SECRETING B CELLS

FIELD OF THE INVENTION

The present invention is in the field of immunotherapy. The present invention relates to the generation of interleukin 10-secreting (ILlO-secreting) B cells wherein said method comprises the step consisting of culturing a population of B cells with a culture medium comprising an amount of an antigen linked to a BcR-binding molecule.

BACKGROUND OF THE INVENTION B cells are cells of the immune system that are responsible for producing antibodies and are important in the development of an immune response through their function of antigen presenting cells (APC). B cells possess specialized cell surface receptors (B-cell receptors also called "BcR"). Therefore, if a B cell encounters an antigen capable of binding to its BcR, the B cell endocytoses the antigen and presents it to CD4+ T cells. In turn, the T cells are activated and fully stimulate B cells to proliferate and produce antibodies specific for the recognized antigen. But recent studies have identified a new role for B cells as potent immunosuppressive cells in vivo. The regulatory functions of B cells are of therapeutic interest in the inhibition of unwanted immune responses, such as pathogenic autoimmune responses, or anti-self or anti-graft reactions (or abnormal immune reactions to foreign antigens as in allergy, or immune responses against therapeutic proteins). In support of this notion, regulatory B cells can protect recipient mice from several experimental autoimmune diseases upon adoptive transfer. Furthermore, there is evidence that human B cells display similar regulatory activities. For instance, using the experimental model of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis in humans (MS), it has been demonstrated that B cells, through their capacity to present the antigen in a particular context, may exert a regulatory role in the autoimmune response directed to myelin. In a model of ΒΙΟ.μΜΤ mice (that lack B cells) immunized with the Acl-11 peptide of myelin basic protein (MBP), Janeway et al showed that mice developed the disease with the same incidence and severity as control BIO mice [Wolf, 1996]. However, in contrast to control mice, ΒΙΟ.μΜΤ mice did not experience spontaneous remission, thus suggesting an important role for B lymphocytes in the control of the disease. Other works have depicted a sub-population of B lymphocytes with a unique CDld ^hlgh CD5+ phenotype in naive wild-type mice; 10 to 15% of this subpopulation of B cells produce IL-10 after stimulation with LPS, and reduce in a significant manner the activation of autoreactive T lymphocytes, as well as inflammatory responses [Yanaba, 2008]. However, the stability with time of the regulatory phenotype of these natural regulatory B cells has recently been questioned [Maseda, 2012] and the mechanisms that lead to IL-10 production remain unclear.

In vivo, regulatory B cells develop not only spontaneously but may also be induced. Kala et al explored the protective potential in EAE of B lymphocytes from glatiramer acetate - treated mice [Kala, 2009]. B lymphocytes from glatiramer acetate-treated mice expressed low levels of the co-stimulatory molecules CD80 and CD86; their adoptive transfer inhibited the proliferation of autoreactive CD4+ T lymphocytes, promoted the production of the antiinflammatory cytokine IL-10, and controlled the initiation of EAE. Similarly, Rafei et al documented that the ex vivo treatment of splenocytes with the 'fusokine' GIFT 15 generates regulatory B cells, that completely corrected EAE upon adoptive transfer [Rafei, 2009].

In 2003, using the experimental model of type II collagen (CTII)-induced arthritis, Mauri et al demonstrated that CTII-specific B lymphocytes may be turned into regulatory B cells upon incubation of the splenocytes with the antigen in vitro concomitant with the stimulation by an agonist anti-CD40 antibody [Mauri, 2003]. Anti-CD40 antibody- induced regulatory B cells were also able to control the development of experimental systemic lupus erythematosus (SLE) [Blair, 2009].

In all these experiments, interleukin-10 (IL-10) was demonstrated to be the critical effector of the regulatory potential of B cells [Kala, 2009; Rafei, 2009]. B cells isolated from IL-10-deficient mice were thus unable to acquire tolerogenic properties and protect against the diseases. Recently, Mauri et al described a population of B cells in healthy humans expressing a particular phenotype (CD19+CD24hiCD38hi) that acquires regulatory properties following challenge with an agonist anti-CD40 antibody [Blair, 2010]. Interestingly, isolation of this population from peripheral blood of SLE patients shows that these B cells fail to exert regulatory effects on autoreactive T cells; the lack of regulatory function was attributed to the reduced capacity to produce IL-10 [Blair, 2010]. Thus, only some activation methods conferred tolerogenic/regulatory properties to B cells, and the mechanisms leading to IL-10 production were then not clearly identified.

Therefore, there is a strong need for an efficient and easy method that generates a large population of IL 10- secreting B cells that can efficiently induce antigen-specific immune tolerance (more particularly by down-regulating CD8+ T-cell responses) for use in the prevention or the treatment of autoimmune diseases, alloimmune responses, allergy graft rejection (or for induction of transplant tolerance) and uncontrolled/exacerbated immune responses following exposure to microbes (e.g., sepsis). SUMMARY OF THE INVENTION

The present invention relates to a method for obtaining a population of ILlO-secreting B cells, wherein said method comprises the step consisting of culturing a population of B cells with a culture medium comprising an amount of an antigen linked to a BcR-binding molecule.

The present invention also relates to a population of ILlO-secreting B cells obtainable by a method of the invention.

The present invention further relates to a population of ILlO-secreting B cells of the invention for use as a medicament, especially for use in the prevention or the treatment of an autoimmune disease, allergy, graft rejection (or for induction of transplant tolerance), unwanted alloimmune responses against exogenous therapeutic proteins and/or proteins expressed in the course of gene therapy, and uncontrolled immune responses following exposure to microbes. DETAILED DESCRIPTION OF THE INVENTION

The inventors have demonstrated that it is possible to obtain a population of IL 10- secreting B cells by culturing a population of B cells obtained from naive mice with a culture medium comprising an amount of an antigen linked to a BcR-binding molecule.

The inventors have indeed shown that a population of ILlO-secreting B cells specific for the MOG antigen obtained by the method of the invention protects mouse model recipients in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis in humans (MS).

This approach is of interest in the fields of autoimmunity, allergy, transplantation, treatment with therapeutic protein and gene therapy, to avoid degradation of self or therapeutic molecules/tissues by the immune system and uncontrolled immune response following exposure to microbes which occur in sepsis, severe sepsis and septic shock.

Methods for obtaining a population of ILlO-secreting B cells

The present invention relates to a method for obtaining a population of ILlO-secreting B cells, wherein said method comprises the step consisting of culturing a population of B cells with a culture medium comprising an amount of an antigen linked to a BcR-binding molecule.

As used herein, the term "B cell" refers to a type of white blood cells (or leukocytes) and, specifically, a type of lymphocytes that plays a major role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells). B cells are an essential component of the adaptive immune system since their principal functions are to make antibodies against antigens, and eventually develop into memory B cells after activation by antigen interaction. B cells are also essential parts of the innate immune system since they perform the role of APCs.

Each B cell has a unique receptor protein (referred to as the B cell receptor; "BcR") on its surface that will bind to one particular antigenic motive. The BcR is an immunoglobulin complex having the function of antigen binding and signaling when an antigen binds the receptor. The BcR is expressed at the plasma membrane of B cells, and in its canonical form is a hetero-oligomeric structure composed of an antigen binding component, a disulfide bond complex consisting of two identical copies of a membrane-bound form of immunoglobulin heavy chains and two identical immunoglobulin light chains, and a signaling subunit, a heterodimer of the Ig-alpha and Ig-beta proteins (CD79a, and CD79b, respectively) non- covalently associated with the membrane -bound immunoglobulin heavy chains.

Each B cell expresses one type of immunoglobulin but the population of B cells in each individual displays a wide variety of antigen specificities. When the BcR binds to antigen, it initiates a signal through the cytoplasmic tails of Ig-alpha and Ig-beta chains that are each associated with distinct sets of downstream signaling/effector molecules.

Signaling through the BcR thus plays an important role in the generation of antibodies and in the establishment of immunological tolerance. These signaling events are responsible for both the B-cell proliferation and increased expression of activation markers (such as major histocompatability complex-II ("MHCII") and CD86) that are required to prime B cells for their subsequent interactions with a sub-group of CD4+ T cells referred to as T-helper ("Th") cells. To generate an efficient response to antigens, BcR-associated proteins and T-cell assistance are also required. The antigen/BcR complex is internalized, and the antigen is proteolytic ally processed. A small part of the antigen remains complexed with MHCII molecules and is expressed at the surface of the B cells where the complex can be recognized by T cells. T cells activated by such antigen presentation secrete a variety of lymphokines that induce B-cell maturation.

As used herein, the term "IL 10- secreting B cell" refers to a B cell that has been activated and that secretes IL-10 into its surrounding medium.

