BRONTE VINCENZO (IT)
| WO2007082177A2 | 2007-07-19 |
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MOVAHEDI KIAVASH ET AL: "Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity.", BLOOD 15 APR 2008, vol. 111, no. 8, 15 April 2008 (2008-04-15), pages 4233 - 4244, XP002553175, ISSN: 0006-4971
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| CLAIMS 1. Procedure for the culture and the differentiation of myeloid- derived suppressor cells characterised by the use of culture media additivated with one or more of the following cytokine mixes: granulocyte macrophage colony-stimulating factor associated with granulocyte colony-stimulating factor/ or granulocyte macrophage colony-stimulating factor associated with interleukin 6; in concentrations and for times needed for the differentiation and the growth of said myeloid-derived suppressor cells. 2. Procedure as claimed in claim 1, comprising a phase of derivation of said myeloid-derived suppressor cells from mammals . 3. Procedure as claimed in claims 1 and 2, comprising a phase of derivation of said myeloid-derived suppressor cells from mice . 4. Procedure as claimed in claims 1 and 2, comprising a phase of derivation of said myeloid-derived suppressor cells from human beings. 5. Procedure as claimed in any of the previous claims, comprising a phase of derivation of said myeloid-derived suppressor cells from bone marrow and/or other organs and/or tissues comprising hematopoietic stem cells. 6. Procedure as claimed in any of the previous claims, in which said bone marrow derives from isolation of the marrow from mouse bone or bone of other mammals and/or from human marrow aspiration. 7. Procedure as claimed in any of the previous claims, in which in which from said bone marrow derives a cellular suspension. 8. Procedure as claimed in any of the previous claims, comprising a phase of elimination from said cellular suspension of the erythrocites contained in it by means of cellular lysis or other procedure. 9. Procedure as claimed in any of the previous claims, in which said cytokine mixes comprise each cytokine in a concentration varying between 20 and 100 ng/ml . 10. Procedure as claimed in any of the previous claims, in which said cytokine mixes comprise each cytokine in a concentration of 40 ng/ml. 11. Procedure as claimed in any of the previous claims, comprising an in vitro culture phase of said cellular suspension obtained from said murine bone marrow in a concentration of 0.1- 0.4 million cells per ml of culture medium. 12. Procedure as claimed in any of the previous claims, in which said concentration of culture of said cellular suspension obtained from said murine bone marrow is 0.25 million cells per ml of culture medium. 13. Procedure as claimed in any of the previous claims, comprising an in vitro culture phase of said cellular suspension obtained from said human bone marrow in a concentration of 0.25- 0.5 million per ml of culture medium. 14. Procedure as claimed in any of the previous claims, in which said concentration of culture of said cellular suspension obtained from said human bone marrow is 0.375 million cells per ml of culture medium. 15. Procedure as claimed in any of the previous claims, comprising a phase of culture of said cellular suspension lasting 3-7 days at 370C and 5% CO2. 16. Procedure as claimed in any of the previous claims, in which said phase of culture lasts 4 days at 37 °C and 5% CO2. 17. Procedure as claimed in any of the previous claims, in which said culture in said culture media additivated with one or more of said cytokine mixes permits the differentiation of said cellular suspension in said myeloid-derived suppressor cells. 18. Procedure as claimed in any of the previous claims, in which said myeloid-derived suppressor cells show a phenotypic profile similar to the myeloid-derived suppressor cells isolated in vivo from tumour bearing individuals. 19. Procedure as claimed in any of the previous claims, in which said murine myeloid-derived suppressor cells show a more expanded cellular population having on its surface the marker GR-I expressed at low intensity. 20. Procedure as claimed in claim 19, in which said cellular population having on its surface the marker GR-I expressed at low intensity has on its surface a marker correlated to the suppressor ability of said myeloid-derived suppressor cells. 21. Procedure according to the claims 19 and 20, in which said marker correlated to the suppressor ability of said myeloid- derived suppressor cells is the receptor alpha for the interleukin 4. 22. Procedure as claimed in any of the previous claims, in which said human myeloid-derived suppressor cells have a more expanded cellular population having on its surface the markers CD16+/CDllb+ and/or CD15+/CD14+. 23. Procedure as claimed in claim 22, in which said cellular population having on its surface the markers CDlβ+/CDllb+ and/or CD15+/CD14+ has on its surface said marker correlated with the suppressor ability of said myeloid-derived suppressor cells. 24. Procedure as claimed in claims 22 and 23, in which said marker correlated with the suppressor ability of said myeloid- derived suppressor cells is the receptor alpha for the interleukin 4. 25. Myeloid-derived suppressor cells characterised by being obtained by means of a procedure that uses culture media additivated with one or more of the following cytokine mixes: granulocyte macrophage colony-stimulating factor associated with granulocyte colony-stimulating factor; or granulocyte macrophage colony-stimulating factor associated with interleukin 6; in concentrations and for times needed for the differentiation and the growth of said myeloid-derived suppressor cells . 26. Myeloid-derived suppressor cells as claimed in claim 25, in which said myeloid-derived suppressor cells derive from mammals. 27. Myeloid-derived suppressor cells as claimed in claims 25 and 26, in which said myeloid-derived suppressor cells derive from mice. 28. Myeloid-derived suppressor cells as claimed in claims 25- 27, in which said myeloid-derived suppressor cells derive from human beings. 29. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-28, in which said myeloid-derived suppressor cells derive from bone marrow and/or other organs and/or tissues comprising hematopoietic stem cells. 30. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-29, in which said bone marrow derives from isolation of the marrow from mouse bone or bone of other mammals and/or from human marrow aspiration. 31. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-30, in which from said bone marrow derives a cellular suspension. 32. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-31, in which from said cellular suspension are eliminated the erythrocytes contained in it by means of cellular lysis or other procedure. 33. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-32, in which said cytokine mixes comprise each cytokine in a concentration varying between 20 and 100 ng/ml . 34. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-33, in which said cytokine mixes comprise each cytokine in a concentration of 40 ng/ml. 35. myeloid-derived suppressor cells as claimed in any of the previous claims 25-34, in which said cellular suspension obtained from said murine bone marrow is cultivated in vitro in a concentration of 0.1-0.4 million cells per ml of culture medium. 36. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-35, in which said concentration of culture of said cellular suspension obtained from said murine bone marrow is 0.25 million cells per ml of culture medium. 37. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-36, in which said cellular suspension obtained from said human bone marrow is cultivated in vitro in a concentration of 0.25-0.-5 million per ml of culture medium. 38. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-37, in which said concentration of culture of said cellular suspension obtained from said human bone marrow is 0.375 million cells per ml of culture medium. 39. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-38, in which said cellular suspension is cultivated for 3-7 days at 370C and 5% CO2. 40. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-39, in which said culture has a duration of 4 days at 370C and 5% CO2. 41. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-40, in which said culture in said culture media additivated with one or more of said cytokine mixes allows the differentiation of said cellular suspension in said myeloid- derived suppressor cells. 42. