WO2002078698A2

WO2002078698A2 - Polarisation of dendritic cells (treatment of infections, cancer and autoimmune diseases) using histamine

Info

Publication number: WO2002078698A2
Application number: PCT/FR2002/001052
Authority: WO
Inventors: Gerssende Caron; Pascale Jeannin; Jean-Yves Bonnefoy
Original assignee: Pierre Fabre Medicament
Priority date: 2001-03-29
Filing date: 2002-03-27
Publication date: 2002-10-10
Also published as: AU2002255075A1; FR2822705A1; WO2002078698A3

Abstract

The invention relates to the use of histamine to induce the polarisation of dendritic cells and, more specifically, to induce the polarisation of dendritic cells into type 2 dendritic cells, which are used, in particular, for the therapeutic and prophylactic treatment of pathologies associated with an excessive Th1 response.

Description

POLARIZATION OF DENDRITIC CELLS BY HISTAMINE

The present invention relates to the use of histamine to induce the polarization of dendritic cells, and more particularly to induce the polarization of dendritic cells into type 2 dendritic cells, useful in particular for the therapeutic and prophylactic treatment of pathologies associated with an excessive Thl response. Histamine is a proinflammatory mediator released by activated mast cells and basophils. This mediator exists preformed in granules present in the cytoplasm of these cells and is released quickly after activation. Histamine is released in particular during the IgE-dependent activation of mast cells and basophils during allergic phenomena. It participates in clinical manifestations associated with the immediate hypersensitivity reaction such as vasodilation and bronchial secretion.

Recently it has been observed that histamine also exhibits immunoregulatory properties. Histamine increases the production of IL-6 and IL-8 by endothelial cells as well as the production of IL-1 and IL-6 by monocytes. Histamine has also been observed to reduce the production of IL-12 by monocytes from patients with atopy (van der Pouw Kraan et al, J Clin Invest 1998 Nov 15; 102 (10): 1866-73) .

Dendritic cells play a role in the development of an immune response and in the initiation of a specific T lymphocyte response (Steinman RM et al Immuno Rev (1997) 156, 25 & Sella M et al Curr Opin Immunol (1997) 9, 10). Immature dendritic cells are located in non-lymphoid tissue. At the level of the skin, in all the epidermis except the intestine, they are then called Langerhans cells; dendritic cells, in smaller quantities, are also located in organs such as the lung, liver, intestine. These immature dendritic cells capture and digest antigens very efficiently. After antigenic stimulation in vivo or stimulation by proinflammatory molecules, the dendritic cells which have captured the antigens migrate into the secondary lymphoid organs. During this migration, the dendritic cells undergo functional and phenotypic modifications which are grouped under the term of maturation. This maturation is characterized by an increase on the CD surface of molecules involved in the activation of T lymphocytes (such as CD40, CD54, CD58, CD86 and MHC class I / II), the production of proinflammatory cytokines (IL-1 and IL-6) and chemokines (such as TNFα, IL-8, MCP-1 and MlP-lalpha) and lose their ability to process ^λ antigen. Chemokines recruit inflammatory cells and promote the initiation of an immune response. In particular, IL-8, MCP-1 and MlP-lalpha attract not only inflammatory cells but also naive and memory T lymphocytes. Thus, CD by secreting chemokines increase their chances of coming into contact with T lymphocytes. The proinflammatory cytokines and in particular IL-1 will also act by directly activating CD.

The establishment of an antibody response directed against an antigen requires a series of complex events. It involves cells presenting the antigen, regulatory T lymphocytes (Th for T "helper") and antibody-producing B lymphocytes. Two types of Th lymphocytes can be distinguished according to the profile of cytokines produced: Th lymphocytes of type 1 producing IFN-γ and IL-2 and promoting the formation of IgG2a in mice, and Th lymphocytes of type 2 producing IL-4, IL-5 and IL-10 with formation of IgG1 in mice (Mosmann, TR and Sad S. Immunol. Today 1996, 17: 138). Furthermore, it has been shown that, for the same given antigen, it is the adjuvant which directs towards the majority isotype during the antibody response (Toellner K.-M. et al. J. Exp. Med. 1998, 187: 1193). Thus, it is known that aluminum salts, such as Alhydrogel, induce in the mouse an essentially Th2 type response (Allison AC In Vaccine design - The role of cytokine networks Vol. 293, 1-9 Plénum Press 1997) .