As used herein, the terms "regulatory B cell" or "tolerogenic B cell" or "Breg cell" refer to a tolerant B cell compared to a naive B cell after activation by a particular antigen. Tolerance is generally defined as a state of altered responsiveness to a particular antigen that prevents the development of either a cellular or antibody-based immune response to that antigen. Thus, regulatory B cells (compared to naive B cells) may have for instance a reduced expression of antigen-presenting molecule (preferably MHC-II or its human counterpart HLA); reduced expression of co-stimulatory molecules (such as CD80 and/or CD86); and/or an increased production of immunosuppressive molecules (such as interleukin 10 (IL-10)). A population of ILlO-secreting B cells according to the invention exhibits the ability to induce immune tolerance and/or to suppress and/or inhibit immune responses, preferably antigen- specific immune response(s) directed against the antigen(s) involved in the disease to be treated, and/or unwanted adaptive immune responses mediated by CD4+ T cells, CD8+ T cells, B cells and/or antibodies, preferably immune T-cell tolerance or reduced T-cell activation (by a decreased/lower IL-2 production). It should further be noted that the suppression and/or inhibition of immune responses obtained by a population of IL10- secreting B cells of the invention may also be associated with the development of IL-10 producing CD4+ T cells, and inhibition and/or prevention of an inflammatory T-cell response, preferably by suppression of IFN- y -, IL-17- and/or TNF-cc-producing CD4+ or CD8+ T cells, especially of TH 1 and/or TH 17 cell types.

The population of B cells that serves as starting material may be isolated according to any technique known in the art. A population of B cells may be isolated from a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate (including human subject). For instance, a population of B cells may be obtained from various biological samples containing lymphocytes. In humans, populations of B cells are typically isolated from peripheral blood. For instance, a population of B cells may be isolated from human peripheral blood mononuclear cells (PBMCs) by positive selection using magnetic immunobeads coated with CD 19 and/or CD20 antibodies. Such a population of B cells encompasses thus memory B cells, activated B cells, plasma cells and naive B cells.

In a particular embodiment, the population of B cells is a population of naive B cells. As used herein, the term "naive B cell" generally refers to a B cell that has not been exposed to an antigen. More particularly, a naive B cell refers to any B cell not activated, especially not deliberately activated by an antigen or via B-cell receptors, toll-like receptors and/or CD40 receptors. Thus, within the context of the invention, naive B cells are B cells that were purified without any prior exposure to any stimulus of B-cell activation.

The population of naive B cells that serves as starting material may be isolated according to any technique known in the art. A population of naive B cells may be isolated from a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate (including human subject). For instance, a population of naive B cells may be obtained from various biological samples containing lymphocytes. In humans, populations of B cells are typically isolated from peripheral blood. For instance, a population of naive B cells may be isolated from human peripheral blood mononuclear cells (PBMCs) by negative depletion using biotinylated CD27 antibodies and magnetic immunobeads (Naive B Cell Isolation Kit II from Miltenyi Biotec) in order to discriminate naive B cells (CD19 ⁺ CD27 ^~ cells) from other B cells, including memory B cells, activated B cells, and plasma cells (CD19 ⁺ CD27 ⁺ cells).

Alternatively, in mice, populations of B cells are isolated from the spleen. Thus, a population of naive B cells may be isolated from splenic B cells using dynabeads Mouse CD43 (Untouched B cells from Invitrogen) or using Pan B Cell Isolation Kit from Miltenyi Biotec.

As used herein, the term "antigen" refers to a substance capable of binding to an antigen binding region of an immunoglobulin molecule (or antibody). Thus, the term "antigen" includes, but is not limited to, antigenic determinants, haptens, and immunogens which may be proteins, polypeptides, peptides, small molecules (including oligopeptide mimics (i.e, organic compounds that mimic the antibody binding properties of the antigen)), carbohydrates e.g. polysaccharides, lipids, nucleic acids, or combinations thereof. It should be further noted that an antigen according to the invention may be a protein which can be obtained by recombinant DNA technology or by purification from different tissue or cell sources. Such proteins are not limited to natural ones, but also include modified proteins or chimeric constructs, obtained for example by changing selected aminoacid sequences or by fusing portions of different proteins. Alternatively, said antigen may be a synthetic peptide, obtained by Fmoc biochemical procedures, large-scale multipin peptide synthesis, recombinant DNA technology or other suitable procedures.

Typically, in one embodiment of the invention, the antigen is selected from the group consisting of auto-antigens, allo-antigens, allergens and foreign antigens. In one embodiment of the invention, the antigen is linked, on one hand to a BcR- binding molecule, and on the other hand to cholera toxin subunit B (CTB). Without wishing to be bound by theory, the inventors believe that ILlO-secreting B cells obtained by culturing B cells in the presence of such an antigen will be able to induce antigen- specific Foxp3+ regulatory cells (see Sun et al. 2012). As used herein, the term "culture medium" refers to any medium capable of supporting the growth and the differentiation of naive B cells into IL 10- secreting B cells. Typically, it consists of a base medium containing nutrients (a source of carbon, aminoacids), a pH buffer and salts, which can be supplemented with growth factors and/or antibiotics. Typically, the base medium can be RPMI 1640, DMEM, IMDM, X-VIVO or AIM-V medium, all of which are commercially available standard media.

Preferred media formulations that will support the growth and the differentiation of naive B cells into IL 10- secreting B cells include chemically defined medium (CDM). As used herein, the term "chemically defined medium" (CDM) refers to a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A chemically defined medium is a serum-free and feeder-free medium.

As used herein, the term "BcR-binding molecule" refers to any molecule capable of binding to the BcR in an antigen-independent manner.

Typically, the BcR-binding molecule according to the invention is a molecule which has a low affinity binding for BcR.

In a preferred embodiment of the invention, the BcR-binding molecule binds to the BcR with an affinity ranging from about ΙΟηΜ to about 10μΜ, preferably from about 100 nM to 10 μΜ, even more preferably around 200 to 1000 nM.

For comparison's sake, the affinity of an anti-IgM for the BcR is in the picomolar range.

In one embodiment, the BcR-binding molecule is fluorescein isothiocyanate (FITC) or a molecule having substantially the same BcR-binding properties.

It falls within the ability of the skilled person in the art to determine the BcR-binding properties of a given molecule.

Typically, the skilled person in the art can screen for BcR-binding molecules according to the present invention as follows:

- Couple the candidate BcR-binding molecule to a model antigen, such as ovalbumin

(OVA);

- incubate the OVA-coupled BcR-binding molecule with a population of splenic cells from naive mice at 4°C; - measure the percentage of CD19-positive cells that are bound to OVA-coupled BcR- binding molecule. (If the BcR-binding molecule is fluorescent, flow cytometry can be used to determine if some B cells (CD19-positive) are labeled (15 to 30%), while the CD19-negative cells are not labeled. If the BcR-binding molecule is not fluorescent, then the determination can be made by detecting the bound OVA using a specific antibody.)

Without wishing to be bound by theory, it is believed that the binding of FITC and other BcR-molecules to the BcR is antigen-independent. In other words, the binding is not restricted to a single set of VH and VL genes.

In one embodiment of the invention, the BcR-binding molecule is a molecule that is capable of inducing a weak activation of B cells.

Typically, the activation of B cells can be measured by a calcium mobilization assay, as explained in the example below.

Typically, a BcR-binding molecule will be deemed to induce a weak activation of B cells if it induces a weak efflux of calcium from the endoplasmic reticulum.

For comparison's sake, an anti-IgM antibody, which induces a strong activation of the BcR and thereby boosts the immune response, induces a strong efflux of calcium from the endoplasmic reticulum under the experimental conditions described below (see Figure 2B).

Examples of BcR-binding molecules according to the invention include, but are not limited to, fluorochromes and heme.

As used herein, the term 'fluorochrome" has its general meaning in the art. Typically, it refers to a fluorescent chemical compound that can reemit light upon light excitation.

Typically, the BcR-binding molecule according to the present invention can be selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives (available from Sigma- Aldrich and Life Technologies), heme and its derivatives (Sigma-Aldrich), hydrocoumarin and its derivatives (Sigma-Aldrich, Life Technologies), cyanine and its derivatives, and AlexaFluor® compounds (Life Technologies).

Techniques for carrying out the FITC labeling reaction of the antigen of interest (or its linkage to any other suitable BcR-binding molecule) are well known in the art. Indeed, FITC conjugation occurs through the free amino groups of proteins or peptides, forming a stable thiourea bond. Suitable kits useful for labeling proteins or peptides are known in the art, for example FluoroTag™ FITC Conjugation Kit purchased from Sigma- Aldrich. As used herein, the term "bound" refers to a binding that may be covalent, e.g., by chemically coupling, or non-covalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc. Covalent bonds can be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. The term "bound" includes terms such as "labeled", "coupled", "attached", "conjugated" and "fused".