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-41, in which said myeloid-derived suppressor cells show a phenotypic profile similar to the myeloid-derived suppressor cells isolated in vivo from tumour-bearing individuals . 43. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-42, in which said murine myeloid-derived suppressor cells show a more expanded cellular population having on its surface the marker GR-I expressed at low intensity. 44. Myeloid-derived suppressor cells as claimed in claim 43, in which said -cellular population having on its surface the marker GR-I expressed at low intensity has on its surface a marker correlated with the suppressing ability of said myeloid-derived suppressor cells. 45. Myeloid-derived suppressor cells as claimed in the claims 43 and 44, in which said marker correlated with the suppressing ability of said myeloid-derived suppressor cells is the receptor alpha for the interleukin 4. 46. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-45, in which said human myeloid-derived suppressor cells show a more expanded cellular population having on its surface the markers CD16+/CDllb+ and/or CD15+/CD14+. 47. Myeloid-derived suppressor cells as claimed in claim 46, in which said cellular population having on its surface the markers CDlβ+/CDllb+ and/or CD15+/CD14+ has on its surface said marker correlated with the suppressing ability of said myeloid-derived suppressor cells. 48. Myeloid-derived suppressor cells as claimed in claims 46 and 47, in which said marker correlated with the suppressing ability of said myeloid-derived suppressor cells is the receptor alpha for the interleukin 4. 49. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-48, in which said myeloid-derived suppressor cells are used as immunosuppressive agents in any method suitable for limiting the excessive immune response. 50. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-49, in which said excessive immune response is mediated by the lymphocytes T. 51. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-50, in which said excessive immune response is mediated by other types of cells or molecules belonging to the immune system. 52. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-51, in which said limitation of the immune response occurs by means of the suppression of the lymphocyte proliferation of mononuclear cells derived from peripheral blood mediated by said myeloid-derived suppressor cells. 53. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-52, in which said suppression of the lymphocyte proliferation mediated by said myeloid-derived suppressor cells occurs in mixed lymphocyte cultures additivated by means of a stimulation with an antigenic peptide. 54. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-53, in which said antigenic peptide is recognised by the lymphocytes T CD8. 55. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-54, in which said lymphocyte suppression mediated by said myeloid-derived suppressor cells occurs in mixed lymphocyte cultures additivated by means of a stimulation with mitogen agents and in an antigen-dependent way. 56. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-55, in which said mitogen agents comprise antibodies that recognise the chain ε of the receptorial complex of the CD3 of the lymphocytes, e.g., OKT3, or which recognise a co-stimulating molecule, e.g., CD28, or other substances whose function is to trigger the development and the proliferation of the lymphocytes T. 57. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-56, in which said lymphocyte suppression mediated by said myeloid-derived suppressor cells occurs in mixed lymphocyte cultures additivated by means of a stimulation with alloantigens . 58. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-57, in which said alloantigens are chosen among the antigens belonging to an individual but recognised as foreign by another individual of the same species, e.g., mononuclear cells of peripheral blood of a different individual compared to the receptor individual. 59. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-58, in which said myeloid-derived suppressor cells are used as immunosuppressive agents in any method suitable for treating autoimmune disorders. 60. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-59, in which said autoimmune disorders are rheumatoid arthritis, type I diabetes, multiple sclerosis, lupus erythematos, rheumatoid arthritis and any other autoimmune disorder. 61. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-60, in which said myeloid-derived suppressor cells are used as immunosuppressive agents in any method suitable for treating alloimmune responses. 62. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-61, in which said alloimmune responses are the rejection of transplants, host-versus-graft disease and any other type of alloimmune response. 63. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-62, in which said myeloid-derived suppressor cells deriving from said mice are syngeneic. 64. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-63, in which said syngeneic myeloid-derived suppressor cells are transferred adoptively for said suppression of the lymphocyte proliferation in diabetic mice receiving a transplant of allogenic pancreatic islets. 65. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-64, in which said adoptive transfer of said syngeneic myeloid-derived suppressor cells prolong the survival of the receptor mice with respect to the control group. 66. Procedure as claimed in any of the previous claims 25-65, in which said adoptive transfer of said myeloid-derived suppressor cells can be used as immunosuppressive agents in other types of allogenic transplant. 67. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-66, in which said lymphocyte suppression mediated by said myeloid-derived suppressor cells occurs in individuals receiving a transplant or presenting an autoimmune disorder or an excessive immune response. 68. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-67, in which said lymphocyte suppression mediated by said transfer of said myeloid-derived suppressor cells occurs in individuals receiving a transplant and/or presenting an autoimmune disorder and/or an excessive immune response and/or any study model. 69. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-68, in which said myeloid-derived suppressor cells are used as study model in vivo in cases of tumorous neoplasia and generalised infections . 70. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-69, in which said myeloid-derived suppressor cells are present in the tumorous microenvironment . 71. Myeloid-derived suppressor cells as claimed in any of the previous claims 25-70, in which said myeloid-derived suppressor cells are used -as a means of evaluating the action of new compounds that inhibit the suppressor action or of any other compound the action of which interferes with the action of said myeloid-derived suppressor cells. 72. Myeloid-derived suppressor cells as claimed in any of the previous claims, in which said myeloid-derived suppressor cells can be obtained in vivo by administering said cytokine mixes. 73. Myeloid-derived suppressor cells as claimed in any of the previous claims, in which said myeloid-derived suppressor cells can be obtained by engineering or other suitable method. 74. Procedure for the treatment of an autoimmune disorder, and/or of an alloimmune response, and/or of an excessive immune response in an individual, characterised by the administering of myeloid-derived suppressor cells obtained by means of a procedure that uses culture media additivated with one or more of the following cytokine mixes: granulocyte macrophage colony-stimulating factor associated with granulocyte colony-stimulating factor; or granulocyte macrophage colony-stimulating factor associated with interleukin 6; in concentrations and for times needed for the differentiation and the growth of said myeloid-derived suppressor cells. 75. Procedure as claimed in claim 74, in which said myeloid- derived suppressor cells derive from mammals. 76. Procedure as claimed in claims 74 and 75, in which said myeloid-derived suppressor cells derive from mice. 77. Procedure as claimed in claims 74-76, in which said myeloid-derived suppressor cells derive from human beings. 78. Procedure as claimed in any of the previous claims 1A-Il , in which said myeloid-derived suppressor cells derive from bone marrow and/or other organs and/or tissues comprising hematopoietic stem cells. 