Because of their capacity to induce the polarization of naive T lymphocytes into effector cells Thl (producers of IFNgamma) or Th2 (producers of IL4), mature CD were called CDl or CD2 respectively (Liu, YJ et al Curr. Top Microbiol Immunol 2000). Myeloid CDs can polarize to CD1 or CD2 depending on the nature of the polarization factors that will influence the production of IL12 (a Thl-oriented cytokine). While IFNgamma induces the polarization of CD during maturation into CD1 (Hilkens, CM et al Blood 1997; Vieira, PL et al J Immunol 2000), prostaglandin E2 (Kalinski P, et al J Immunol 1997; Kalinski P , et al J Immunol 1998), cholera toxin (Gagliardi MC, et al European J Immunol 2000), and ATP (La Sala A, et al J Immunol 2001) promote the development of CD2. These mature dendritic cells present the antigens to the T lymphocytes and initiate the specific T responses very efficiently, the costimulation molecules being expressed in large quantities on these cells. As cells mature, they lose their ability to capture and process the antigen. In the thy o-dependent areas of the lymphoid organs, the migrated dendritic cells have acquired powerful immunostimulatory properties and will therefore very effectively activate the circulating naive T lymphocytes. The authors of the present invention have demonstrated for the first time the link between histamine and the polarization of dendritic cells. More specifically, they demonstrated that histamine promotes the polarization of dendritic cells into CD2-type dendritic cells. This is all the more surprising since the person skilled in the art cannot distinguish the molecules promoting a Th2 response acting directly on naive T lymphocyte cells or acting via dendritic cells. In the remainder of this description, the expression “histamine” will be used to name histamine, but also the histamine salts such as histamine dihydrochloride, histamine phosphate or other salts, esters or precursors histamine and histamine receptor agonists (Hl, H2, H3). HTMT, Amthamine or Dimaprit, as well as other analogs or agonists of the histamine receptors described in US Pat. No. 5,728,378 are also included in the term "histamine".

The present invention thus relates to the use of histamine and / or histamine receptor agonists to induce the polarization of dendritic cells, and more particularly to polarize dendritic cells into type 2 dendritic cells.

The present invention relates more particularly to the use of histamine and / or agonists of the histamine receptors for the manufacture of a medicament intended to induce the polarization of dendritic cells, preferably in dendritic cells of type 2 (DC2) .

In a preferred embodiment of the invention, the histamine and / or the histamine receptor agonists according to the invention can serve to promote the polarization of the dendritic cells in vitro, the mature polarized cells can then be reinjected in vivo.

In an even more preferred embodiment of the invention, this medicament can serve to promote the polarization of dendritic cells in vitro, after contact with a biological agent. Thus, the invention relates to the use according to the invention of histamine and / or agonists of histamine receptors and in addition of at least one agent biological for the manufacture of a medicament. The latter may for example increase the immunological response to this biological agent.

This biological agent can be chosen from nucleic acids, proteins, lipids, lipopeptides or polysaccharides.

It can more particularly be chosen from vaccinating antigens and / or antigens from bacteria, viruses, yeasts, parasites, fungi. More preferably, the biological agent is a tumor antigen. For the purposes of the present invention, a tumor antigen is defined as a tumor protein or peptide, and in particular as an epitope, in particular a CTL epitope (peptide sequences interacting with class I molecules and presented to CD8 + T lymphocytes) or as the nucleic acid sequence encoding such proteins, peptides or epitopes. The following tumor antigens may be mentioned, without limitation: MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, GD2, carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigens OV- TL3 and MOV18, TUAN, alpha-feto protein (AFP), OFP, CA-125, CA-50, CA- 19-9, renal tumor-associated antigen G250, EGP-40 (or EpCAM), S100 (malignant melanoma - associated antigen), p53, prostate tumor-associated antigens (eg, PSA and PSMA) and p21ras.