In one embodiment, the method comprises a step of culturing a population of B cells with a culture medium comprising an amount of an oxidizing agent, prior to the step of culturing said population of B cells with a culture medium comprising an amount of antigen linked to a BcR-binding molecule.

The inventors have indeed observed that such as pretreatment step increases the proportion of B cells which bind to the antigen linked to a BcR-binding molecule. Therefore, it may increase the yield of the method, resulting in a bigger population of ILlO-secreting B cells.

As used herein, the term "oxidizing agent" refers to any compound that is capable of altering the redox state of a biological molecule. More specifically, the oxidizing agent is a molecule that has the ability to be reduced by acting as an electron acceptor for other molecules that act as electron donors. Examples of oxidizing agents include, but are not limited to, "ferrous ions" (also known as [Fe(II)] ions or Fe2+ ions) which may be added to culture medium in the form of a ferrous salt. Other oxidizing agents are sodium periodate (NaI04) or potassium permanganate (KMn04).

As used herein, the term "ferrous ions" (also known as [Fe(II)] ions or Fe2+ ions) refers to ferrous ions in the form of a ferrous salt added to culture medium.

For instance, ferrous salt may be added to the culture medium under different forms selected from the group consisting of ammonium ferrous sulphate, ferrous bromide, ferrous chloride, ferrous fluoride, ferrous iodide, ferrous fumarate, ferrous gluconate, ferrous lactate, ferrous oxalate, ferrous succinate, ferrous thiocynate and ferrous sulphate.

In a particular embodiment, ferrous salt is added to the culture medium under the form of ferrous sulphate or FeS04. Ferrous ions are added to the culture medium in a final concentration ranging from 1 to 500 μ M, preferably ranging from 30 to 300 μ M, still preferably at about 100 μ Μ. For instance, ferrous salt is added to the culture medium under the form of ferrous sulphate or FeS04 under excess. In a particular embodiment, the method may comprise an additional step of isolating a population of ILlO-secreting B cells obtained after the step of culturing said population of B cells with a culture medium comprising an amount of antigen linked to a BcR-binding molecule. Thus, in one particular embodiment, the method for obtaining a population of ILlO- secreting B cells specific for an antigen, wherein said method comprises the steps consisting of:

i) culturing said population of B cells with a culture medium comprising an amount of antigen linked to a BcR-binding molecule;

ii) isolating a population of ILlO-secreting B cells from the cells obtained at step i).

Typically, said isolation step can comprise selecting ILlO-secreting B cells for the presence or absence of the BcR-binding molecule on the cell surface. When the BcR-binding molecule is a fluorochrome, this can readily be done, for instance, by Fluorescence- Associated Cell Sorting (FACS).

Within the context of the invention, the population of B cells is pulsed with an antigen-linked to the BcR-binding molecule of interest in order to achieve antigen- presentation by the population of ILlO-secreting B cells, said antigen being provided in an amount effective to "prime" the population of B cells and thus obtain a population of ILlO- secreting B cells presenting said antigen. In one embodiment, the culture medium may comprise a mixture of several antigens (involved in the disease to be treated). For instance, when the disease to be prevented or to be treated is graft rejection, the culture medium may comprise a lysate from the graft to be transplanted to the patient. When the disease to be prevented or to be treated is multiple sclerosis, the culture medium may comprise a mixture of myelin-related antigens (e.g. myelin basic protein (MBP) (e.g. MBP83-102 peptide), myelin oligodendrocyte glycoprotein (MOG) (e.g. MOG35-55 peptide) and proteolipid protein (PLP) (e.g. PLP139-151 peptide), all linked to the BcR-binding molecule. Accordingly, the population of IL 10- secreting B cells thus obtained may be specific for several antigens involved in the disease to be treated in order to obtain a more efficient tolerance. In others words, said population of ILlO-secreting B cells may be heterogeneous, being composed of several populations of IL 10- secreting B cells, each pulsed for a given antigen of interest.

Within the context of the invention, populations of ILlO-secreting B cells of the invention are useful in the prevention or treatment of unwanted immune responses, such as those involved in autoimmune disorders, immune reactions to therapeutic proteins, and/or allergies.

Populations of ILlO-secreting B cells specific for an antigen associated with the disease to be treated (pathogenic antigen) should be obtained. Therefore, the antigen of interest is selected from the group consisting of auto-antigens, allo-antigens, allergens and foreign antigens such as bacterial antigens.

For instance, when the autoimmune disease is multiple sclerosis, the autoantigen is selected from the group consisting of myelin-related antigens (e.g. myelin basic protein (MBP) (e.g. MBP83-102 peptide), myelin oligodendrocyte glycoprotein (MOG) (e.g. MOG35-55 peptide) and proteolipid protein (PLP) (e.g. PLP139-151 peptide).

When the autoimmune disease is Type I diabetes, the autoantigen is selected from the group consisting of insulin, insulin precursor proinsulin (Prolns), glutamic acid decarboxylase 65 (GAD65), glial fibrillary acidic protein (GFAP), islet- specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulinoma-associated antigen-2 (IA-2) and zinc transporter 8 (ZnT8).

When the autoimmune disease is rheumatoid arthritis, the autoantigen is type II collagen (CTII).

It is also intended that alloantigens include, but are not limited to, antigens expressed by the allograft, proteins expressed in the course of gene therapy (and also viral antigens issued from the viral vector used) as well as therapeutic proteins.

As such an "allograft" is a transplant between two individuals of the same species having two genetically different MHC haplotypes.

The term "therapeutic proteins" refers to proteins or peptides and their administration in the therapy of any given condition or illness. Therapeutic proteins relate to any protein or peptide, such as therapeutic antibodies, cytokines, enzymes or any other protein, that is administered to a patient. Examples of protein therapy relate to treatment of hemophilia via administration of plasma-derived or recombinant clotting factor concentrates (e.g. factor VIII and factor IX), the treatment of cancer or cardiovascular disease using monoclonal antibodies or the treatment of metabolic or lysosomal disease by enzyme replacement therapy.

Without wishing to be bound by theory, it is believed that the antigen-coupled BcR- binding molecule binds to the BcR through its BcR-binding molecule moiety, rather than by the antigen moiety. This is the case for instance for ovalbumin (OVA) or MOG35-55 peptide.

In contrast, some antigens (e.g. factor IX (FIX) or type 2 collagen (CTII)) may bind directly to the BcR following exposure to ferrous salt.

Populations of ILlO-secreting B cells according to a method of the invention and pharmaceutical compositions comprising them

In another aspect, the present invention also relates to a population of IL 10- secreting B cells obtainable by a method as defined above. In one embodiment, the BcR-binding molecule is FITC and the population of IL-10 secreting B cells is fluorescent.

The present invention also provides a pharmaceutical composition comprising the population of IL 10- secreting B cells according to the invention. The pharmaceutical composition may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. This pharmaceutical composition can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e. g. human serum albumin) suitable for in vivo administration.

As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

Therapeutic methods and uses:

As above-mentioned, populations of IL 10- secreting B cells of the invention are of interest in the fields of autoimmunity, allergy, transplantation, gene therapy and treatment with therapeutic protein, to avoid degradation of self tissues or therapeutic proteins by the immune system.

Another aspect of the invention thus relates to a population of ILlO-secreting B cells of the invention for use as a medicament.

More particularly, an aspect of the invention relates to a population of ILlO-secreting B cells of the invention for use in the prevention or the treatment of an autoimmune disease. The invention also relates to a method for preventing or treating an autoimmune disease comprising the step of administering a pharmaceutically effective amount of a population of ILlO-secreting B cells of the invention to a patient in need thereof. An "autoimmune disease" is a disease in which the immune system produces an immune response (for example, a B-cell or a T-cell response) against an antigen that is part of the normal host (that is, an autoantigen), with consequent injury to tissues. In an autoimmune disease, the immune system of the host fails to recognize a particular antigen as "self" and an immune reaction is mounted against the host's tissues expressing the antigen.

Exemplary autoimmune diseases affecting mammals include rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (for example, Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, acquired hemophilia, thrombotic thrombocytopenic purpura and the like. Another aspect of the invention relates to a population of ILlO-secreting B cells of the invention for use in the prevention or the treatment of allergy.

The invention also relates to a method for preventing or treating allergy comprising the step of administering a pharmaceutically effective amount of a population of IL10- secreting B cells of the invention to a patient in need thereof.

As used herein, the term "allergy" or "allergies" refers to a disorder (or improper reaction) of the immune system. Allergic reactions occur to normally harmless environmental substances known as allergens; these reactions are acquired, predictable, and rapid. Strictly, allergy is one of four forms of hypersensitivity and is called type I (or immediate) hypersensitivity. It is characterized by excessive activation of certain white blood cells called mast cells and basophils by a type of antibody known as IgE, resulting in an extreme inflammatory response. Common allergic reactions include eczema, hives, hay fever, asthma, food allergies, and reactions to the venom of stinging insects such as wasps and bees. Another aspect of the invention relates to a population of ILlO-secreting B cells of the invention for use in the prevention or the treatment of graft rejection (or for induction of transplant tolerance).

