter, experiments were conducted assuming that for respective M-MDSCs induced by differentiation upon treatment with a TLR2 or TLR9 agonist, differences in phenotype, T cell inhibition capacity, regulatory T cell formation capacity, and differentiation into dendritic cells are due to different cytokine secretion induced by each TLR stimulation. Therefore, cytokines induced at early differentiation stages (8, 18, and 36 hours after stimulation with TLR agonist) after treatment of bone marrow cells with a TLR2 or TLR9 agonist were analyzed. As a result, as illustrated in FIG. 45, it was found that TNF-α, IL-6, and PGE2 are induced in a case of being treated with the TLR2 agonist, and TNF-α, IL-6, IFN-α, and IFN-β are induced in a case of being treated with the TLR9 agonist; and in particular, it was found that IL-6 is remarkably induced in a case of being treated with the TLR2 agonist as compared with a case of being treated with the TLR9 agonist. Here, although not shown by data, it was found that IFN-γ, IL-15, IL-1β, TGF-β, IL-4, IL-12p70, IL-10, and FLT2L are not induced upon TLR2 or TLR9 stimulation.
[Example 11] Analysis of Association of Cytokines with Differentiation into M-MDSCs
(86) In order to identify association of TLR stimulation-induced cytokines with formation of M-MDSCs and inhibition of secondary differentiation as identified in Example 10, the 3 method in Example 1 was performed, except that TNF-α, IL-6, PGE2, IFN-α, or IFN-β is applied in place of the TLR agonist. On day 6 of culture, it was identified whether M-MDSCs and dendritic cells are formed. As illustrated in FIG. 46, it was found that in a concentration-dependent manner, M-MDSCs are formed and differentiation into dendritic cells is inhibited in a case of being treated with IL-6, PGE2, IFN-α, or IFN-β.
(87) Additionally, in order to identify whether formation of PDCA-1.sup.+ M-MDSCs, which had been induced in a case of being treated with a TLR9 agonist, was caused by such a cytokine, distribution of CD11c.sup.−CD11b.sup.+Ly6G.sup.−Ly6C.sup.+PDCA-1.sup.+ and CD11c.sup.−CD11b.sup.+Ly6G.sup.−Ly6C.sup.+PDCA-1.sup.− was checked for the cells obtained on day 6 of culture as described above. As a result, as illustrated in FIG. 47, it was found that M-MDSCs having the phenotype CD11c.sup.−CD11b.sup.+Ly6G.sup.− Ly6C.sup.+PDCA-1.sup.− are induced in large quantities, in particular, upon stimulation with IL-6.
[Example 12] Evaluation of M-MDSC Induction Capacity Caused by TLR9 and IL-6
(88) In order to evaluate differentiation into M-MDSCs caused by IL-6 and functions of the M-MDSCs, recombinant IL-6 or an IL-6 receptor blocker was applied together with a TLR9 agonist using the 3 method in Example 1, and then the cells were harvested on day 6 of culture. Among the cells harvested according to the respective treatments, proportions of MDSCs and dendritic cells were checked. As a result, as illustrated in FIG. 48, it was found that in a case of being treated with IL-6, differentiation into PDCA-1.sup.+ M-MDSCs is increased and differentiation into dendritic cells is further decreased; and it was found that in a case of being treated with an IL-6 receptor blocker, differentiation into dendritic cells is increased and differentiation into PDCA-1.sup.+ M-MDSCs is decreased.
(89) In order to identify whether the M-MDSCs induced according to respective treatments as described above are secondarily differentiated into dendritic cells, culture was performed for 5 days in the same manner as the method of inducing differentiation into dendritic cells in Example 8. As a result, as illustrated in FIG. 49, it was found that degree of differentiation of M-MDSCs into dendritic cells is remarkably decreased and thus M-MDSCs are maintained in a case of being further treated with IL-6, as compared with a case of being treated with only TLR9 agonist, and that the degree of differentiation is increased in a case of being treated with an IL-6 receptor blocker.
(90) From there results, it can be seen that in a case where bone marrow cells are treated with an agonist of an intracellular toll-like receptor such as a TLR9 agonist, combined treatment with IL-6 makes it possible to obtain stable tolerogenic myeloid-derived suppressor cells that are not induced to differentiate into dendritic cells.
[Example 13] Evaluation of M-MDSCs' Capacity to Differentiate into Dendritic Cells, Induced by Type I IFN
(91) The 3 method in Example 1 was performed, except that a TLR2 agonist, a TLR9 agonist, or IFN-β is applied, and then only Ly6C.sup.+ M-MDSCs are collected on day 6 of culture. Then, GM-CSF was applied in the same manner as in Example 8 and culture was performed for 3 days. Subsequently, proportions of CD11c.sup.+ cells were checked for the respective treatments. As a result, as illustrated in FIG. 50, it was found that in a case of being treated with IFN-β which is type I IFN, differentiation into dendritic cells is induced similarly to a case of being treated with a TLR9 agonist.
(92) Additionally, a TLR2 agonist was applied using the 3 method in Example 1, with IFN-β being further applied in an amount of 5 ng/ml or 25 ng/ml. Then, on day 6 of culture, only Ly6C.sup.+ M-MDSCs were collected. Subsequently, GM-CSF was applied in the same manner as in Example 8 and culture was performed for 3 days. Then, proportions of CD11c.sup.+ cells were checked for the respective treatments. As a result, as illustrated in FIG. 51, it was found that differentiation into dendritic cells is hardly induced in a case of being treated with the TLR2 agonist, whereas differentiation into dendritic cells is increased in a concentration-dependent manner in a case where the TLR2 agonist is applied in combination with IFN-β which is type I IFN.
(93) Finally, in order to accurately check the function of type I IFN, bone marrow cells harvested from wild-type mice and type I IFN receptor knockout mice were treated with a TLR9 agonist using the 3 method in Example 1; and on day 6 of culture, degree of formation of PDCA-1.sup.+ M-MDSCs and dendritic cells was evaluated. As a result, as illustrated in FIG. 52, it was found that MDSCs themselves are not induced from type I IFN receptor knockout mice.
(94) From these results, it can be seen that in the present invention, the type I IFN receptor plays an important role in inducing differentiation of bone marrow cells into PDCA-1.sup.+ M-MDSCs and inducing differentiation of the PDCA-1.sup.+ M-MDSCs into immunogenic dendritic cells.
(95) As stated above, specific parts of the present invention have been described in detail. However, it is apparent to those skilled in the art that such specific description is only for illustrating preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
INDUSTRIAL APPLICABILITY
(96) According to an embodiment of the present invention, there are provided a method for producing myeloid-derived suppressor cells, a myeloid-derived suppressor cell produced thereby, and uses thereof.
(97) According to another embodiment of the present invention, there are provided a method for producing dendritic cells, a dendritic cell produced thereby, and uses thereof.
