TREATMENT OF INFLAMMATORY AND DYSIMMUNE RESPONSE

Abstract

A drug and, more particularly, to a drug for treating inflammatory and dysimmune response. The present invention also relates to a drug for treating graft-versus-host disease. Thus, the present invention relates in particular to a cell expressing CD33, CD11b, CD14, CD163, CD206, HLA-DR, CD44, CD31, CCR5 and CD105.

Claims

1-17. (canceled)

18. Cell comprising it expresses CD33, CD11b, CD14, CD163, CD206, HLA-DR, CD44, CD31, CD105 and CCR5.

19. Cell according to claim 18, wherein it does not express one or a plurality of molecules chosen in the group comprising CD1a, CD80, CD86, CD16, CD56, CD3, CD19, CD66b, CCR7 and PDL1.

20. Cell according to claim 18, wherein it expresses one or a plurality of molecules chosen in the group comprising CCL2 and IL-6.

21. Cell according to claim 18, for use as a medicinal product.

22. Cell according to claim 18, for treating graft-versus-host disease, autoinflammatory diseases, giant cell arteritis (Horton disease), rheumatoid arthritis, autoimmune diseases and transplant rejection.

23. Cell according to a claim 18, for inducing an increase in CD8 regulatory T cells.

24. Cell according to claim 18, for inducing inhibition of the proliferation of effector T lymphocytes.

25. Composition comprising a cell according to claim 18 and a pharmaceutically acceptable vehicle.

26. Method for preparing a cell according to claim 18, comprising the step consisting of: (i) culturing monocytes in the presence of IL-6 and GM-CSF.

27. Method for preparing a cell according to claim 26, comprising the steps consisting of: (i) culturing monocytes in the presence of IL-6 and GM-CSF. (ii) isolating from the cells obtained following the preceding step, the cells expressing CD33.

28. Preparation method according to claim 26, wherein the cells deriving from at least one haematopoietic cell are PBMC.

29. Preparation method according to claim 27, wherein the step consisting of isolating in the cells expressing CD33 is performed via a cell sorter.

30. Preparation method according to claim 27, wherein the step consisting of isolating the cells expressing CD33 is performed via magnetic beads.

31. Preparation method according to claims 26, wherein the IL-6 present in the culture medium in step (i) is between 5 and 15 ng/ml.

32. Preparation method according to claim 26, wherein the GM-CSF present in the culture medium in step (i) is between 5 and 15 ng/ml.

33. Preparation method according to claim 26, wherein step (i) is performed for a period between 4 and 10 days.

34. Preparation method according to claim 26, wherein step (i) is performed at 37° C.

Description

DESCRIPTION OF EMBODIMENTS

Materials and Methods

[0030] Preparation of the Cells According to the Invention

[0031] Peripheral blood mononuclear cells were isolated in healthy donors or in patients to be treated using two techniques. The first technique consists of centrifuging peripheral blood on Ficoll gradient, retrieving the PBMC (peripheral blood mononuclear cells), and isolating the monocytes by magnetic sorting using anti-CD14 antibody coupled with a magnetic bead. The second consists of isolating, from peripheral blood, cells expressing CD34 by magnetic sorting, and culturing these cells in the presence of medium promoting the multiplication thereof (CD34 expansion medium), and differentiating same by culturing same in the presence of M-CSF.

[0032] The monocytes were then cultured at a concentration of 5.10.sup.6 cells/ml in RPMI 1640, supplemented with 10% Foetal calf serum, 10 ng/ml GM-CSF and 10 ng/ml IL-6 for 7 days, the medium being replaced every 3 days. The cells according to the invention were then purified, after labelling with a specific CD33 marker, via a cell sorter. [0033] Cell Proliferation Test

[0034] T lymphocytes, CD4+CD25− T lymphocytes and CD4+CD25+T lymphocytes were labelled with the “Cell trace Violet cell proliferation kit” (Cell Trace, Carlsbad, Calif.). The labelled cells are cultured in the presence of beads coated with anti-CD3/anti-CD28 (Dynabeads, Invitrogen, Cergy Pontoise, France) with or without the cells according to the invention. The proliferation of the T lymphocytes was detected by flow cytometry. [0035] Morphological Analysis