The invention also relates to a method for preventing or treating graft rejection (or inducing transplant tolerance) comprising the step of administering a pharmaceutically effective amount of a population of ILlO-secreting B cells of the invention to a patient in need thereof.

Another aspect of the invention relates to a population of ILlO-secreting B cells of the invention for use in the prevention or the treatment of any unwanted immune reaction directed to proteins expressed in the course of gene therapy, and/or therapeutic proteins, such as factor VIII (hemophilia A) and other coagulation factors, enzyme replacement therapies, monoclonal antibodies (e.g. natalizumab, rituximab, infliximab) or cytokines (e.g. IFN β ).

The invention also relates to a method for preventing or treating any unwanted immune reaction directed to proteins expressed in the course of gene therapy, and/or therapeutic proteins, comprising the step of administering a pharmaceutically effective amount of a population of ILlO-secreting B cells of the invention to a patient in need thereof.

This approach can indeed be applied to suppress an immune response, especially to prevent immune reactions to specific proteins when their expression is restored by gene therapy in individuals with corresponding genetic deficiencies. For example the IL10- secreting B cells of the present invention can be used to prevent immune reactivity towards proteins normally absent in the patient due to mutations, while their reconstitution is achieved by gene therapy. For example, a gene therapy patient can be additionally treated with modified naive B cells specific for a protein antigen, which is also the therapeutic gene product to be expressed via gene therapy. Through this approach the gene therapy product is less likely to be degraded by specific immune reactions upon expression in vivo.

Moreover, protein therapy is an area of medical innovation that is becoming more widespread, and involves the application of proteins, such as enzymes, antibodies or cytokines, directly to patients as therapeutic products. One of the major hurdles in delivery of such medicaments involves the immune responses directed against the therapeutic protein themselves. Administration of protein-based therapeutics is often accompanied by administration of immune suppressants, which are used in order to facilitate a longer lifetime of the protein and therefore increased uptake of the protein into the cells and tissues of the organism. General immune suppressants can however be disadvantageous due to the unspecific nature of the immune suppression that is carried out, resulting in unwanted side effects in the patient. Therefore, this approach can be applied to suppress an immune response against therapeutic proteins and peptides, such as therapeutic antibodies, cytokines, enzymes or any other protein administered to a patient.

Another aspect of the invention relates to a population of ILlO-secreting B cells of the invention for use in the prevention or the treatment of any uncontrolled immune response to exposure to microbes and microbial products, such as bacterial antigens. The invention also relates to a method for preventing or treating any uncontrolled immune response to exposure to microbes and microbial products, comprising the step of administering a pharmaceutically effective amount of a population of ILlO-secreting B cells of the invention to a patient in need thereof. This approach can indeed be applied to suppress an immune response, especially to prevent uncontrolled immune reactions which occur in sepsis, severe sepsis and septic shock. Thus, in a particular embodiment, a population of ILlO-secreting B cells of the invention may be used in the prevention or the treatment of sepsis, severe sepsis and septic shock. In the context of the invention, the terms "treating" or "treatment", as used herein, refer to a method that is aimed at delaying or preventing the onset of a pathology, at reversing, alleviating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the symptoms of the pathology, at bringing about ameliorations of the symptoms of the pathology, and/or at curing the pathology.

As used herein, the term "pharmaceutically effective amount" refers to any amount of a population of ILlO-secreting B cells according to the invention (or a pharmaceutical composition thereof) that is sufficient to achieve the intended purpose. As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human.

Effective dosages and administration regimens can be readily determined by good medical practice based on the nature of the pathology of the subject, and will depend on a number of factors including, but not limited to, the extent of the symptoms of the pathology and extent of damage or degeneration of the tissue or organ of interest, and characteristics of the subject (e.g., age, body weight, gender, general health, and the like). For therapy, populations of ILlO-secreting B cells and pharmaceutical compositions according to the invention may be administered through different routes. The dose and the number of administrations can be optimized by those skilled in the art in a known manner.

Methods for obtaining a population of regulatory T cells

by using a population of ILlO-secreting B cells

An aspect of the present invention relates to a method for obtaining a population of regulatory T cells specific for an antigen comprising a step of culturing a population of regulatory T cells with a population of ILlO-secreting B cells specific for said antigen.

The invention also relates to a method for obtaining a population of regulatory T cells specific for an antigen comprising the steps of:

culturing a population of B cells with a culture medium comprising an amount of said antigen linked to a BcR-binding molecule in order to obtain a population of ILlO-secreting B cells specific for said antigen,

optionally, isolated said population of ILlO-secreting B cells specific for said antigen,

culturing a population of regulatory T cells with said population of ILlO-secreting B cells specific for said antigen.

In another embodiment, the ILlO-secreting B cells are not specific for a given antigen, but they serve to activate certain T cells which are specific for the antigen. In one embodiment, the population of regulatory T cells is a population of CD4+CD25+ regulatory cells.

In a particular embodiment, the population of regulatory T cells is a population of naive CD4+CD25+CD45RA+ regulatory T cells.

In another embodiment, the population of regulatory T cells is a population of IL10- secreting Type 1 T regulatory (Trl) cells (e. g., CD3+CD4+CD18brightCD49b+ T cells) or a population of TGFB-secreting Th3 cells.

The population of regulatory T cells that serve as starting material may be isolated according to any technique known in the art. For instance, the population of regulatory T cells may be obtained from various biological samples containing lymphocytes. Typically, they are isolated from peripheral blood or from the spleen. They may be isolated by a combination of negative and positive selection with beads labelled with different ligands (eg, CD4, CD25, CD45RA). Such labelled cells may then be separated by various techniques such as cell sorting.

Another aspect of the present invention relates to a method for obtaining a population of regulatory T cells specific for an antigen comprising a step of culturing a population of total CD4+ T cells with a population of regulatory B cells specific for said antigen.

The population of total CD4+ T cells that serves as starting material may be isolated according to any technique known in the art. For instance, the population of total CD4+ T cells may be obtained from various biological samples containing lymphocytes. Typically, they are isolated from peripheral blood or from the spleen. They may be isolated by positive selection with beads labelled with different ligands (eg, CD3 or CD4). Such labelled cells may then be separated by various techniques such as cell sorting.

Within the context of the present application, the term "a population of regulatory T cells" refers to a population of T cells characterized by an ability to suppress or downregulate immune reactions mediated by effector T cells, such as effector CD4+ or CD8+ T cells.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1. Naive B cells bind FITC in a BcR-dependent manner. Total cells (10 ⁶ cells/ml) from the spleen (A and E), the lymph nodes or the peritoneum (E) of naive C57BL/6 mice were incubated alone or in presence of FITC-labeled ovalbumin (200 μg/ml, OVA- FITC) on ice for 1 hr in serum free medium. (B and C) Purified B cells from naive C57BL/6 mice were incubated with FITC-labeled or biotinylated-antigens (0 to 800 μg/ml) on ice for 1 hr in serum free medium. (D) Purified B cells from naive Balb/c mice or naive BcR ^_/~ mice (V _H -LMP2A mice) were incubated alone or in presence of OVA-FITC (200 μg/ml) for lh at 4°C. (E) The transitional 1 and 2 (Tl and T2), the marginal zone (MZ) and the follicular B cells (FO) were also identified according to the level of expression of the surface markers CD21, CD23 and CD93. The graphs depict the percentage of antigen-positive cells evaluated either by the direct fluorescence of the FITC moiety or using Pacific Blue-labeled streptavidin of viable cells by flow cytometry (cell viability was >85%). Percentage of positive cells from at least three independent experiments are presented. Error bars represent SEM.