The biological agent can also be chosen from a lysate of autologous and / or heterologous tumor cells. For the purposes of the present invention, the term “autologous tumor cells” means tumor cells belonging to the subject who will receive the compositions according to the invention. The term “heterologous tumor cells” should be understood to mean cells originating from tumors originating from an individual different from that for which the composition according to the invention is intended. The use of heterologous cells makes it possible to obtain pharmaceutical compositions which make it possible in particular to treat patients suffering from cancer from whom the removal of tumor cells is not possible. The use of heterologous cells also makes it possible to obtain compositions according to the invention standardized comprising antigens found in many types of cancer and thus usable in a majority of patients. Tumor cells can be obtained from a sample of cancerous tissue, for example following a biopsy or surgical resection. These cells can then be used as they are or can be cultured before being lysed. A cell lysate can be defined within the meaning of the present invention as a mixture of intracellular and / or membrane antigens, preferably intra and membrane. Said autologous and / or heterologous tumor cell lysate according to the invention can be obtained by mechanical, chemical or enzymatic lysis of tumor cells. In order to lyse the cells mechanically, mention may in particular be made of the techniques known to those skilled in the art, namely in particular sonication, ultrasonication or freezing / thawing. Particularly preferred is freeze / thaw, and most particularly the use of multiple freeze / thaw cycles. The cells can also be lysed using chemical compounds or enzymes, such as for example digitonin lysis buffer, triton X-100 or Nonidet P40. Any method of breaking the cell membrane of tumor cells can be used to obtain a lysate.

The dendritic cells are thus modified using these antigens or cell lysates so that they express tumor antigens. Thus, said drug can be injected at the same time as the dendritic cells or preferably said drug can be used in vitro, in order to promote the polarization of the dendritic cells before reinjecting them into patients.

Thus, thanks to their effectiveness in presenting antigens and stimulating the immune system, dendritic cells in the presence of histamine and / or histamine receptor agonists may be used to generate anticancer CTL responses (Nestlé FO et al., 1998, Nat. Med., 4, 328-332). This approach, called "ex vivo" therefore consists in loading the dendritic cells ex vivo with the antigen of interest (peptides or cell lysate) in the presence of histamine and / or agonists of the histamine receptors and in reimplanting these cells in the patient. A step of washing the dendritic cells so as to remove the histamine and / or the agonists of the histamine receptors from the medium containing the dendritic cells can be carried out before reimplanting these cells in the patient.

Another approach according to the invention consists in transfecting ex vivo the dendritic cells with the gene coding for the antigen of interest and in particular with a gene coding for a bacteria, virus, yeast, parasite, fungus antigens, or for a tumor antigen, such as those described above, and / or with a gene encoding a cytokine or a growth factor, such as those described below, and to put the dendritic cells, before and / or during and / or after transfection, in the presence of histamine and / or histamine receptor agonists and to reinject these transfected cells (Gilboa E. et al., 1998, Cancer Immunol. Immunother., 46, 82- 87). A step of washing the dendritic cells so as to remove the histamine and / or the histamine receptor agonists from the medium containing the dendritic cells can be carried out before re-implanting these cells in the patient. Such approaches have been successfully used in mice and in humans (Hsu F.J. et al., 1996, Nat. Med., 2, 52-58).