[0036] The morphological analysis of the cells according to the invention was performed after Wright/Giemsa labelling. [0037] Cytokine Assay The concentration of IFN-γ, TNF-α, IL-10, IL-6 and TGF-β was determined in the culture supernatant of the cells cultured using the ELISA technique. [0038] Animal Model of Graft-Versus-Host Disease

[0039] NOD/SCID/IL2Rγc-/- mice (Jackson Laboratory), aged 8 to 12 weeks, received peripheral blood mononuclear cells intravenously (20.10.sup.6 cells per mouse) with or without cells according to the invention (5.10.sup.6 cells per mouse). The cells are mixed just before injection. The signs of graft-versus-host disease were detected blind every 3 days. [0040] Histological Analysis of Lesions of Graft-Versus-Host Disease

[0041] The organs removed from the treated mice were fixed in formaldehyde and included in paraffin. Sections of 5 μm are prepared and stained with eosin and haematoxylin.

Results

[0042] The cells obtained in this way referred to as HuMoSC have the physical, phenotypic and functional characteristics as described below. [0043] Physical Nature of the Cells According to the Invention

[0044] After staining with Wright/Giemsa stain, the HuMoSC cells appear as a homogeneous population of large mononuclear cells with a basophilic cytoplasm. [0045] Phenotype of the Cells According to the Invention

[0046] The cells according to the invention have a CD33.sup.+CD11b.sup.+CD14.sup.+CD163.sup.+CD206.sup.+ HLA-DR.sup.+CD44.sup.+CD31.sup.+CD105.sup.+ CCR5.sup.+ phenotype, and weakly expressing CCR6.

[0047] The cells according to the invention do not express CD1α, CD80, CD86, CD16, CD56, CD3, CD19 and CCR7. [0048] Effect of the Cells According to the Invention on T Lymphocyte Proliferation

[0049] Stimulated autologous T lymphocytes were co-cultured with the cells according to the invention with a ratio of 2 T lymphocytes/cells according to the invention. It was observed that the T lymphocytes, stimulated and co-cultured with the cells according to the invention, proliferate less than T lymphocytes cultured alone. A separate analysis of the T lymphocytes demonstrated that the proliferation of CD4+ T lymphocytes and CD8+ T lymphocytes was also inhibited by the cells according to the invention. Moreover, the antiproliferative effect of the cells according to the invention is also observed on the autologous T lymphocytes and on the allogenic T lymphocytes. The T lymphocytes co-cultured with the cells according to the invention do not express the CD25+ marker unlike the T lymphocytes cultured alone. The analysis of the culture supernatants of the various samples made it possible to demonstrate that the cells according to the invention inhibit the production of proinflammatory cytokine (INF-γ and TNF-α) by T lymphocytes. As such, the cells according to the invention are characterised in that they inhibit cellular activation and cellular proliferation of autologous and allogenic CD4+ and CD8+ T lymphocytes and the cytokine secretion thereof. [0050] The Suppressant Effect of the Cells According to the Invention is STATS-Dependent

[0051] The suppressant effect of the cells according to the invention is not dependent on direct cell-to-cell contacts but on one or more soluble factors. Pre-treating the cells according to the invention with an inhibitor of the phosphorylated form of STATS induces loss of the suppressant effect, reduction of CCL2 and IL-6 secretion without interfering with the viability of the cells according to the invention. [0052] The Cells According to the Invention Protect Against the Onset of Graft-Versus-Host Disease

[0053] The mice having received human peripheral blood cells develop the clinical signs of graft-versus-host disease between 20 and 30 days post-injection and die before the 50.sup.th day post-injection. On the other hand, the mice having received a co-injection of human peripheral blood cells with the cells according to the invention merely exhibit slight signs of graft-versus-host disease and survive this injection. In particular, histological lesions of GvHD in the liver are markedly reduced in the group receiving the cells according to the invention [0054] Effect of the Cells According to the Invention on Regulatory T Lymphocytes.

[0055] Regulatory T lymphocytes represent a population of suppressor cells capable of promoting specific alloantigen tolerance.

[0056] The mice having received the cells according to the invention exhibit a greater quantity of CD8+ T lymphocytes expressing FoxP3 compared to the control mice.

[0057] The same results were observed in vitro after co-culture of a total T lymphocyte population with the cells according to the invention.