79. Procedure as claimed in any of the previous claims 74-78, in which from said bone marrow derives a cellular suspension. 80. Procedure as claimed in any of the previous claims 74-79, in which said cytokine mixes comprise each cytokine in a concentration varying between 20 and 100 ng/ml. 81. Procedure as claimed in any of the previous claims 74-80, in which said cytokine mixes comprise each cytokine in a concentration of 40 ng/ml. 82. Procedure as claimed in any of the previous claims 74-81, in which said cellular suspension is cultivated for 3-7 days at 370C and 5% CO2. 83. Procedure as claimed in any of the previous claims 74-82, in which said culture has a duration of 4 days at 370C and 5% CO2. 84. Procedure as claimed in any of the previous claims 74-83, in which said culture in said culture media additivated with one or more of said cytokine mixes permits the differentiation of said cellular suspension in said myeloid-derived suppressor cells. 85. Procedure as claimed in any of the previous claims 74-84, in which said myeloid-derived suppressor cells show a phenotypic profile similar to the myeloid-derived suppressor cells isolated in vivo from tumour bearing individuals. 86. Procedure as claimed in any of the previous claims 25-85, in which said murine myeloid-derived suppressor cells have a more expanded cellular population having on its surface the marker GR-I expressed at low intensity. 87. Procedure as claimed in claim 86, in which said cellular population having on its surface the marker GR-I expressed at low intensity has on its surface a marker correlated with the suppressor ability of said myeloid-derived suppressor cells. 88. Procedure as claimed in claims 86 and 87, in which said marker correlated with the suppressor ability of said myeloid- derived suppressor cells is the receptor alpha for the interleukin 4. 89. Procedure as claimed in any of the previous claims 74-88, in which said human myeloid-derived suppressor cells have a more expanded cellular population having on its surface the markers CDl6+/CDllb+ and/or CD15+/CD14+. 90. Procedure as claimed in claim 89, in which said cellular population having on its surface the markers CD16+/CDllb+ and/or CD15+/CD14+ has on its surface said marker correlated with the suppressor ability of said myeloid-derived suppressor cells. 91. Procedure as claimed in claims 89 and 90, in which said marker correlated with the suppressor ability of said myeloid- derived suppressor cells is the receptor alpha for the interleukin 4. 92. Procedure as claimed in any of the previous claims 74-91, in which said myeloid-derived suppressor cells are administered as immunosuppressive agents in any method suitable for limiting the excessive immune response. 93. Procedure as claimed in any of the previous claims 74-92, in which said excessive immune response is mediated by the lymphocytes T. 94. Procedure as claimed in any of the previous claims 74-93, in which said excessive immune response is mediated by other types of cells or molecules belonging to the immune system. 95. Procedure as claimed in any of the previous claims 74-94, in which said autoimmune disorders are rheumatoid arthritis, type I diabetes, multiple sclerosis, lupus erythematos, rheumatoid arthritis and any other autoimmune illness. 96. Procedure as claimed in any of the previous claims 74-95, in which said alloimmune responses are transplant rejection, host-versus-graft disease and any other type of alloimmune response . 97. Procedure as claimed in any of the previous claims 74-96, in which said lymphocyte suppression mediated by said administering of said myeloid-derived suppressor cells occurs in individuals receiving a transplant and/or having an autoimmune disorder and/or an excessive immune response and/or any study model. 98. Procedure as claimed in any of the previous claims 74-97, in which said myeloid-derived suppressor cells are used as study model in vivo in cases of tumorous neoplasia and generalised infections . 99. Procedure as claimed in any of the previous claims 74-98, in which said myeloid-derived suppressor cells are used as a means for evaluating the action of new compounds that inhibit the suppressor action or of any other compound the action of which interferes with the action of said myeloid-derived suppressor cells. 100. Procedure as claimed in any of the previous claims 74-99, in which said myeloid-derived suppressor cells can be obtained in vivo by administering the said cytokine mixes. 101. Procedure as claimed in any of the previous claims 74-100, in which said myeloid-derived suppressor cells can be obtained by means of engineering or other suitable method. 102. Use of inhibitors of the proliferation and the differentiation of the suppressor cells for the production of a medication for the treatment of a tumorous neoplasia characterised by the elimination of myeloid-derived suppressor cells and/or by the elimination/silencing of the function of myeloid-derived suppressor cells at systemic level and/or at tumorous microenvironment level. 103. Use of myeloid-derived suppressor cells obtained by means of a procedure that uses culture media additivated with one or more of the following cytokine mixes: granulocyte macrophage colony-stimulating factor associated with granulocyte colony-stimulating factor; or granulocyte macrophage colony-stimulating factor associated with interleukin 6; in concentrations and for times needed for the differentiation and the growth of said myeloid-derived suppressor cells for the establishment of a system suitable for highlighting the effectiveness and/or the effects of drugs and/or treatments with antineoplastic action. |
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a population of suppressor cells of myeloid origin and the procedure for obtaining it in vitro starting with mice marrow cells or human marrow aspiration.
BACKGROUND ART
The immune system is composed of cells and molecules responsible for protecting a body from illnesses in general. Their response, coordinated with the introduction of foreign substances in the body, is the so-called immune response.
Nevertheless, the mechanisms which normally protect an individual from infections and from foreign substances at the same time can, in some circumstances, cause damage to the tissues in which the immune response occurs, causing a consequent illness. The best known pathologies of this type are: anaphylactic shock, allergies, self-immune illnesses and other pathological situations involving an exalted immune response.
The immune system has therefore evolved, developing a series of mechanisms able to prevent the damage caused by excessive or prolonged inflammation in the host. Some of these mechanisms which defend the host from the damage caused by the immune system itself comprise the generation and/or the expansion of cellular populations which, for example, negatively regulate the functions of one of the central components of the cell-mediate immune responses: the T. lymphocytes.
One of these populations with negative regulatory function, recently called "suppressor cells of myeloid derivation", is able to successfully inhibit the expansion of the T lymphocytes, both CD4 + and CD8 + , and induces major dysfunctions of the immune system, both in the tumorous pathology context and in the course of acute and chronic infections.
The term "designation cluster" or "differentiation cluster", abbreviated in CD, identifies a protocol used to identify molecules present on the cellular surface of different types of cells.
The name CD is used to classify such surface molecules, to each of which is attributed a number, and to identify cell markers that enable the cell to be classified according to the presence of such molecules on its surface.
The myeloid-derived suppressor cells are immature myeloid cells of hematopoietic derivation and can be traced in the blood, in the bone marrow, in the spleen and in the lymph nodes, but also in the tumorous micro-environment and in a context of strong immune activation, where they are able to suppress the immunity through complex paths of molecular activation that call for an increase of the metabolism of the amino acid L-arginine.
In mice, the characteristic markers on the surface of the myeloid-derived suppressor cells are Gr-I (an epitope common to the proteins Ly6G and Ly6C) and CDlIb. In human beings on the other hand the myeloid line is mainly distinguished by the markers CD14 and CD15, though a general consensus has not yet been achieved regarding their fine characterisation.