The medicament according to the invention can comprise, in a preferred embodiment of the invention, also a factor for the maturation of dendritic cells. The latter may in particular be chosen from LPS, TNFα, and all compounds bacterial or viral inducing the maturation of immature dendritic cells, such as for example LPS (lipopolysaccharides), OmpA, oligonucleotides or monophosphoril A. This maturation factor may in particular be used prior to histamine, so as to mature the immature dendritic cells. It is also possible, preferably, to simultaneously use histamine and the maturation factor to mature and polarize the dendritic cells. The medicament according to the invention can also further comprise a cytokine or a growth factor, in particular interferon alpha or gamma, GM-CSF, IL-2, IL-12, IL-4, IL-6 and IL-18, an HSP (Heat Shock Protein) such as for example hsp70, hsp90, hsp96, which makes it possible to potentiate the immune response; and / or fibroblasts genetically modified so as to release a cytokine or a growth factor. Mention may be made of fibroblasts expressing GM-CSF sold by the company Immune Response Corporation. Histamine and / or histamine receptor agonists with at least one biological agent associated with TNF alpha can be used for the manufacture of a medicament intended to increase the proliferation of T lymphocytes.

The medicament according to the invention may also comprise an adjuvant making it possible to increase the immune response, in particular chosen from the group of adjuvant comprising MPL-A, Quil-A, ISCOM, CpG, Leif, CT (cholera toxin) or LT (Heat labile enterotoxin), just like the detoxified versions of CT or LT, or bacterial membrane proteins such as OMPC from Neisseria meningitidis (Vella et al ., Infect. Immun. 60, 1992, 4977-4983), TraT of Escherichia coli (Croft et al., J. Immunol. 146, 1991, 793-798) or PorB of Neisseria meningitidis (Fusco et al., J Infect. Dis. 175, 1997, 364-372), and preferably an OmpA of a bacteria of the genus Klebsiella, major protein of the outer membrane called P40, having an adjuvant activity for peptide subunit antigens (WO 95/27787 and WO 96/14415; Haeuw et al., Eur. J. Biochem. 255, 1998, 446-454; Plotnicky-Gilquin et al., J. Virol. 73, 1999, 5637-5645).

The medicament according to the invention can also comprise a pharmaceutically acceptable medium. Within the meaning of the present invention, the pharmaceutically acceptable medium is the medium in which the compounds of the invention are administered, preferably a medium injectable into humans. It can consist of water, an aqueous saline solution or an aqueous solution based on dextrose and / or glycerol.

The medicament according to the invention can also be conveyed in a form making it possible to improve its stability and / or its immunogenicity; thus, it can be conveyed in the form of liposomes, virosomes, nanospheres, microspheres or microcapsules. The drug or the combination of histamine and / or histamine receptor agonists - biological agent may also be in an easily administered form such as an ointment, a lotion, a solution or even in the form of an adhesive composition: plaster or "patch".

The medicament according to the invention may be used in particular for the treatment of: - microbial infections, in particular chronic type infections associated with the development of an ineffective specific immune response (virus: for example the human immunodeficiency virus (HIV) ), the hepatic viruses, in particular the hepatic viruses A, B, C and D and the parainfluenza virus, bacteria, parasites and yeasts),

- cancers, in particular in subjects carrying HIV and / or suffering from myelomas, lymphomas, leukemias, melanomas, carcinomas, of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, shed, for example.

As described above, the invention particularly relates to cellular immunotherapy using anti-cancer and anti-infectious vaccines in particular, by generating in vitro autologous polarized dendritic cells, by introducing a tumor antigen there and by reinjecting them after a step optional washing. Exposure of dendritic cells in vitro to histamine and / or histamine receptor agonists and optionally to a maturation factor, will increase the polarization of dendritic cells and therefore increase the effectiveness of the vaccine .

Also, the present invention relates particularly to the use according to the invention of histamine and / or histamine receptor agonists for the preparation of a medicament for increasing the immunological response vis-à-vis an agent biological as defined above. This medication can be a vaccine for the treatment or prevention of infectious diseases of viral origin - such as in particular HIV, hepatic viruses and parainfluenza virus -, bacterial, fungal, or caused by a yeast or a parasite.

The present invention also relates to the use according to the invention of histamine and / or histamine receptor agonists with at least one biological agent for the manufacture of a medicament for the treatment or prevention of cancers and particularly cancers among myelomas, lymphomas, leukemias, carcinomas of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, and for the manufacture of a medicament for the treatment or prevention of skin cancers chosen from keratinomas and carcinomas.

