WO2013050413A1

WO2013050413A1 - Methods for obtaining a population of regulatory t cells

Info

Publication number: WO2013050413A1
Application number: PCT/EP2012/069532
Authority: WO
Inventors: Jean-Pierre Gorvel; Anna Martirosyan
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Cnrs (Centre National De La Recherche Scientifique); Université D'aix-Marseille
Priority date: 2011-10-03
Filing date: 2012-10-03
Publication date: 2013-04-11

Abstract

The present invention relates to a method for obtaining a population of regulatory T cells, said method comprising the steps of (i) coculturing a population of dendritic cells with a population of naïve CD4+ T cells in a culture medium having an amount of at least one tetraacylated lipopolysaccharide (LPS), and (ii) isolating the population of regulatory T cells as obtained in step i).

Description

METHODS FOR OBTAINING A POPULATION OF REGULATORY T CELLS

FIELD OF THE INVENTION:

The present invention relates to methods for obtaining a population of regulatory T cells.

BACKGROUND OF THE INVENTION:

Foxp3+ CD25+ CD4+ T cells, known as regulatory T cells, or 'Tregs' are fundamental in controlling various immune responses in that Tregs can rapidly suppress the activity of other immune cells. In particular, Tregs are crucial for maintaining tolerance by downregulating undesired immune responses to self and non-self antigens.

For instance, Treg defects have been discovered in patients with multiple sclerosis (MS), type I diabetes (T1D), psoriasis, myasthenia gravis (MG) and other autoimmune diseases. Similar links may also exist for atopy and allergic diseases. For all these diseases reports exist pointing to a reduced in vitro immune suppression of the patient's Treg cells. This has led to an increasing interest in the possibility of using Tregs in immunotherapy to treat or prevent chronic infections, autoimmune diseases, allergies and transplantation-related complications, such as graft rejection or graft- versus-host disease (GvHD).

Despite evidence of success in numerous animal models, then therapeutic use of Treg cells for autoimmunity has been severely limited clinically. Part of the reason for this is the inability to induce high level of Tregs that remain functional for extended periods of time.

LPS (lipopolysaccharide), also referred to as "endotoxin", is the major surface component of gram negative bacteria. Under normal conditions, LPS is inserted in the outer surface of the outer membrane of gram negative bacteria. LPS is composed of three main domains called lipid A, the O-antigen (also called the O-polysaccharide) and the core region, which creates an oligosaccharide link between lipid A and the O antigen. The biologically active component of LPS is lipid A. For example, activity analysis of lipid A biosynthesis precursors or synthetic intermediates showed that various elements of lipid A are essential for pyrogenicity. A number of lipid A structures have been identified that lack pyrogenic activity and are tetraacylated LPSs (i.e., they effectively block the pyrogenic activity of LPS from gram negative bacteria, e.g., E. coli LPS). For example, LPS structures predominated by tetraacyl-lipid A (etraacylated LPSs) are non pyrogenic and display tetraacylated LPS activity. However, it has never been demonstrated that ttetraacylated LPS can be used for inducing high levels of Treg cells.

SUMMARY OF THE INVENTION:

DETAILED DESCRIPTION OF THE INVENTION:

Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. The inventors investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4+ T and CD8+ T cell responses and importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, the data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

An object of the present invention relates to a method for obtaining a population of regulatory T cells, said method comprising the steps of (i) coculturing a population of dendritic cells with a population of naive CD4+ T cells in a culture medium having an amount of at least one tetraacylated lipopolysaccharide (LPS), and (ii) isolating the population of regulatory T cells as obtained in step i).

The term "CD4+ T cells" refers to lymphocytes that produce the CD4 protein, and are able to interact with dendritic cells. Such CD4+ T cells include, but are not limited to, cells isolated from natural sources such as blood, cell lines grown in culture, and CD4+ T cell clones. Typically, a CD4+ T cell according to the invention is a human CD4+ T cell. As used herein, the term "naive CD4+ T cells" refers to a CD4+ T cell that is functionally defined by the expression of cell surface markers of naivety that include CD45RA+ CD25-HLA-DR-. As used herein, the term "Treg cell" is intended to describe the subpopulation of T cells that may be characterised by cell surface expression of CD4, CD25 and Foxp3 and act to "suppress" effector T cells in vitro and/or in vivo.

As used herein the term "dendritic cell" or "DC" is an antigen presenting cell which can be derived from a hematopoietic stem cell or a monocyte. Dendritic cells and their precursors can be isolated from a variety of lymphoid organs, e.g., spleen, lymph nodes, as well as from bone marrow and peripheral blood. The DC has a characteristic morphology with thin sheets (lamellipodia) extending in multiple directions away from the dendritic cell body. DCs express constitutively both MHC class I and class II molecules, which present peptide antigens to CD8+ and CD4+ T cells respectively. Generally, DCs express CD85, CD 180, CD 187 CD205 CD281, CD282, CD284, CD286 and in a subset manner CD206, CD207, CD208 and CD209.

In some embodiments, the population of dendritic cells is a population of human dendritic cells, and in particular a population of human myeloid dendritic cells.