During the maturing process, the myeloid-derived suppressor cells can differentiate in a number of different cell types, such as, for example, macrophages, neutrophilic, monocytes, dendric cells, etc.
The main functions performed by this cell population concern the suppression of the immune response mediated by the T lymphocytes. Consequent to the action of the suppressor cells, the incapacity of the effector T cells occurs to respond to the antigene, along with the increase in regulatory T cells and the production of growth factors, cytokines and other substances that regulate the growth and expansion cycle of other cells responsible for immune activity. The suppression of the responses of the T lymphocytes is also associated with tumorous growth and the myeloid-derived suppressor cells are involved in this process (Sica A. and Bronte V. J. Clin. Invest., 117:1155-66, 2007; Marigo, I., et al., Immunol Rev., 222:162-79, 2008).
For this reason and for their importance, the study of the myeloid-derived suppressor cells is undergoing strong expansion along with research, in human beings, for markers that can establish their definitive characterisation. Furthermore, the scientific community has recognised the clinical importance of the myeloid-derived suppressor cells and is therefore starting to invest in the cross research associated with the possible use of these cells. Various pathologies do in fact exist that could take advantage of the use of these cells and consequently the development of a procedure able to obtain myeloid-derived suppressor cells in a systematic way could be advantageously exploited for patients with autoimmune complaints or patients undergoing heterologous organ transplant.
OBJECTS OF THE INVENTION
One object of the invention is to upgrade the state of the previous art.
Another object of the invention is to characterise in an in-depth way a population of myeloid-derived suppressor cells.
Another object of the invention is to characterise a population of myeloid-derived suppressor cells generated in vitro.
A further object of the present invention is to obtain a procedure for obtaining myeloid-derived suppressor cells in vitro.
Another object of the present invention is to obtain myeloid-derived suppressor cells starting with mouse marrow cells or human marrow aspiration. A further object of the present invention is to use the myeloid-derived suppressor cells as immunosuppressive agents when the need exists to limit excessive immune response.
A further object of the present invention is to reduce the immune response mediated by the T lymphocytes. A further object of the present invention is to use the myeloid-derived suppressor cells in case of autoimmune illnesses such as, for example but not limited to, type I diabetes (in the event of the pancreatic damage not being irremediable) , rheumatoid arthritis, lupus erythematos, vasculitis, autoimmune thyroiditis, multiple sclerosis or transplant rejection.
A further object of the present invention is the development of an effective iminunotherapeutic approach to combat the above illnesses by administering myeloid-derived suppressor cells to patients suffering- from such illnesses.
Another object is to use the present invention as a model to study the differentiation of myeloid-derived suppressor cells in vivo, in the cases of tumours or generalised infections.
A further object of the present invention is the development of an effective immunotherapeutic approach to combat the above illnesses through the inhibition of the differentiation and of the immunosuppressive activity of the myeloid-derived suppressor cells.
A further advantage of the present invention is the possibility of using the myeloid-derived suppressor cells as a useful instrument for evaluating new compounds that inhibit the suppressor action.
According to an aspect of the present invention, the characterisation is envisaged of a population of myeloid-derived suppressor cells as specified in the claim 25, and of a procedure for obtaining them as specified in the claim 1 and of a procedure for using them as indicated in the claim 74.
The dependent claims refer to preferred and advantageous forms of the invention.
SHORT DESCRIPTION OF THE DRAWINGS
Further characteristics and advantages of the invention will be more evident from the detailed description of a cell population of myeloid-derived suppressor cells, illustrated by way of example and not limited to, the attached drawings in which: the figure 1 represents the phenotype profile (obtained by means of cytofluorimetric analysis) of the murine myeloid-derived suppressor cells obtained from mouse bone marrow cultures of the
C57BL/6 strain normally treated for four days with the cytokine combination Mixl (Fig. 1C) or Mix2 (Fig. ID) compared to the myeloid-derived suppressor cells isolated from the spleen of tumour-bearing mice MCA203 (fibrosarcoma) (Fig. IA, positive control) and to the cells of mouse bone marrow of the C57BL/6 strain not treated with cytokine (Fig. IB, negative control) . In the axis Y of the left panel (indicated by 1) is indicated the marker Gr-I, in the axis Y of the right panel (indicated by 2) is represented the marker lymphocyte antigen 6 complex, locus G Ly6G, in the axis X of the left panel are indicated the markers CDlIb (3), CD62 Ligando (4), receptor alpha for the interleukin 4 (IL-4R alpha) (5) and F4/80 (6); in the axis x of the right panel is indicated the marker lymphocyte antigen 6 complex, locus C LyβC; the figure 2 is a graph representing the suppressor activity of the murine myeloid-derived suppressor cells, according to the present invention, in mixed lymphocyte cultures activated by means of peptide stimulation (MLPC, represented in A) or by means of halogenic stimulation (MLR, represented in B) . The cytotoxic activity of the activated lymphocytes was assayed by means of the 51 Cr release test. The left panel shows the graphs corresponding to 12% of myeloid-derived suppressor cells, in the central panel 6% of myeloid-derived suppressor cells and in the right panel 3% of myeloid-derived suppressor cells. In each graph, in the axis X, is indicated the dilution of the effector cells while in axis Y is indicated the 51 Cr release percentage. The lines interspersed with black circles refer to MLPC, those interspersed with white circles refer to the myeloid-derived suppressor cells cultivated with the Mixl and those interspersed with triangles refer to the myeloid-derived suppressor cells cultivated with the Mix2 ; the figure 3 represents the phenotypic analysis of human bone marrow cells after cell culture, according to the present invention.
In particular in (A) is represented the untreated marrow and in (B) the marrow treated with cytokine. In the graphs of the left panel, in the axis X is indicated the marker CD16 (8) and in the axis Y the marker CDlIb (3) , in the central panel of the axis X is indicated the marker CD15 (9) and in the axis Y the marker CD14 (10) , in the right panel in the axis X is indicated the marker CD15 (9) and in the axis Y the marker IL-4R alpha (11); the figure 4 is the representation of the suppression of the proliferation of the PBMC responder marked Carboxy Fluorescein Succinimidyl Ester (CFSE) and stimulated with OKT3 and anti-CD28 in the presence of cells taken from marrow, according to the present invention. In particular in (A) are represented the data relating to the culture of the PBMC responder stimulated with 0KT3 and anti-CD28; in (B) the PBMC responder stimulated by OKT3 and anti-CD28, with the addition of marrow cells cultivated in vitro without the addition of cytokine in a ratio of 1:1; in (C) the PBMC responder stimulated by OKT3 and anti-CD28, with the addition of marrow cells cultivated in vitro with the addition of cytokine granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) (Mixl) . The gate was positioned on the cells CD3+/CFSE+. In each graph, in the axis X is indicated the fluorescence intensity of the Carboxy Fluorescein Succinimidyl Ester (CFSE) and in the axis Y the number of cells; the figure 5 represents the experimental transplant pattern of pancreatic islets from syngeneic or allogenic mice and the subsequent adoptive transfer of myeloid-derived suppressor cells, according to the present invention. The numbers present inside the figure refer to the days on which the various actions are performed and on day 0 the transplant is made; the figure 6 represents the effect of the treatment with myeloid-derived suppressor cells, according to the present invention, on survival after the allogenic transplant of pancreatic islets (indicated by the Kaplan-Meier curves) . In the axis X is indicated the time expressed in days and in the axis Y the survival percentage. The lines interspersed with circles refer to the myeloid-derived suppressor cells cultivated with Mixl, those interspersed with squares to the myeloid-derived suppressor cells cultivated with the Mix2 and those interspersed with triangles to the control; the figure 7 represents the histological evaluation of the transplants of allogenic islets after therapy with myeloid- derived suppressor cells, according to the present invention.