As described above, the treatment of these cancers can also be envisaged by injecting dendritic cells. autologous, including autologous dendritic cells that have been modified to express tumor antigens. The polarization of the dendritic cells will be ensured by histamine and / or the histamine receptor agonists. If immature dendritic cells are used, a maturation factor may also be used.

Another subject of the invention is the use of histamine and / or histamine receptor agonists for the preparation of a pharmaceutical composition intended to generate or increase an immune response against pathologies associated with an excessive Th1 response. .

Examples of such pathologies include Hashimoto's thyroiditis, multiple sclerosis, type I diabetes, transplant rejection, Crohn's disease, rheumatoid arthritis, sarcoidosis and autoimmune diseases . Certain DC2-based immunotherapies are more particularly envisaged in the article by Yong-Jun et al, Blood, April 15, 2000, volume 95, number 8, 2482-2483.

The invention also relates to the use of histamine and / or histamine receptor agonists to induce polarization of dendritic cells in vitro, and more particularly to polarize dendritic cells into type 2 dendritic cells.

In a particularly preferred embodiment of the invention, histamine and / or agonists of the histamine receptor, a maturation factor, are also used to induce in polarization of dendritic cells, and more particularly for polarizing dendritic cells into type 2 dendritic cells. The invention also relates to a device for polarizing dendritic cells, such as for example a kit, comprising at least histamine and / or receptor agonists. histamine. In a particularly preferred embodiment of the invention, the device for the polarization of dendritic cells, such as for example a kit, further comprises a maturation factor. This kit can be adapted for the polarization of dendritic cells in vitro and can be used for example in research laboratories. This kit may also contain histamine and / or histamine receptor agonists a maturation factor, immature dendritic cells and / or the means necessary to isolate immature dendritic cells, such as for example means of purification of blood mononuclear cells. It may thus include for example histamine and / or histamine receptor agonists, a maturation factor, different culture media, washing solutions, culture plates, reagents, controls such as by example of the antibodies, and an explanatory note for the implementation of the method according to the invention of maturation of the dendritic cells. A kit according to the invention can also be adapted to the implementation of the therapeutic method mentioned above. This kit can also contain histamine and / or histamine receptor agonists, a maturation factor, immature dendritic cells and / or the means necessary to isolate immature dendritic cells, such as for example means purifying blood mononuclear cells. It may thus include, for example, histamine and / or histamine receptor agonists, a maturation factor, different culture media, washing solutions, culture plates, reagents, controls, and a explanatory note for the implementation of the therapeutic method mentioned above. If necessary, it may also contain heterologous antigens or means for obtaining a lysate of autologous cells. The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope. These examples demonstrate the action of histamine on dendritic cells: histamine induces the maturation of immature human dendritic cells and gives them powerful stimulatory and antigen-presenting properties.

In these examples, reference is made to the following figures:

Figure 1: Histamine prevents the production of IL12 by mature CDs.

A&B: CD maturation is induced by 10 ng / ml of LPS, in the absence or presence of increasing doses of IFNg and / or histamine. After 48 hours, mature CDs are stimulated with soluble CD40 ligand (A) or LPS + IFNg (B). The concentration of IL12 p70 in the supernatants was measured by ELISA after 24 hours. The results are representative of one of 3 experiments. C: immature CDs were incubated with LPS (10 ng / ml) + IFNg (100 ng / ml) and treated or not with histamine (10 ^{~ 5} M), H1, H2 or H3 receptor antagonists (Mepyramine, cimetidine, and thioperamide, respectively) (all at 5x10 ^"5 M) 1 hour before the addition of histamine (10 ^{~ 5} M), or were treated with H1 and H2 receptor agonists (HTMT and Amthamine, respectively) (10 ^{~ 5} M). After 2 days the mature CDs were restimulated by LPS + IFNg and the production of IL12 p70 was assayed The results are expressed in pg / ml as the mean ± SD of four experiments.