Figure 2. Optimal stimulation of BcR by OVA-FITC leads to the production of IL-10 by natural FITC-binding naive B cells. (A) Negatively selected splenic B cells (10 ⁶ cells/ml) from naive C57BL/6 mice were incubated alone, with F(ab') ₂ fragments of an anti- IgM antibody (10 μ^πύ), with FITC-labeled or biotinylated-OVA (200 μ^πύ) for 2 days at 37 °C in complete medium. B cells were then washed and stimulated for 6 hr at 37 °C in the presence of phorbol 12-myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml) and Monensin. The production of IL-10 was evaluated on fixed permeabilized cells by flow cytometry. Values in quadrants (A, upper panels) depict the percentages of cells positive for CD 19 and IL-10 and the graphs (A, bottom panels) represent the concentrations of TNFcc, IL-10 and MCP-1 secreted by the B cells. Data are representative of three independent experiments. (B) Negatively selected splenic B cells (10 ⁶ cells/ml) from naive C57BL/6 mice were loaded with the calcium sensitive dye Indo-1 (2 μΜ). Calcium mobilization was assessed after stimulation of B cells with F(ab') ₂ fragments of an anti-IgM antibody (10 μg/ml), or with OVA-FITC (200 μg/ml) at 37°C. The graph depicts the calcium efflux (Krebs-Ringer solution containing 0.5 mM EGTA) and the calcium influx (Krebs-Ringer solution containing 1 mM CaCl ₂ ) in real-time following stimulation of BcR. Data are representative of two independent experiments. Figure 3. Natural FITC-binding naive B cells endocytose FITC-labeled OVA and induce IL-10-producing CD4 ⁺ T cells. (A) Purified splenic B cells (10 ⁶ cells/ml) from naive C57BL/6 mice were incubated with FITC-labeled (full squares) or biotinylated-OVA (empty circles) at 0 to 800 μg/ml, for 1 hr on ice or at 37°C in serum free medium. Endocytosis of OVA was evaluated either by direct fluorescence of FITC or using Pacific Blue-labeled streptavidin on fixed and permeabilized cells by flow cytometry. The graph depicts the difference of mean fluorescence intensity at between 37 °C and 4°C. The MFI was normalized with respect to FITC-labeled anti-CD 19 antibody or Pacific Blue-labeled anti-CD 19 antibody, used as positive controls. Data are representative of three independent experiments. (B) Purified splenic B cells (2.10 ⁵ cells) from naive Balb/c mice were co-incubated with the OVA ₃₂ 3-339-specific hybridoma (DO54.8 , 2.10 ⁵ cells) alone, in the presence of FITC-labeled (full bars) or biotinylated-OVA (empty bars) at 10 to 200 μg/ml for 48 hr at 37°C in complete medium. The graph depicts the concentration of IL-2 secreted by the DO54.8 in the supernatant evaluated by ELISA. Data are obtained from three independent experiments. Error bars represent SEM. The statistical significance of the observed differences between B cells incubated with OVA-FITC or OVA-biotin was determined by the double-sided non- parametric Mann- Whitney U test: **p<0.01 and ***p< 0.001. (C and D) Negatively selected splenic CD4 ⁺ T cells (1.5xl0 ⁵ cells) from OTII C57BL/6 mice were stained with cell trace violet (CTV) and co-incubated with purified splenic B cells (1.5xl0 ⁵ cells) from naive C57BL/6 mice alone or in the presence of OVA-FITC (5-20 μg/ml) for 6 days at 37°C in complete medium. As a positive control, CD4 ⁺ T cells were incubated with 2 μg/ml of Concanavalin A (ConA). Cells were then washed and stimulated for 6 hr at 37 °C in the presence of phorbol 12-myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml) and Monensin. The proliferation (C, top panels) and polarization (C, bottom panels; D) of CTV-stained CD4 ⁺ T cells were evaluated on fixed permeabilized cells by flow cytometry. Values in quadrants depict the percentages of cells positive for the different markers: CTV, IFN-gamma and/or IL- 10, gated on the CD4-positive population. Data are representative of three independent experiments.

Figure 4. Adoptive transfer of MOG ₃₅ -55-FITC-loaded naive B cells protects mice from EAE. (A) EAE was induced in 8 to 9 week old female wild-type C57BL/6 mice by immunization with MOG in Freund's adjuvant and injection of pertussis toxin. EAE mice were intravenously administered with negatively selected splenic B cells from naive C57BL/6 mice (10 ⁶ B cells/mouse) five days and one day before induction of EAE, as well as 5 days after injection of EAE. Mice received B cells that had been incubated alone (full circles), with OVA-FITC (200 μg/ml, empty squares), with biotinylated (4 μΜ, empty circles) or FITC- labeled MOG ₃₅ _55 (4 μΜ, full squares), for 3 hr at 37°C in serum free medium. The graph depicts the mean clinical scores over a period of 30 days. Error bars represent SEM (n=10 mice per group). Statistical significance of differences in EAE scores between groups were analyzed by the two-way ANOVA with Bonferroni's post-hoc t test: *p<0.05, **/?<0.01 and ***/?< 0.001. (B) Mice that received MOG ₃₅ _55-biotin loaded-B cells (empty circles) or MOG ₃₅ -55-FrrC loaded-B cells (full squares) were sacrificed at day 18 after induction of EAE. The graphs depict percentages of CD4 ⁺ T cells from blood, draining lymph nodes (DLN) and spleen. The studied T-cell populations include CD4 ⁺ T cells that are positive for IL-10 or for IFNy, and CD4 ⁺ IL-10 ⁺ T cells that are positive for FoxP3 expression. Horizontal lines depict mean values and SEM. Statistical significance was assessed using the double- sided non-parametric Mann Whitney U test: ns: not- significant; *: /?<0.05.

Figure 5: Effect of a preincubation with ferrous ions. Naive splenic B lymphocytes were exposed (full bars) or not (empty bars) to Fe(II) ions for 10 min at 4°C and incubated with FITC-labelled or unlabelled antigens for 1 hr at 4°C. The percentage of antigen-positive B cells was then assessed by flow cytometry.

Figure 6: Human CD20 ⁺ B cells from human PBMCs and splenocytes bind FITC.

Total PBMCs or splenocytes (10 ⁶ cells/ml) from immunologically healthy donors were incubated alone or in presence of FITC-labeled ovalbumin (200 μg/ml, OVA-FITC) on ice for 1 hr in serum free medium. The graphs depict the percentage of OVA-FITC-positive CD20 ⁺ B cells evaluated by the direct fluorescence of the FrfC moiety of viable cells by flow cytometry (cell viability was >90 ).

Figure 7: In vitro stimulation of human CD20 ⁺ splenic B cells with OVA-FITC leads to the production of IL-10. Total splenocytes (10 ⁶ cells/ml) from immunologically healthy donors were incubated alone, with F(ab') ₂ fragments of an anti-IgM antibody (10 μ^πύ) or with FITC-labeled OVA (200 μ^πύ) for 2 days at 37°C in complete medium. B cells were then washed and stimulated for 6 hr at 37 °C in the presence of phorbol 12- myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml) and Monensin. The production of IL-10 was evaluated on fixed permeabilized cells by flow cytometry. Values in quadrants depict the percentages of cells positive for the expression of CD20 and IL-10.

EXAMPLES:

Example 1 - Production of ILlO-secreting B cells from splenocytes of naive mice using OVA-FITC as a model antigen linked to a BcR-binding molecule

Materials and methods

Animals, antigens and cell clones. Eight to 9-week-old Balb/c mice (males), C57BL/6 mice (females and males) and OTII mice (males) were obtained from Charles River (France). V _H -LMP2A mice (males) on Balb/c background were provided by Dr Christophe Sirac (UMR CNRS 6101, Limoges, France). All animal studies were performed according to the guidelines of Charles Darwin ethical committee for animal experimentation (UPMC Paris) at the pathogen-free animal facility of Cordeliers Research Center, Paris. FITC-labeled and biotinylated MOG ₃₅ -55 peptides were purchased from Polypeptide Laboratories (Strasbourg, France) and FITC-labeled ovalbumin from Invitrogen (OVA-FITC, life technologies, Saint Aubin, France). Ovalbumin (OVA, Sigma- Aldrich, Saint-Quentin Fallavier, France) was labeled with biotin using EZ-Link* NHS-LC-Biotin (Thermo scientific, Illkirch, France); the excess of free biotin was removed by dialysis against Phosphate-buffered saline (PBS). Cells were cultured in RPMI medium (Lonza, Levallois-Perret, France) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin, 50 μΜ 2-ME, 0.1 mM non essential amino acids and 10% of heat inactivated fetal bovine serum. DO54.8 is an OVA ₃₂₃ - ₃₃₉ -specific mouse CD4 ⁺ T hybridoma restricted to I- A ^d (C57BL/6) .

Purification of B and T cells. B cells were purified from the spleen of Balb/c, V _H - LMP2A or C57BL/6 mice using either the "Pan B cell isolation kit" or CD 19 MicroBeads (MACS, Miltenyi Biotec, Paris, France). Briefly, splenocytes were isolated after mechanical dilaceration of spleens of naive mice and splenocytes were incubated 90 s in ACK lysis buffer (life technologies™, Invitrogen) at room temperature. Cells were then passed through 70 μιη nylon membrane filters and suspended in PBS supplemented with 2 mM EDTA and 0.5% bovine serum albumin (BSA, Sigma- Aldrich) as recommended by the manufacturer. CD4+ T cells were purified from splenocytes of OTII and wild-type C57BL/6 mice using Dynabeads® Mouse CD4 (life technology , Invitrogen). Total cells were also purified from the blood and lymph nodes of mice. In brief, blood was collected after perfusion of the mice with 40 ml of 0.2 mM EDTA in PBS through the left ventricle. Draining lymph nodes were collected and mechanically dilacerated. Single cell suspensions were prepared as described above.