In particular in 1-3 is represented the syngeneic transplant, in 4-6 the allogenic transplant on untreated receptors and in 7-9 the allogenic transplant on receptors treated with myeloid-derived suppressor cells.
EMBODIMENTS OF THE INVENTION The present invention relates to a population of myeloid- derived suppressor cells and to the procedure for obtaining them in vitro.
In particular, such cells can be obtained from bone marrow or from other organs and tissues containing hematopoietic totipotent cells starting with mice and other mammals, including human beings .
Such cells are cultivated in the presence of various cytokine combinations in concentrations and for times long enough for the differentiation of the myeloid-derived suppressor cells.
The culture conditions occur mainly in the presence of suitable selected ' growth factors present in determinate conditions inside the culture medium.
The great interest shown for this type of cells stems from the possibility of using them to treat various pathologies including autoimmune pathologies and allogenic responses, such as transplant rejection.
One of the main characteristics of these cells in fact is their capacity to reduce a lymphocyte response of T lymphocytes following the administering of myeloid-derived suppressor cells, and in particular to reduce the immune response of the T cells with respect to the host itself.
According to the present invention, such myeloid-derived suppressor cells are, in fact, suitable for use in the treatment of autoimmune illnesses, alloimmune responses or other pathologies involving a T lymphocyte response. Some examples of such pathological conditions are: type I diabetes, multiple sclerosis, lupus erythematos, rheumatoid arthritis, transplant rejection, etc. The experiments included below by way of explanation but not limited to the present invention, concern in particular the use of bone marrow of mice and human beings, but the marrow or other tissues or organs of other animals can be used.
Furthermore, in agreement with the present invention, various conventional cellular biology, molecular biology, microbiology, recombinant DNA and immunology techniques can be used, together with other techniques commonly present in laboratory practice.
GENERATION OF MYELOID-DERIVED SUPPRESSOR CELLS The haematopoietic cells are isolated from bone marrow of animals or human beings and are stimulated to differentiate in myeloid-derived suppressor cells through culture with different combinations of cytokines in particular concentrations.
The cells can be isolated and cultivated using different techniques known to the experts of the sector
PHENOTYPIC AND FUNCTIONAL VERIFICATION OF THE MYELOID- DERIVED SUPPRESSOR CELLS
To make sure the cells obtained in vitro correspond to myeloid-derived suppressor cells, a phenotypic test is performed (which allows ascertaining the degree of differentiation of the myeloid-derived suppressor cells) and a function in vitro test to show the actual suppressing capacity of the cells thus obtained.
The tests shown below, in an exemplary but not limitative way, of the present invention, are based on the knowledge and experience acquired during the numerous years of study on this type of cells and on their in vivo generation following the onset of tumour in a host.
It appears clear nevertheless that such tests are not the only ones that can be performed to evaluate the phenotypic and functional characteristics of a cell population and that the most common procedures of molecular biology, cellular biology and genetic and biomolecular analysis can be applied with similar results (by way of example, analysis of transcriptome by means of genetic chip, microRNA profile analysis, analysis of metabolites produced by means of nuclear magnetic resonance,
Elisa immunoenzymatic assays, Western blot protein analysis, secondary mediator flow analysis, etc.).
Such tests all fall within the protection scope of the present invention.
The present invention goes on to describe a number of examples which are purely illustrative and not limitative of the present invention.
Changes and variations may be known to the experts in the sector as regards the culture and cellular analysis procedure and all fall within the protection scope of the present invention.
EXAMPLES Example 1
In vitro culture of murine bone marrow.
The bone marrow is recovered using known methods such as, for example, by means of "flushing" (which consists in inserting a syringe needle of a gauge corresponding to the medullary canal followed by forced injection of the liquid medium contained in the syringe to dislocate all the contents of the medullary canal itself) different strains, such as BALB/c or C57BL/6, from the tibias of mice. The most commonly used needles are the type 23G but different types of needles can also be used.
The cellular suspension thus obtained, including hematopoietic stem cells, undergoes the lysis of the red blood cells using a hypotonic solution. The lysing solutions that can be used, being careful of the type of cells being treated, are known and commonly used.
Some : examples of the most commonly used lysing solutions are: the solution consisting of NH 4 Cl 15.4 mM, KHCO 3 0.1 mM, 0.01 mM EDTA, the ACK Lysing Solution (BioWhittaker, Walkersville, MD, USA), etc..
The cellular suspension from which the red blood cells have been eliminated is then resuspended in a suitable culture medium, preferably RPMI 1640 to which has been added 10% of fetal bovine serum (BioWhittaker) , 2 mM of L-glutamine, ImM of sodium pyruvate, 1000 U/ml of streptomycin, 100 U/ml of penicillin and 20 μM of 2-β-mercaptoethanol,
Less additivated media can also.be used; nevertheless, the cellular culture will be affected by this in terms of growth and survival .
To the cellular suspension so obtained are added the cytokines needed for cell differentiation and growth. The cytokines used are of two different mixes: the Mixl containing 20-100 ng/rαl (preferably 40 ng/ml) of recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) and 20-100 ng/ml (preferably 40 ng/ml) of recombinant murine granulocyte colony-stimulating factor (rmG-CSF) and the Mix2 containing 20-100 ng/ml (preferably 40 ng/ml) of recombinant murine granulocyte macrophage colony-stimulating factor (rmGM- CSF) and 20-100 ng/ml (preferably 40 ng/ml) of recombinant murine interleukin 6 rmIL-6) .
The cells are cultivated at the concentration of 1-4 million per plate with a preferred concentration of 2.5 million per petri plate (100 mm 2 ) in 10 ml of above-described medium additivated with the mixes of cytokines at the above-mentioned concentrations, and incubated for 4 days at 37 0 C and 5% CO 2 .
Such culture concentrations, media and conditions merely represent a non-limitative example of the present invention.