Figure 2: Histamine is a factor of polarization from CD to CD2.

Naive T cells (CD4 ⁺ CD45Ra ⁺ ) were cultured for

5 days with mature allogeneic CDs. The maturation of these

CD was induced by a 2-day treatment with LPS (10 ng / ml), LPS (10 ng / ml) + INFg (100 ng / ml), LPS (10 ng / ml) + histamine (10 ^{~ 5} M), or LPS (10 ng / ml) + INFg (100 ng / ml) + histamine (10 ^{~ 5} M). After an expansion induced by IL2, the T lymphocytes were stimulated with PMA + ionomycin for 5 hours and the production of IL4 and of IFNg was evaluated by FACS, after intracellular labeling. The percentage of positive cells is indicated in each dial. The data are representative among 3.

Example 1

Histamine decreases the capacity of dendritic cells during maturation to produce IL12 after restimulation, even in the presence of IFNγ.

1 / Materials and Methods

Human dendritic cells are generated from monocytes isolated from peripheral blood. The blood is taken by leukopheresis in the presence of an anticoagulant such as for example lithium heparinate. Mononuclear cells (CMN) are isolated from healthy subjects by centrifugation on a Ficoll-Hypaque gradient (density = 1.077) (Amersham Pharmacia Biotech, Uppsala, Sweden). Briefly, the blood cells are centrifuged at 1500 rpm for 30 minutes at room temperature. The CMNs, located at the Ficoll-plasma interface, are recovered and washed twice in the presence of RPMI 1640 medium

(Life technologies, Cergy Pontoise, France). Monocytes are purified by positive selection using a magnetic cell separator (MACS ™; Miltenyi Biotec, Bergisch

Gladbach, Germany) in accordance with the manufacturer's instructions. Briefly, the CMNs are incubated for 20 minutes at 4 ° C. with magnetic beads on which are fixed an anti-human CD14 monoclonal antibody. After washing, the cell suspension plus beads is placed on a column and subjected to a magnetic field. After three washes, the column is no longer subjected to the magnetic field and the monocytes are collected by gravitation. The purity of the monocytes is evaluated by cytofluorometry (FACScan cytofluorometer; Becton Dickinson, Erembodegem, Belgium) on the basis of the size-granulosity parameters of the cells. The purity is greater than 98%. The monocytes are then cultured at a concentration of 10 ⁶ cells / ml in the following medium (hereinafter called complete culture medium): RPMI 1640 medium supplemented with 10% fetal calf serum (heating at 56 ° C. for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ug / ml of streptomycin (Life technologies) in 6-well culture plates (Nunc, Roskilde, Denmark) at a rate of 5 ml of medium per well. The cells are activated with 20 ng / ml of recombinant human IL-4 and 20 ng / ml of recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom). After 5 to 7 days of culture (37 ° C, 5% C0 ₂ in a humid atmosphere), the phenotype of the cells is defined by cytofluorometry. Briefly, an aliquot of the cell suspension is removed. The cells are washed in FACS buffer (10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide) and then distributed in wells of a 96-well culture plate at the bottom conical (Nunc) at the rate of 2 × 10 ⁴ cells in a volume of 50 μl of FACS buffer. In each well is added either an anti-human CDla antibody labeled with fluorescein (Becton Dickinson) or an unlabeled human anti-CD83 antibody revealed by an anti-mouse immunoglobulin antibody labeled with fluorescein (Becton Dickinson). After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 μl of FACS buffer and then are resuspended in 200 μl of this same buffer. The analysis of the expression of CDla versus CD83 is evaluated by FACS. Only immature dendritic cells characterized by expression of the CDla molecule (mean fluorescence intensity (IMF)> 100) and the absence of expression of the CD83 molecule were used. After 7 days, immature CDs were stimulated with LPS (from Escherichia coli isotype 0111: B4, Sigma, Saint Louis, MO) alone, or in combination with IFNg (R&D Systems, Abingdon, UK) and or histamine (Sigma). After 2 days, the mature CDs were washed and recultivated to 10 ⁵ cells / 200 μl / well in 96-well culture plates and were or were not stimulated with 5 μg / ml of CD40 recombinant soluble ligand (Peprotech, Rocky Hill, NJ) or with LPS (10 ng / ml) + IFNg (100 ng / ml). After 24 hours, the IL12 was determined in the supernatants by ELISA (R&D Systems; sensitivity 0.5 pg / ml) the results are expressed in pg / ml as the mean ± SD of four experiments