Binding and endocytosis tests.

Binding of antigens to cell surface was evaluated using total or negatively selected B cells from splenocytes of naive mice. Cells were incubated 1 hr on ice alone or in the presence of antigens. After incubation, cells were washed and stained with a PE-labeled anti-mouse CD19 antibody (1D3; BD biosciences, Le Pont de Claix, France) for 25 min at 4°C. Cells were then incubated for 25 min with APC labeled annexin V (Invitrogen), washed and incubated with propidium iodide (Invitrogen) prior to FACS analysis. Binding of antigens was evaluated by flow cytometry (BD LSRII) either by direct fluorescence of FITC or using Pacific Blue-coupled streptavidin (Molecular Probes, Invitrogen) on the viable cell population (i.e., negative for annexin V and propidium iodide). Endocytosis of antigens was perform using negatively selected B cells purified as described above. B cells were incubated alone or in the presence of antigens for 1 hr at 4°C or 37 °C. After incubation, B cells were washed, stained with a PE-labeled anti-mouse CD19 antibody (1D3; BD biosciences) for 25 min at 4°C, and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences, Le Pont de Claix, France). When indicated, the FITC and Pacific blue fluorescences were normalized using FITC-labeled and Pacific Blue-labeled anti-CD19 IgG (clone 1D3). Endocytosis of antigens was evaluated by subtracting the normalized mean of fluorescence intensity (MFI) obtained at 37 °C from that obtained at 4°C. Cells were acquired using a BD LSR II and analyzed with FACS Diva or FlowJo software (BD Biosciences).

Analysis of T-cell activation and of T-cell polarization. The D054.8 T-cell hybridoma (2.5.10 ⁵ cells) was incubated alone or with negatively selected B cells (2.5.10 ⁵ cells) from Balb/c mice in complete medium with or without FrfC-labeled or biotinylated- OVA (10-200 μg/ml) for 2 days at 37°C. The supernatant was collected and the production of IL-2 by D054.8 was quantified using a mouse IL-2 ELISA Set (BD OptEIAlO™, BD bioscience). For T cell polarization, purified CD4 ⁺ T cells from OTII mice were labeled with CellTrace™ violet (life technology™, Invitrogen). Briefly, purified CD4 ⁺ T cells (10 ⁶ cells/ml) were incubated with CTV (3 μΜ) for 20 min at 37 °C in complete medium and washed to remove the excess of free dye. B cells (10 ⁵ cells) from C57BL/6 mice were co- incubated with purified CD4 ⁺ T cells (10 ⁵ cells) with or without OVA-FITC (5-20 μ^πύ) for 6 days at 37°C. For intracellular cytokine staining, cells were stimulated in vitro with phorbol 12-myristate 13- acetate (50 ng/ml, Sigma-Aldrich) in the presence of Ionomycin (1 μg/ml, eBioscience, Paris, France) and Monensin (GolgiStop®, BD biosciences) for 6 hr at 37°C. The T-cell polarization was evaluated using PE-Cy5-labeled anti-CD4 antibody (H129.19, BD biosciences), PE-labeled anti-IL-10 antibody (JES5-16E3, BD biosciences), APC-labeled anti-IFNy antibody (XMG1.2, BD biosciences), Alexa 700-labeled anti-FoxP3 antibody (FJK- 16s, eBioscience) and the FoxP3 staining buffer set (ebioscience) by flow cytometry. Analysis of B-cell subpopulations. Total splenocytes were isolated from naive mice as previously described. Cells were surface stained with a combination of PE-labeled anti- CD21/ CD35 antibody (4E3, ebioscience), PE-Cy7-labeled anti-CD23 antibody (B3B4, ebioscience), APC-labeled anti-CD19 antibody (1D3), and biotin-labeled anti-CD93 antibody (AA4.1, ebioscience) associated with the Alexa- fluor® 700 labeled streptavidin (Molecular probes®, Invitrogen).

Cytokine production by B cells. Purified B cells were obtained as described above. B cells (10 ⁶ cells/ml) were incubated alone or in the presence of antigens (200 μg/ml) for 2 days at 37°C. Supernatant was stored at -80°C and B cells were stimulated in vitro for intracellular cytokine staining as previously described. Intracellular staining was evaluated using APC- labeled anti-CD19 antibody (clone 1D3), PE-labeled anti-IL-10 antibody (JES5-16E3, BD biosciences) and the Cytofix/Cytoperm kit (BD Biosciences) by flow cytometry. Quantification of cytokines secreted by B cells in the supernatant was assessed using the BD CBA Mouse Inflammation Kit (BD biosciences).

Induction of EAE. Female C57BL/6 mice were immunized with 100 μg MOG ₃₅ _55 peptide emulsified in complete Freund's adjuvant (CFA, Sigma-Aldrich, v/v 1: 1) containing 400 μg of Mycobacterium tuberculosis (Difco Laboratories, L'Arbresle, France). A final volume of 200 μΐ was injected subcutaneously at 2 sites over the flanks. In addition, 150 ng of Pertussis toxin (List Biologic Laboratories, Meudon, France) was given intravenously on the same day and 2 days later. Clinical signs of EAE were assessed daily by the following scoring system: 0, no sign; 1, hindlimb weakness; 2, hindlimb weakness and tail paralysis; 3, hindlimb and tail paralysis; 4, hindlimb and tail paralysis and forelimb weakness; 5, moribund. B-cell transfer experiments. On day five and day one before EAE induction, as well as on day five after induction of the disease, mice received intravenously B cells (10 ⁶ cells) from naive animals incubated alone or in the presence of FITC-labeled or biotinylated antigens for 3 hr at 37 °C in HL-1 medium (Lonza).

Measurement calcium mobilization. Negatively selected splenic B cells (10 ⁷ cells/ml) from naive C57BL/6 were incubated in complete medium in the presence of 2 μΜ Indo-l-AM (Invitrogen) and 0.015% PluronicF127 (Invitrogen) at 37°C for 30 min. The B- cell suspension was then washed twice with complete medium and once with PBS. B cells were then incubated with APC-labeled anti-CD19 antibody (clone 1D3) for 25 min on ice. The cells were washed twice with Krebs-Ringer solution containing 10 mM HEPES, 10 mM glucose, 4 mM KC1, 140 mM NaCl, 1 mM MgCl ₂ and 1 mM CaCl ₂ , pH 7.0, at room temperature. Immediately before FACS analysis, B cells were washed and diluted in Krebs- Ringer solution containing 0.5 mM EGTA to a concentration of 10 cells per ml. The baseline was recorded for 30 seconds prior to addition of the BcR stimulus. Calcium efflux was assessed after stimulation of the B cells with F(ab') ₂ fragments of an anti-IgM antibody (10 μg/ml, Jackson ImmunoResearch Laboratories, Suffolk, United Kingdom) or with OVA- FrrC (200 μg/ml) for 6 min. Calcium influx was then evaluated for 6 min following the addition of CaCl ₂ (1 mM). Intracellular calcium mobilization was performed in real-time using FACSAria II (BD Biosciences). Data files were analyzed using FlowJo software and are presented as a median in comparative overlay analyses.

Statistics. All data are shown as mean + SEM (n between 3 and 10, as indicated). Statistical significances were assessed using the double-sided non-parametric Mann Whitney U test. We used the two-way ANOVA with Bonferroni's post-hoc t test for comparing EAE scores.

Results

B cells from naive mice bind FITC in a BcR-dependent manner.