Such culture procedure, thanks to the cytokines used in the above-described concentrations, permits the development and the differentiation of myeloid-derived suppressor cells according to the present invention. ' Variations of these protocols which comprise different concentrations of cytokines or use of different plastics for in vitro culture are equally acceptable and concern the present invention.
Example 2 In vitro culture of cells from human marrow aspiration.
The marrow blood used is a cytoaspiration with cytologic characteristics within normal limits. The marrow blood contains numerous erythroblasts and therefore undergoes the lyses of the red blood cells using a lysing solution. Some examples of the most used lysing solutions are: the hypotonic solution consisting of NH 4 Cl 15.4 mM, KHCO 3 0.1 mM, 0.01 mM EDTA, the BD FACS™ Lysing Solution (BD), etc.. The cellular suspension from which the red blood cells have been eliminated is then re-suspended in a suitable culture medium, e.g., IMDM additivated with 15% FBS (Fetal Bovine Serum) or human serum, hepes buffer 0.01M (in 0.85% NaCl), penicillin 200 ϋ/ml and streptomycin 200 U/ml.
Less additivated media can also be used, however the cellular culture is affected in terms of growth and survival.
The cells are then plated at the concentration of 0.5-1 million/ml with a preferred concentration of 0.75 million/ml in 24-well culture plates, in an end volume of 2 ml.
Such culture procedure, thanks to the cytokines used in the above-described concentrations, permits the development and the differentiation of myeloid-derived suppressor cells according to the present invention. To the cellular suspension thus obtained are added the cytokines required for cellular differentiation and growth. The cytokines used are: human recombinant granulocyte colony- stimulating factor (rhG-CSF) at the concentration of 20-100 ng/ml (preferably 40 ng/ml) associated with human recombinant granulocyte macrophage colony-stimulating factor (rhGM-CSF) at the concentration of 20-100 ng/ml (preferably 40 ng/ml) , or granulocyte macrophage colony-stimulating factor (rhGM-CSF) 20- 100 ng/ml (preferably 40 ng/ml) associated with interleukin 6 (IL-6) 20-100 ng/ml (preferably 40 ng/ml) . The cells are cultivated at 37 0 C, 8% CO 2 for a period of time varying between 3 and 5 days, even though preferably the phenotypic and functional assays are made on the 4 th culture day.
Example 3 Phenotypic analysis by means of flow cytofluorimetry of the murine myeloid-derived suppressor cells.
The murine myeloid-derived suppressor cells obtained from the cultures are evaluated for the expression profile of a number of surface markers which is compared to the expression profile of the myeloid-derived suppressor cells obtained from the spleen of tumour-bearing animals (used as positive control) and fresh untreated marrow- (used as negative control) . To prevent the non-specific binding of the antibodies, the most common laboratory procedures are applied. In particular, the cells can be pre-incubated for about 10 minutes at room temperature with the antibody 24G2 (ATCC, clone HB-197) mouse anti-receptor Fc-γ, that recognises the extracellular domain of Fc-γRIII and murine RII.
Subsequently, the marking of the appropriate antibodies is performed.
The markers used in mice are (prevalently but not only) : Gr- 1, CDlIc, CD62 Ligand, alpha receptor for interleukin 4 (IL4R alpha) , F4/80, lymphocyte antigen 6 complex, locus C Ly6C, lymphocyte antigen 6 complex, locus G Ly6G, CD115, CD68,
Arginase 1.
Example 4 Phenotypic analysis by means of flow cytofluorimetry of the human myeloid-derived suppressor cells.
The human myeloid-derived suppressor cells obtained from the cultures are evaluated for the expression profile of a number of surface markers. To saturate the non-specific attack sites of the antibodies the most common laboratory procedures are used. An example of such methods could be incubating with human poly IgG (0.2 mg/ml) depending on the indications of the producer.
The markers used in human beings to analyse the populations of immature myeloid-derived suppressor cells are: CD-14; CDlIb; CD15; CD16; CD124 (IL4R alpha); CD115, Arginase 1; CD33; CD34, and the correct isotypic controls.
Marker and fluorochrome coupling is due solely to the fact of managing to perform multiple and contemporaneous markings inside the same sample and does not represent a limitative character of the present invention.
Such marking has made it possible to identify the populations present in the marrow samples, before and after treatment with cytokines, and to so assess which were the maturative profiles induced by the different cytokines and whether there was any expansion of an immature myeloid population expressing IL4-R alpha (fig. 3) . Example 5
Analysis of the suppressor activity of the myeloid-derived suppressor cells generated from cultures of lymphocite murine bone marrow stimulated by peptides (MLPC) or by alloantigens . The suppressor capacity on the lymphocytes T CD8+ of the murine myeloid-derived suppressor cells derived from the above- described bone marrow cultures can be evaluated in vitro by adding these cells as third part to mixed leukocytic cultures stimulated by peptide (MLPC) . In these MLPC the lymphocytes T CD8+ specific for the antigen gplOO from p-mel transgenic mice with specific TCR for gplOO, were stimulated in vitro for 5 days and then tested as effector cells in a typical 51 Cr release assay.
The suppressor capacity was also evaluated with regard to lymphocytes T CD8+ specific for other antigens such as HA (using transgenic mice C14) and for the antigen OVA (using transgenic mice OT-I) .
The myeloid-derived suppressor cells were added as third part to the MLPC in different concentrations: 12%, 6% and 3% (Fig. 2A) .
Furthermore, the suppressor capacity of the myeloid-derived suppressor cells derived from bone marrow cultures on the lymphocytes T CD8+ was evaluated in vitro by adding these cells as third part to mixed leukocytic cultures (MLR) , where splenocytes from C57BL/6 were stimulated in vitro for 5 days by gamma-irradiated allogenic splenocytes from mice BALB/C and then tested as effector cells in 51 Cr release assay.
The myeloid-derived suppressor cells were added as third part to the MLR in different concentrations: 12%, 6% and 3% (Fig. 2B) .
Example 6
Analysis of the suppressor activity of the myeloid-derived suppressor cells in lymphocyte cultures stimulated by mitogens or by alloantigens. To evaluate the suppressor activity of the cells of marrow aspiration cultivated in vitro with the above-mentioned cytokines, two lymphocyte proliferation assays were made. In the first assay, cultures were set up with mononuclear cells taken from peripheral blood (Peripheral Blood Mononuclear Cells, PBMC) , allogenic and stimulated with two monoclonal antibodies: 1) OKT3, a monoclonal antibody that recognises the epsilon chain of the receptor complex of the CD3 whose function it is to stimulate the activation and the proliferation of the lymphocytes T in an independent way from the antigen; 2) a monoclonal antibody anti-CD28 directed against the co- stimulating molecule CD28. These antibodies allow the perfect stimulation of the lymphocytes and are both necessary to induce a good suppression on the part of the myeloid-derived suppressor cells.