2 / Results

The maturation of dendritic cells is induced by a 2-day incubation with LPS in the presence or absence of IFNg and or histamine.

As described previously (Vieira et al JI 2000), CD matured with LPS produce low levels of IL12 after stimulation with CD40 ligand or with LPS + IFNg. As described previously (Vieira et al JI 2000), the addition of IFNg generates mature CDs producing high levels of IL12 after restimulation.

Our results show that whatever the concentration of IFNg used, histamine prevents the capacity of restimulated CD (by CD40 ligand or by IFNg + LPS) to produce IL12.

Example 2

The inhibitory effect of histamine on the production of IL12 by CD is mediated by the H1 and H2 receptors.

1 / Materials and Methods

The CDs were generated and stimulated as before with the following modification: - in some experiments the CD were preincubated for 1 hour with H1, H2 and H3 receptor antagonists (mepyramine, cimetidine and thioperamide, respectively) used at 10 ^"5 M before addition of histamine - in other experiments the CDs were incubated not with histamine or H1 and H2 receptor agonists (HTMT dimaleate and amthamine dihydrobromide, respectively) (both marketed by Biomol, Plymouth Meeting, PA).

2 / Results

The results show that the inhibitory effect of histamine on the production of IL12 is mediated by the H1 and H2 receptors but not H3. Indeed, the Hl and H2 antagonists inhibit the effect of histamine: moreover these results are confirmed by the fact that the Hl and H2 receptor agonists mimic the inhibitory effect of histamine on the production of IL12.

Example 3

Histamine polarizes the CD during maturation towards CD2 and this even in the presence of IFNγ.

1 / Materials and Methods The CDs were generated and stimulated as before.

The T cells were prepared and stimulated as follows. After rosetting and depletion of CD8 ⁺ T lymphocytes, the naive CD4 ⁺ CD45RA ⁺ T lymphocytes were purified by MACS (Miltenyi biotec, Bergisch Gladbach, Germany) by positive selection.

Purified naive T lymphocytes (5 × 10 ⁴ ) were cultured from irradiated allogeneic CD (2 × 10 ⁴ ), the maturation of which was induced by LPS (10 ng / ml), LPS (10 ng / ml) plus IFNg (100 ng / ml ), LPS (10 ng / ml) + histamine (10 ^{~ 5} M) or LPS (10 ng / ml) + IFNg (100 ng / ml) + histamine (10 ^{~ 5} M). After 5 days, 50 U / ml of recombinant human IL2 (R&D Systems) were added and the cultures were maintained for 7 days. Then the T lymphocytes were washed and restimulated with 10 ng / ml of PMA (Sigma) + 1 μg / ml of ionomycin (Calbiochem, San Diego, CA) for 5 hours.

Brefeldin A (10 μg / ml, Sigma) was added during the last 2 hours of culture.

The cells were then fixed, permabilized and labeled with an FITC coupled anti-IFNg mAb (Pharmingen, San Diego, CA) and with a PE coupled anti-IL4 mAb (BD biosciences, Franklin Lakes, NJ), then analyzed with a FACScan. (Beckton Dickinson)

2 / Results

As expected (Vieira et al JI 2000), the naive T lymphocytes stimulated with mature CDs in the presence of LPS differentiate into ThO, characterized by a small percentage of cells producing IL4 and IFNg.