We first investigated the capacity of splenic B cells from naive wild-type mice to bind FITC. In order to detect FITC binding, we used FITC coupled to OVA as a carrier protein. Splenocytes purified from the spleen of naive C57B1/6 mice were incubated with 200 μg/ml of FITC-labeled OVA at 4°C. The percentage and mean fluorescence of OVA-FITC ⁺ cells was analyzed by flow cytometry. There was a general right shift of both CD19 ⁺ and CD 19 ^" splenocytes upon incubation with OVA-FITC (Figure 1A) due either to the background fluorescence of the FITC moiety (a FITC-labeled isotype control being irrelevant in our experiment), or to the binding of OVA-FITC at different degrees to the different cell types included in the splenocyte preparation. Since binding of FITC at the surface of B lymphocytes has been documented previously (Scott, 1976) we placed the threshold of FITC positivity based on the CD19 ^" population. In such conditions, OVA-FITC-positive B cells represented 9.9+1.3% (mean+SEM) of the total splenocytes (Figure 1A) and 16.4+2.26% of the CD19 ⁺ cells. Accordingly, the mean fluorescence intensities of FITC among the CD 19 negative and positive populations were 84+10 and 263+21.7, respectively. The incubation of purified naive CD19 ⁺ splenic B cells with varying amounts of OVA-FITC revealed a dose-dependent binding of OVA-FITC to the cell surface (Figure IB). The percentage of B cells able to bind OVA-FITC reached a plateau of about 20% in the presence of a large excess of OVA-FITC. Interestingly, biotinylated OVA failed to bind the surface of CD 19+ B cells, indicating that binding was mediated by the FITC moiety rather than by OVA. Importantly, 15 to 25% of purified CD19 ⁺ B cells could bind FITC-coupled MOG ₃₅ -55 peptide, pro-coagulant factor IX and factor VIII (Figure 1C and data not shown, all at 200 μg/ml), while no binding was observed with the biotinylated forms. We then investigated whether the binding of OVA- FrrC to CD 19+ cells is mediated by the BcR. LMP2A BCR-deficient mice (BcR ^"/_ ) mice possess normal percentages of mature splenic B cells that do not express surface IgM and IgG (Figure ID, upper panel, and data not shown). As seen in the case of splenocytes from C57B1/6 mice, 20.3+1.7% of splenic CD19+ B cells from naive Balb/c mice were able to bind FrrC-labeled OVA (Figure ID). In striking contrast, B cells from naive BcR ^_/" Balb/c mice failed to bind OVA-FITC, indicating that the binding of FITC to the naive B lymphocytes occurs through the BcR.

We further investigated whether the binding of OVA-FITC by the CD19 ⁺ cells was a reflect of the high natural poly/self-reactivity of some B-cell sub-populations (Casali, 1989; Chen, 1995; Zhou, 2004) Bl B cells (CD19 ⁺ CDl lb ⁺ CD5 ⁺ ) were isolated from the peritoneum and B2 B cells from the inguinal lymph nodes and the spleen of naive mice. The different subpopulations of splenic B cells were identified based on the expression level of the surface marker CD93, CD21 and CD23. The marginal zone B cells (MZ) were referred as CD93 ^" CD21 ⁺⁺ CD23 ^low , the follicular B cells (FO) as CD93 ^" CD21 ^low CD23 ⁺ , the transitional 1 B cells (Tl) as CD93 ⁺ CD21 ^" CD23 ^" and the transitional 2 B cells (T2) as CD93 ⁺ CD21 ⁺ CD23 ⁺ (Figure IE, upper panels). As previously observed, about 20% of total splenic CD19 ⁺ B cells were positive for OVA-FITC (19.6+1.2%). The same results were obtained when CD19 ⁺ cells were isolated from the inguinal lymph nodes (21.1+2.2%) while 60.8+4.7% of peritoneal B cells from naive mice were able to bind to OVA-FITC. We then analyzed the reactivity with FITC of the different splenic subpopulations of B cells. Interestingly, all the subpopulations of B cells were able to bind to OVA-FITC, but to different extents. Thus, FITC-positive B cells accounted for 17.8+1.7, 10.9+0.1, 33.5+2.3 and 26+2.4% of Tl, FO, T2 and MZ B cells, respectively (Figure IE, bottom panels). Binding of FITC-labeled OVA to B cells induces IL-10 production in vitro.

We then evaluated the cytokine profile secreted by purified splenic B cells incubated in vitro either with an anti-IgM antibody or with OVA-FITC. To this end, purified CD19 ⁺ splenic B cells from naive C57B1/6 mice were incubated for 48 hrs alone, with F(ab') ₂ fragments of an anti-mouse IgM antibody (10 μg/ml) or with OVA-FITC (200 μg/ml); the concentrations of IL-10, TNFa and MCP-1 secreted in the supernatant were measured (Figure 2A, upper panels). The in vitro stimulation of purified splenic B cells with F(ab') ₂ fragments or with OVA-FITC induced different cytokine profiles. When stimulated in vitro with F(ab') ₂ fragments, the purified splenic B cells secreted high amounts of TNFa and MCP-1 as compared to control B cells (TNFa: 231.7+46.9 versus 3.5+2.5 pg/ml, P=0.039; MCP-1: 125.6+89.5 versus 18.4+0.9 pg/ml, P=0.006, respectively). In striking contrast, purified splenic B cells stimulated with OVA-FITC secreted high amounts of IL-10 as compared to cells incubated alone or with F(ab') ₂ fragments (6670.4+483.7 versus 38.4+2.7 and 62.7+5.5 pg/ml, P=0.005, respectively). B cells incubated with OVA-biotin failed to produce cytokines.

While stimulation of the B cells with the anti-IgM F(ab') ₂ fragments lead to 1.3+0.2% of IL-10 ⁺ B cells, 14.2+1.3% of the cells were positive for IL-10 following incubation with OVA-FITC (Figure 2A, bottom panels). Importantly, purified B cells from LMP2A BcR-/- mice failed to produce IL-10 after incubation with OVA-FITC (data not shown). Moreover, analysis of the expression of the B-cell-specific surface marker CD19 showed that 54.4+2.3% of the B cells were activated upon incubation with the anti-IgM F(ab') ₂ fragments, which was not the case when B cells were incubated alone, with OVA-biotin or with OVA-FITC.

In order to decipher whether the fate of the B cells is dictated by the strength of BcR signaling, we compared calcium fluxes in B cells following stimulation with anti-IgM F(ab') ₂ fragments or with OVA-FITC. Purified CD19 ⁺ splenic B cells were loaded with the calcium sensitive probe Indo-1 and the strength of BcR stimulation was determined according to the calcium mobilization in the cells in real time. The mobilization of intracellular calcium from the endoplasmic reticulum (calcium efflux), evaluated in buffer containing 0.5 mM EGTA, was seen upon stimulation with the anti-IgM F(ab') ₂ fragments but not upon addition of OVA-FITC (Figure 2B). Conversely, the influx of extracellular calcium, evaluated after addition of 2 mM of CaCl ₂ , was more pronounced in the case of incubation with OVA-FITC that in that of anti-IgM F(ab') ₂ fragments.

Endocytosis and presentation of FITC-labeled OVA by naive splenic B cells polarizes CD4 ⁺ T cells.

We explored the consequences of BCR-mediated FITC binding to the BcR of naive B cells on their antigen presenting capacity. Purified CD19 ⁺ splenic B cells from naive C57BL/6 mice were incubated with increasing concentrations of FITC or biotin-labeled OVA at 4°C or 37°C to evaluate endocytosis. Naive B cells endocytosed FITC-labeled OVA in a dose- dependent manner, but failed to endocytose biotin-labeled OVA (Figure 3A) In order to study the presentation of the endocytosed FITC-labeled OVA to CD4+ T lymphocytes, we co- incubated naive splenic B cells from Balb/c mice with the D054.8 T-cell hybridoma, an OVA ₃₂ 3_339-specific CD4 ⁺ T-cell hybridoma with the I-A ^d restriction, together with increasing amounts of OVA-FITC or OVA-biotin. Co-incubation of purified CD19 ⁺ splenic B cells with D054.8 in the presence of OVA-FITC lead to a dose-dependent secretion of IL-2 by the T- cell hybridoma (Figure 3B, full bars). Importantly, co-incubation of the B cells and D054.8 in the presence of biotinylated-OVA, or unlabeled OVA, failed to induce IL-2 secretion in the supernatant (Figure 3B, empty bars and data not shown). Accordingly, T cells incubated with B cells in the absence of antigen were not activated.

We then evaluated the proliferation and the polarization of primary OVA ₃₂ 3_33 ₉ - specific CD4+ T cells following activation by OVA-FITC-"educated" B cells. Naive CD4+ splenic T cells purified from OTII mice were stained with cell trace violet (CTV) and co- incubated alone or with splenic B cells from naive C57B1/6 and varying amounts of OVA- FITC. Incubation of CD4 ⁺ OTII T cells with ConA, used as a positive control, induced the proliferation of 38.2+1.7% of the total CD4+ T cells after 6 days of incubation (Figure 3C, upper panels). The percentage of proliferating T cells increased in a dose-dependent manner (6.3+0.8 to 27.8+0.98%, respectively) when OTII T cells were incubated in the presence of increasing amounts of OVA-FITC (5 to 20 μ^πύ). Conversely, only 4.5+0.7% of CD4+ T cells proliferated when incubated with B cells alone. When CD4+ OTII T cells were incubated in presence of ConA, 0.6+0.1% of the cells were positive of intracellular IFNy after 6 days while 2.3+0.2% were positive for IL-10 (Figure 3C, bottom panels). Most of the OTII CD4 ⁺ T cells incubated alone, as a negative control, were negative for IFNy and IL-10 (0.9+0.2 and 0.6+0.3%, respectively). Interestingly, the co-culture of OTII CD4 ⁺ T cells and B cells in the presence of OVA-FITC lead to a dose-dependent generation of IL-10 ⁺ CD4 ⁺ T cells (17.4+0.6% and 27.8+2.7% for 10 and 20 μg/ml OVA-FITC, respectively). In contrast, the percentage of IFNy ⁺ CD4 ⁺ T cells only marginally increased as compared to the negative control (1.3+0.2 and 2.2+0.4% for 10 and 20 μg/ml OVA-FITC, respectively).