Such cultures, in fact, occur in the presence or not of the cells derived from the marrow. The proliferation of the PBMC is determined by means of marking with Carboxy Fluorescein Succinimidyl Ester.
By means of the cytofluorimetric analysis of the fluorescence of the PBMC and the consequent analysis of the data using specific software (ModFiT, Verity House) , the number of cellular divisions can be monitored.
The PBMC responders to be marked with Carboxy Fluorescein Succinimidyl Ester are resuspended at the concentration of 20 7 /ml in PBS (Phosphate Buffered Saline) and mixed with a solution of Carboxy Fluorescein Succinimidyl Ester in PBS at the variable final concentration between 4 and 7 μM.-
Such cells are then re-suspended at the desired concentration and plated in culture in 96 flat-bottom well plates previously covered with lμg/ml of OKT3 to which is added the antibody anti-CD28 at the concentration of 1 μg/well (1 mg/ml) , in a final volume of 200 μl/well in complete culture medium.
The PBMC responders marked with Carboxy Fluorescein Succinimidyl Ester are then cultivated on the wells recovered with OKT3 and in the presence of anti-CD28 in the presence or not of the marrow cells treated with the cytokines, at the concentration of 10 5 per population in each well (in a ratio of 1:1). The cellular culture, is incubated for 4 days in an incubator at 37° C, with a concentration of CO 2 of 5%. The lymphocyte proliferation is evaluated on the third or fourth day of the culture by means of cytofluorimetry, evaluating the incorporation and the reduction in intensity of the Carboxy Fluorescein Succinimidyl Ester.
A second assay made to highlight the suppression mediated by myeloid-derived suppressor cells uses mixed lymphocyte cultures (MLR) . In this way, the proliferation activity is evaluated of PBMC undergoing an allogenic stimulation. The PBMC responders are stimulated by the gamma-irradiated PBMC of another donor (called PBMC stimulator) , which therefore have alloantigens able to trigger the lymphocyte response of the responders. In all the experiments carried out, a same pool of γ-irradiated PBMC was used, taken from three healthy donors. The cells derived from marrow in the above-described conditions are gamma-irradiated and added as third part so as to determine their possible interference in the generation of an allogenic response by the PBMC responders. The third part is added in dilution starting with a proportion 1: 1 (responder: stimulator) up to a proportion 1:1/32. At the sixth day of culture the cells are marked with tritiated thymidine ( 3 HTdR) and after 20-24 hours of incubation, the proliferation of the PBMC responders is quantified.
These two types of experiments allow us to analyse the effects of the myeloid-derived suppressor cells both in an activation model dependent on the alloantigen, and in an independent antigen activation model. Both the assays evidence the suppression, even though the assay with the mixed lymphocyte cultures shows a greater suppression compared to the activation with mitogens.
Example 7
Myeloid-derived suppressor cells generated in vitro to limit the immune response against an allogenic transplant. The tolerogenic ability in vivo of the myeloid-derived suppressor cells derived from bone marrow was then evaluated by examining their therapeutic effect in mice BALB/c made diabetic and transplanted with pancreatic islets from animals of strain BALB/c (syngeneic) or C57BL/6 (allogenic) .
The glycaemia was measured three times a week and the animals were sacrificed when this parameter exceeded 250 mg/dL for at least three consecutive measurements. The difference between the control mice that do not receive myeloid-derived suppressor cells and those receiving syngeneic myeloid-derived suppressor cells generated with the Mix2 is statistically significant (P<0.001); Upon rejection, or at determinate days after the transplant, the kidneys containing allogenic islets were histologically examined to determine the state of the transplant at the time of the explant and the insulin content in the transplant itself (Fig. 7). These experiments can also be performed with other strains of animals that are allogenic with one another, such as, for example, donors BALB/c and receptors 57BL/6.
As indicated above, such possibilities fall within the protection scope of the present invention.
RESULTS AND DISCUSSION
The figure 1 shows the phenotypic profile of the murine myeloid-derived suppressor cells obtained from cultures of bone marrow of mice C57BL/6 normally treated for four days with the cytokine combination Mixl (Fig. 1C) or Mix2 (Fig. ID) compared to the myeloid-derived suppressor cells isolated from the spleen of MCA203 tumour-bearing mice (Fig. IA, positive control) and to the bone marrow cells of C57BL/6 mice normally not treated with the cytokines (Fig. IB, negative control) . Analysing the phenotypic profiles obtained and comparing them with the characteristic profile of the myeloid-derived suppressor cells, the cytokine concentrations to be used were established as well as the culture times needed for the culture to obtain the greatest expansion of the myeloid-derived suppressor cells, phenotypically similar to the myeloid-derived suppressor cells of tumour-bearing mice.
As appears evident from the example shown in figure IC-D, by means of the cytokine treatments we have managed to obtain a phenotype identical to that of the myeloid-derived suppressor cells obtained from the tumour, shown in figure IA. In particular, the expression of the examined markers shows a profile identical to the percentage of expression of the myeloid-derived suppressor cells obtained from mice with tumour and above all identical is the distribution of the various markers with respect to the GR-I marker. Compared to the untreated marrow (figure IB) the Gr-l low population appears more expanded (low intensity of the Gr-I) .
The expression of IL-4R alpha is more evident in the Gr-l low fraction of the treated marrow compared to the untreated marrow. The expression of this marker is not only phenotypically but also functionally correlated to the suppressing ability of the myeloid-derived suppressor cells, as previously demonstrated (Gallina et al . J. Clin. Invest., 116:2777-2790, 2006).
Such data go to show that four days of treatment with the growth factors are perfect for obtaining a profile of markers of this type, and that along with the increase of culture days, there is also an increase in the expression of markers that correspond with dendritic cells, while there is a decrease in the expression of the marker CD62L, important for homing to the lymph nodes of these cells. The suppressing ability evaluated in vitro of the myeloid-derived suppressor cells obtained from bone marrow in various types of assays shows that these cells could be used in immunosuppression therapy.
The figure 2 shows the suppressing activity of the murine myeloid-derived suppressor cells added in gradually reduced percentages (12%, 6% and 3%) in mixed lymphocyte cultures activated by means of peptide stimulation (MLPC, as shown in Fig. 2A) or by means of allogenic • stimulation (MLR, as shown in Fig. 2B) .
Such cytotoxic activity with respect to the activated lymphocytes was assayed by means of the 51 Cr release test. These two types of experiments allow analysing the effects of the myeloid-derived suppressor cells both in an activation model dependent on the alloantigen, and in an activation model with tumour antigen.
It can be seen that in both the experiments (Fig. 2A and B) , with the greater concentration of effector cells we find the maximum release of chromium, relating therefore to the higher percentage of lysed target cells. The inhibition percentage of the lysis after adding suppressor cells decreases along with a decrease in the percentage of myeloid-derived suppressor cells present in the culture. The situation is comparable for the two different cytokine combinations Mixl and Mix2. In all these cases therefore, it can be seen that the myeloid-derived suppressor cells obtained from the cultures of both the cytokine mixes induce immunosuppression in vitro.