The results show that the addition of histamine during the maturation of the CD generates CD2 which favor the development of lymphocytes of type Th2, characterized by an increase in the percentage of cells producing IL4 and a decrease in the percentage of cells producing IFNg.

As expected (Vieira et al JI 2000), T cells stimulated by DC matured in the presence of LPS and IFNg differentiate into Thl, characterized by a large percentage of cells producing IFNg and a very low percentage of cells producing IL4.

The results show that the addition of histamine in addition to LPS and IFNg to immature CD leads to the generation of mature CD2 type CD: indeed, after addition of histamine, a decrease in the percentage of IFNg producing cells.

Thus the addition of histamine during the maturation of the CD leads to CD2 which induce the differentiation of naive T lymphocytes into effector lymphocytes of the Th2 type. This is observed even in the presence of IFNg, a powerful Thl polarization factor.

Claims

1. Use of histamine and / or histamine receptor agonists for the manufacture of a medicament intended to induce the polarization of dendritic cells, and more particularly to polarize dendritic cells into type 2 dendritic cells.

2. Use according to claim 1, characterized in that the polarization of the dendritic cells is carried out in vitro.

3. Use according to claim 1 or 2, characterized in that the polarization of the dendritic cells is carried out in vitro, the mature polarized cells then being reinjected in vivo.

4. Use according to one of claims 1 to 3 and in addition of at least one biological agent for the manufacture of a medicament.

5. Use according to claim 4, characterized in that the biological agent is chosen from bacteria, virus, yeast, parasite, fungus antigens, tumor antigens, and lysates of autologous tumor cells and / or heterologous.

6. Use according to one of claims 1 to 5, characterized in that the dendritic cells are transfected ex vivo with a gene coding for an antigen and / or a cytokine or a growth factor.

7. Use according to one of claims 1 to 6, characterized in that the medicament also contains a factor for the maturation of dendritic cells, preferably chosen from LPS and TNFα.

8. Use according to one of claims 1 to 7, characterized in that the medicament also contains a cytokine or a growth factor, preferably interferon alpha or gamma, GM-CSF, IL-2, l 'IL-4, IL-6 and IL-18 or an HSP.

9. Use according to one of claims 1 to 8 for the manufacture of a medicament for the treatment or prevention of infectious diseases of viral, bacterial, fungal origin or caused by a yeast, or a parasite.

10. Use according to one of claims 1 to 8 for the manufacture of a medicament for combating a virus chosen from HIV, hepatic viruses and parainfluenza virus.

11. Use according to one of claims 1 to 8 for the manufacture of a medicament for the treatment or prevention of cancers.

12. Use according to one of claims 1 to 8 for the manufacture of a medicament for the treatment or prevention of cancer among myelomas, lymphomas, leukemias, carcinomas of the kidney, brain, prostate, rectum , pancreas, ovaries, lung, keratinomas and carcinomas.

13. Use of histamine and / or histamine receptor agonists for the preparation of a pharmaceutical composition intended to generate or increase an immune response against pathologies associated with an excessive Th1 response.

14. Use of histamine and / or histamine receptor agonists for the preparation of a pharmaceutical composition intended to generate or increase an immune response against Hashimoto's thyroiditis, multiple sclerosis, type 2 diabetes I, transplant rejection, Crohn's, rheumatoid arthritis, sarcoidosis and autoimmune diseases.

15. Use of histamine and / or histamine receptor agonists to induce polarization of dendritic cells in vitro, and more particularly to polarize dendritic cells into type 2 dendritic cells.

16. Use according to claim 15 and in addition to a factor for maturing dendritic cells, preferably chosen from LPS and TNFα.

17. Kit for the polarization of dendritic cells characterized in that it contains at least histamine and / or agonists of histamine receptors.

18. Kit according to claim 17, characterized in that it also contains a maturation factor for dendritic cells, preferably chosen from LPS and TNFα.

19. Kit according to claim 17 or 18, characterized in that it also contains heterologous antigens or means for obtaining a lysate of autologous cells.