To further study the polarization of OTII CD4+ T cells, we determined the percentage of IL-10-positive cells among non dividing (CTV ^hlgh ) and dividing (CTV ^low ) cells, incubated with ConA, with B cells alone or with B cells and 200 μg/ml OVA-FITC. In the presence of ConA, both dividing and non-dividing cells were negative for IL-10 expression (0.8+0.2 and 0.4+0.1%, respectively). In contrast, in the presence of OVA-loaded B cells, the percentages of IL-10 ⁺ dividing and non-dividing CD4 ⁺ T cells increased in a dose-dependent manner from 13.7+1.2 to 36.8+2.8% in the case of dividing cells, and from 2.6+07 to 16.1+1.4% in case of non-dividing cells (Figure 3D). Further, analysis of total IL-10 ⁺ CD4 ⁺ T cells demonstrated that the cells are negative for FoxP3 expression (data not shown).

Adoptive transfer of MOG35 -55-FITC-loaded naive B cells protects mice from

EAE.

We then investigated whether the transfer of naive splenic B cells pre-incubated with

FITC-labeled antigens is beneficial in EAE, a Thl -mediated experimental autoimmune model. EAE was induced in mice by the subcutaneous injection of the MOG ₃₅ -55 peptide (100 μg) emulsified in CFA associated with the intravenous injection of Pertussis toxin (150 ng). Recipient mice received intravenously purified splenic B cells (10 ⁶ cells) from naive mice pre-incubated alone, with OVA-FITC, with biotin- MOG ₃₅ _55 or with FITC-MOG ₃₅ _ ₅₅ . Mice injected with untouched purified B cells developed the first clinical signs at day 10, with a peak of the disease at day 17, when the mean clinical score was 2.85+0.38 (Figure 4A, full circles). Mice that received B cells incubated with MOG-biotin, developed EAE with the same kinetics and intensity as control mice (empty circles, mean clinical score: 2.89+0.27). Interestingly, the transfer of B cells pre-incubated with MOG ₃ 5_55-FITC not only delayed the onset of the disease (day 14) but also significantly decreased the severity of the disease. In such conditions, the peak of severity of the disease was detectable at day 18 with a mean of clinical score of 1.22+0.31 (full squares). Mice treated with OVA-FITC-"educated" B cells developed the first clinical signs at day 10, like control mice. The peak of severity was reached at day 19 with a mean clinical score that did not differ from that of control mice (empty squares, 2.55+0.14), thus indicating the antigen- specificity of the protection conferred by adoptively transferred FITC- stimulated B cells.

At day 18, representative mice that received B cells pre-incubated with FITC- or with biotin-labeled MOG ₃₅ -55 (n=4 per group) were sacrificed and the cells from the spleen, the blood and the draining lymph nodes (DLN) were isolated. We analyzed the percentages of IFNy, FoxP3 and IL-10-positive CD4 ⁺ T cells in each organ (Figure 4B). The transfer of naive B cells pre-incubated with MOG ₃₅ _55 -FITC to EAE mice lead to a statistically significant increase in the percentage of IL-10 ⁺ CD4 ⁺ T cells as compared to the EAE mice that received naive B cells pre-incubated with MOG ₃₅ _55-biotin. This was evidenced in the blood (17.2+3.4% versus 7.08+0.66%, respectively), in the DLN (12.6+1.1 versus 6.5+0.6%) and in the spleen (21.9+1.5 versus 11.3+1.8%). The absolute number of IL-10 ⁺ CD4 ⁺ T cells was also significantly different between the two groups of mice in the spleen and in the DLN but not in the blood (data not shown). Analysis of the IL-10 ⁺ CD4 ⁺ T-cell populations for the expression of FoxP3 showed no statistical difference in any of the tested organs between the 2 groups of mice (Figure 4B). The percentage of IFNy ⁺ CD4 ⁺ T cells in mice that received B cells pre-incubated with MOG ₃₅ -55-FITC was marginally although statistically higher (P=0.042) than mice that received B cells pre-incubated with MOG ₃₅ _55-biotin in the case of the DLN (5.4+0.4% versus 3.3+0.3%, respectively (Figure 4B). However, the absolute numbers of IFNy ⁺ CD4 ⁺ T cells were not statistically different between the 2 groups of mice in any of the organs tested (data not shown).

Example 2- Effect of a pre-treatment with ferrous ions on the production of ILlO-secreting B cells.

As shown in Figure 5, pre-incubation with ferrous ions led to an increase in the percentage of B cells that bound OVA-FITC. Naive splenic B lymphocytes were exposed (full bars) or not (empty bars) to Fe(II) ions for 10 min at 4°C and incubated with FITC-labeled or unlabelled antigens for 1 hr at 4°C. The percentage of antigen-positive B cells was then assessed by flow cytometry.

Example 3- Production of ILlO-secreting B cells from human PBMCs or splenocytes Material and methods

Cells and antigens. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy bags of healthy donors purchased from Hopital Hotel Dieu, Etablissement Francais du Sang, Paris, France. Institutional Review Board (INSERM-EFS) approval was obtained for use of buffy bags of healthy donors. Informed consent was obtained in accordance with the Declaration of Helsinki. Human splenocytes from immunologically healthy donors were provided by Dr Claude-Agnes Reynaud (UMR INSERM 783, Paris, France). FITC-labeled ovalbumin was purchased from Invitrogen (OVA-FITC, life technologies, Saint Aubin, France).

Binding test. Binding of OVA-FITC to cell surface was evaluated using total PBMCs or splenocytes from immunologically healthy donors. Cells were incubated 1 hr on ice alone or in the presence of OVA-FITC. After incubation, cells were washed and stained with a Alexa 700-labeled anti-human CD20 antibody (2H7; ebioscience, Paris, France) for 25 min at 4°C. Cells were then incubated for 25 min with APC labeled annexin V (Invitrogen), washed and incubated with propidium iodide (Invitrogen) prior to FACS analysis. Binding of antigens was evaluated by flow cytometry (BD LSRII) by direct fluorescence of FITC on the viable cell population (i.e., negative for annexin V and propidium iodide).

IL-10 production. Human splenocyte (10 ⁶ cells/ml) were incubated alone, in the presence of F(ab)'2 fragments of an anti-human IgM antibody (10 μg/ml, Jackson ImmunoResearch Laboratories) or OVA-FITC (200 μg/ml) for 2 days at 37°C. For intracellular cytokine staining, cells were stimulated in vitro with phorbol 12-myristate 13- acetate (50 ng/ml, Sigma- Aldrich) in the presence of Ionomycin (1 μg/ml, eBioscience, Paris, France) and Monensin (GolgiStop®, BD biosciences) for 6 hr at 37°C. Intracellular staining was evaluated using Alexa 700-labeled anti-human CD20 antibody (Clone 2H7), PE-labeled anti-IL-10 antibody (JES3-19F1, BD biosciences) and the Cytofix/Cytoperm kit (BD Biosciences) by flow cytometry.

Results

Circulating and resident B cells from immunologically healthy donors bind

FITC. The capacity of B cells from PBMCs or splenocytes to bind FITC has been investigated. In order to detect FITC within cell populations, FITC coupled to OVA as a carrier protein has been used. Human PBMCs and splenocytes were incubated alone or with 200 μg/ml of FITC-labeled OVA at 4°C. The percentage of OVA-FIT C ⁺ CD20 ⁺ B cells was analyzed by flow cytometry. In such conditions, OVA-FITC -positive B cells represented 24.6+1.3% (mean+SEM) of the CD20 ⁺ splenocytes and 94.4+1.1% of the CD20 ⁺ PBMCs (Figure 6).

In vitro stimulation of human CD20 ⁺ splenocytes with FITC-labeled OVA induce IL-10 production.

It has been then investigated whether in vitro incubation of human CD20 ⁺ splenocytes with OVA-FITC induced the production of IL-10 by the B cells. To this end, human splenocytes from immunologically healthy donors were incubated for 48 hrs alone, with F(ab)'2 fragments of an anti-human IgM antibody (anti-IgM, 10μg/ml) or in presence of FrrC-labelled OVA (200μg/ml). The incubation of splenocytes alone or in presence of an anti-IgM failed to induce IL-10 production in the CD20 ⁺ B cells. However, when B cells were stimulated with OVA-FITC, 15.6+2.4% of the cells were positive for IL-10 production (Figure 7).

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