A used herein, the term "culture medium" is recognized in the art, and refers generally to any substance or preparation used for the cultivation of living cells. Typically, the culture medium is a serum free culture medium. As used herein, the term "serum-free culture medium" is defined as serum-free cell culture medium that has a defined chemical composition and supports proliferation of human T cells A list of serum- free culture medium useful in the present invention include but are not limited to LONZA XVIVO-5, XVIVO-10, XVIVO-20, Sigma StemLine I, StemLine II, Yssel's media and AimV media. A skilled practitioner could easily select the appropriate serum-free media capable of supporting T cell proliferation following addition of required growth factors. Such serum-free media contain specific and defined growth factors that are required for T cell proliferation. In one embodiment, the culture medium may further comprise at least one cytokine for maintaining proliferation of T cell, and in particular T regulatory cells such as IL-2 and/or TGFbeta. The term "LPS" or "lipopolyssaccharide" or "endotoxin" , which are used interchangeably herein, are used as known in the art, and includes both wildtype LPS, analogs, and derivatives thereof. Thus, LPS is a molecule comprising an O-specific polysaccharide; a common core region; and a lipid component called lipid. The glycosidic units can be glycopyranosyl or glycofuranosyl, as well as their amino sugar derivatives. The residues may be homopolymers, random, or alternating or block copolymers thereof. The glycosidic units have free hydroxy groups, or acylated hydroxy groups. The glycosides can comprise up to 20 glycosidic units. Preferred, however, are those having less than 10, most preferred, those having 3 or less than 3 glycosidic units. Specific examples are those containing 1 or 10 glycosidic units in the glycoside residue. Among the possible glycopyranosyl structures are glucose, mannose, galactose, gulose, allose, altrose, idose, or talose. Among the furanosyl structures, the preferred ones are those derived from fructose, arabinose or xylose. Among preferred diglycosides are sucrose, cellobiose, maltose, lactose, trehalose, gentiobiose, and melibiose. Among the triglycosides, the preferred ones may be raffinose or gentianose. Among the amino derivatives are N-acetyl-D-galactosamine, N- acetylD-glucosamine, N-acetyl-D-mannosamine, -acetyineuraminic acid, D-glucosamine, lyxosylamine, D-galactosamine, and the like.

The term "lipid A" refers to the hydrophobic portion of an LPS molecule that is linked to the inner core of the LPS molecule through an ester bond. Lipid A, as used herein includes both wildtype lipid A, analogs, derivatives and precursors thereof.

As used herein, the term "tetraacylated lipopolysaccharide" or "tetraacylated LPS" has its general meaning in the art and refers to LPS having a tetraacylated lipid A (Flad HD, Loppnow H, Rietschel ET, Ulmer AJ. Agonists and antagonists for lipopolysaccharide- induced cytokines. Immunobiology. 1993 Apr;187(3-5):303-16. Review.).

Any gram negative bacterial strain for which an accumulation of tetraacylated LPSs has been observed can be used as a source of tetraacylated LPS in the practice of the present invention. The specific culture conditions for the growth of the gram negative bacterial strains for the preparation of a tetraacylated LPS are not critical to the present invention. For illustrative purposes, bacteria can be grown in any standard liquid medium suitable for bacterial growth, such a LB medium (Difco, Detroit Mich.), Nutrient broth (Difco), Tryptic Soy broth (Difco), or M9 minimal broth (Difco), using conventional culture techniques that are appropriate for the bacterial strain being grown. The temperature at which the bacterial strains are cultured is not crucial to the present invention. However, individual bacterial strains may produce a non tetraacylated LPS at one temperature and at other temperatures produce a tetraacylated LPS. For example, Yersinia pestis, the causative agent of plague, showed a temperature-dependent change in lipid A composition, with a reduced degree of acylation when bacteria were grown at 37°C (tetraacylated) versus ambient temperature (hexaacylated) (Telepnev MV, Klimpel GR, Haithcoat J, Knirel YA, Anisimov AP, Motin VL.Tetraacylated lipopolysaccharide of Yersinia pestis can inhibit multiple Toll-like receptor-mediated signaling pathways in human dendritic cells. J Infect Dis. 2009 Dec 1 ;200(1 1): 1694-702.). A straight forward approach to identifying the optimal temperature for the culture of a particular bacterial strain is to grow the bacteria over a range of culture temperatures, isolate LPS from each culture (as described herein below) and determine the nature of lipid A of the LPS produced. In this manner, culture temperatures can be identified that result in the production of tetraacylated LPS by the bacterial strains.

The method used to extract LPS is not critical to the practice of the present invention and can be any one of the well-known methods for LPS extraction. For example, the tetraacylated LPS can be extracted using the well-known hot phenol-water extraction procedure.

Various methods well known in the art can be used to determine the molecular structure of an LPS molecule isolated and purified from bacteria. Exemplary methods are described, e.g, in U.S. Pat. No. 5,648,343. Such methods may involve NMR Spectroscopy and high resolution mass spectrometry, e.g., fast atom bombardment mass spectrometry (FAB-MS).