Figure 3 shows the phenotypic analysis of cells of human bone marrow after cellular culture ' in the presence (Fig. 3B) or absence (Fig. 3A) of the cytokines indicated in the text of the description. The maturation profile of mielo-monocytic populations was obtained using the marker pairs CDlIb and CD16, or CD14 and CD15.
In fact, the combined use of different markers of the human myeloid cells, such as CD14 with CD15 and CDlIb with CD16 (Fig. 3) , allows characterising the maturation stage of the analysed cells, evaluating both the expression and the intensity with which these are expressed on the cellular surface (Terstappen, L. W., M. Safford, and M. R. Loken. Leukemia 4:657-663, 1990). Marking with monoclonal antibodies (πiAb) anti-CD14 and anti-CDl5 divides the mielo-monocytic populations into four distinct areas: the immature cells such as CFU-GM (units forming granulo- monocytic colonies) , CFU-GEMM (units forming mixed granulocytic, erythroides, mielomonocytic and megakaryocytic colonies), hematopoietic stem cells (HSC) , and also all the cells not belonging to the myeloid line such as the lymphocytes, do not express the molecules CD14 and CD15. Maturation in a mielo- monocytic sense leads to an increase in the expression of such markers, until differentiation into mature granulocytes and monocytes. The monocytes express high-intensity CD14, while CD15 is not expressed or low intensity. On the contrary, the granulocytes show a phenotypic profile opposite to that of the monocytes for these markers: in fact they express high-intensity CD15 while CD14 is not expressed or low intensity.
As illustrated in the representative experiment shown in figure 3, the marrow cultures without the addition of cytokines show a low expression of IL4R alpha (9.0%), while the addition to the cultures of marrow of the combination of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces a significant expansion of myeloid cells that express IL-4R alpha (30.8%) . It is interesting to observe that the phenotype obtained shows characteristics similar to the myeloid-derived suppressor cells expanded in patients with neoplasia (Mandruzzato et al . , manuscript sent for publication) , thus indicating that the expansion of marrow cells in vitro with the growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) induces the mobilization of immature myeloid cells with the same phenotype as those observed in patients with carcinoma of the colon and melanoma (Mandruzzato et al . , manuscript sent for publication). As regards the other two monoclonal antibodies on the other hand (CDlIb and CD16) which are used to study the maturation of the myeloid cells, these allow identifying in a more detailed way the various stages of maturation of the myelocyte sub- populations . In fact, the myeloblast, progenitor cell of the myelocyte line, does not express the markers CDlIb and CD16. The differentiation of the myeloblasts in mature neutrofil granulocytes contemplates an increase of the intensity of expression of the marker CD16. This increase is in relation to the maturating stage, in fact, the granulocytes-neutrophils, which represent the terminal stage of the differentiation of these cells, express CD16 at higher intensity compared to the intermediate maturating stage cells (Terstappen et al., 1990). All the differentiating stages of the granulocyte commitment (from the promyelocytes to the granulocytes) are not characterised by changes in intensity of expression for the marker CDlIb; nevertheless, it can be seen that the intensity of fluorescence of the CDlIb of the granulocyte precursors is lower in the marrow cultures not treated with the cytokines compared to those additivated with the growth factors granulocyte colony- stimulating factor (G-CSF) and granulocyte macrophage colony- stimulating factor (GM-CSF) .
In the second suppression evaluation assay on lymphocyte proliferation (Fig. 4) it appears evident how the addition of cells derived from bone marrow cultivated for 4 days with the combination of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) (Fig. 4C) induces a considerable reduction in the percentage of cells that enter the cycle and, conversely, increases the percentage of cells that do not proliferate (evident in the first peak of each box on the right). The addition on the other hand of untreated marrow (Fig. 4B) does not change the proportion between cells in cycle and quiescent cells, but does delay the progression of the proliferating cells that enter the cycle.
The evaluation of the immunosuppression capacity of the myeloid-derived suppressor cells was then verified in allogenic transplants, the pattern of which is represented in figure 5. In particular, the mice BALB/c were made diabetic by means of two injections of streptozotocin (150 mg/kg) . Once hyperglycemia had been induced in the mice, a subcapsular transplant of pancreatic islets was performed. The experimental groups included the transplant of pancreatic islets from syngeneic animals (BALB/c) or allogenic animals (C57BL/6) . The animals transplanted with allogenic islets also received the endovenous adoptive transfer of 10 x 10 6 syngeneic myeloid-derived suppressor cells derived from bone marrow. The adoptive transfer of myeloid-derived suppressor cells was performed once a week for 5 weeks starting on the fourth day after the transplant. The glycemia was measured three times a week and the animals were sacrificed when this exceeded 250 mg/dL. The administration schedule can vary.
As shown in figure 6, the survival of the transplanted mice is indicated by the Kaplan-Meier curves. The glycemia was measured three times a week and the animals were sacrificed when this parameter exceeded 250 mg/dL (for at least three consecutive measurements).
In this model, in the group of animals transplanted with allogenic islets, the adoptive transfer of myeloid-derived suppressor cells derived from the marrow of animals BALB/c and obtained with the Mixl and Mix2, (5 weekly injections starting on the same day as the transplant) increases long-term survival in a statistically significant way compared to the control group transplanted with allogenic islets but in which the adoptive transfer does not occur. The syngeneic myeloid-derived suppressor cells generated with the Mix2 produce a highly significant statistical increase (P<0.001) compared to the control mice.
All the control animals transplanted with syngeneic islets (syngeneic transplant) remain normoglycemic during the period of observation.
The treatments with myeloid-derived suppressor cells to suppress the autoimmune response can be reasonably extended to other types of allogenic transplant and other models, e.g., the model EAE of multiple sclerosis in mice, models of arthritis from autoimmunity towards collagen, models of autoimmune colitis, etc.
In figure 7, the histological evaluation can be displayed of the transplants of allogenic islets after therapy with myeloid-derived suppressor cells. In particular, after the syngeneic transplant (1-3) a strong coloration was found for the insulin in all the times evaluated after 166 days (represented in B) . The receptors of untreated allogenic transplant (4-6) have an average transplant survival of 17 days. Histologically, these transplants present an intense lymphocyte infiltrate and little or no coloration for the insulin. After treatment with the Mix-1 and 2 (7-9) , the histological results vary between complete rejection
(represented in 7), similar to untreated allogenic transplant, and long-term survival after the transplant, with widespread coloration for the insulin, in the absence of lymphocyte infiltrate (8-9) .
It can therefore be seen that the kidneys of animals treated with myeloid-derived suppressor cells derived from the bone marrow and which have survived allotransplant show widespread coloration for the insulin in the absence of any evident lymphocyte infiltrate.