Synthetic tetraacylated LPS may also be synthesized by a variety of organic chemistry synthetic techniques. For an overview of the synthesis of LPS and lipid A structures, see, e.g., Raetz, 1993, J. Bacteriology 175:5745-5753. (See also U.S. Pat. Nos. 5,593,969 and 5,191,072). Methods for determining the structure of an LPS or lipid A molecule are well known in the art (as above described) and can be used to first determine the structure of an LPS or lipid A molecule of interest, prior to synthesizing the same or similar LPS or lipid A molecules using organic chemistry methods.

Methods for isolating Treg cells are well known in the art. For example said methods employ positive selection of T cells expressing the surface markers of Treg. Typically Treg cells are isolated by using antibodies for Treg associated cell surface markers, CD4, CD25 and Foxp3. Commercial kits, e.g. CD4+CD25+ Regulatory T Cell Isolation Kit from Miltenyi Biotec or Dynal® CD4+CD25+ Treg Kit from Invitrogen are available.

According to the present invention, regulatory T cells can be easily and effectively generated in vitro. The ability to obtain a large number of in vitro produced regulatory T cells opens new opportunities for the therapeutic field. The population of regulatory T cells obtainable by the method of the invention may be subsequently injected into the patient suffering from a disorder in need of immune modulation. Such a disorder is characterized in that its clinical picture can be influenced positively by an increase of activated Treg cells. Medical conditions in which treatment with the invention disclosed may be useful include: Thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rheumatoid arthritis, lupus erythematosus, immune hyperreactivity, insulin dependent diabetes mellitus, anemia (aplastic, hemolytic), autoimmune hepatitis, skleritis, idiopathic thrombocytopenic purpura, inflammatory bowel diseases (Crohn's disease, ulcerative colitis), juvenile arthritis, scleroderma and systemic sclerosis, Sjogren's syndrom, undifferentiated connective tissue syndrome, antiphospholipid syndrome, vasculitis (polyarteritis nodosa, allergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Henoch-Schoenlein purpura, Behcet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangiitis obliterans), polymyalgia rheumatica, essentiell (mixed) cryoglobulinemia, Psoriasis vulgaris and psoriatic arthritis, diffus fasciitis with or without eosinophilia, polymyositis and other idiopathic inflammatory myopathies, relapsing panniculitis, relapsing polychondritis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrome, inflammatory dermatitis, unwanted immune reactions and inflammation associated with arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity and allergic reactions, systemic lupus erythematosus, collagen diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of strokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery or organ, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

The invention also provides a pharmaceutical composition comprising a population of regulatory T cells obtainable by the method of the invention, in combination with a pharmaceutically acceptable carrier. Such compositions comprise a therapeutically effective amount of a population of regulatory T cells produced according to the invention, and a pharmaceutically acceptable carrier or excipient. By a "therapeutically effective amount" of a population of cells as above described is meant a sufficient amount of said population of cells to treat a disease or disorder at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific cells employed; and like factors well known in the medical arts.

Pharmaceutically acceptable carrier or excipient includes but is not limited to saline, buffered saline, dextrose, water, glycerol and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, or emulsion. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. A further object of the invention relates to a culture medium having an amount of at least one tetraacylated lipopolysaccharide (LPS) according to the invention. Typically, the culture medium is a serum free culture medium as above defined and supports proliferation of human T cells. In one embodiment, the culture medium may further comprise at least one cytokine for maintaining proliferation of T cell, and in particular T regulatory cells such as IL-2 and/or TGFbeta.

A further object of the invention relates to kit comprising a culture medium as above described and means for isolating a population of regulatory T cells as obtained (e.g. antibodies for Treg associated cell surface markers, CD4, CD25 and Foxp3). In one embodiment, the kit may also comprise a population of dendritic cells and/or a population of na^'ive CD4+ T cells

A further object of the invention relates to a tetraacylated LPS according to the present invention for use in a method for inducing in a subject in need thereof high level of T regulatory cells. In a particular embodiment, the subject is afflicted with a disease as above described. The tetraacylated LPS may be formulated in a pharmaceutical composition.

A further object of the invention relates to a vaccine composition comprising an amount of tetraacylated LPS as adjuvant and an amount of at least one antigen of interest.

As used herein, the terms "antigen" or "Ag" refer to a substance capable of eliciting an immune response, e.g., a T-cell-mediated immune response by the presentation of the antigen on Major Histocompatibility Antigen (MHC) cellular proteins and causing an antigen-specific T-cells response. In the case of a regulatory T-cell (Treg) response to the antigen is a decrease or amelioration of the immune response by other effector cells, e.g., helper T-cells (Th) and/ or cytotoxic T-cells (Tc). The skilled immunologist will recognize that when discussing antigens that are processed for presentation to T-cells, the term "antigen" refers to those portions of the antigen (e.g., a peptide fragment) that is a T-cell epitope presented by MHC to the T-cell receptor. When antigen is modified by self- or auto-, this refers to self or auto antigens that are commonly present in MHC molecules but that also trigger a T-cell response. In certain cases, the antigens delivered by the vaccine are internalized and processed by antigen presenting cells prior to presentation, e.g., by cleavage of one or more portions of the antigen. The vaccine composition according to the invention is particularly suitable for inducing in a subject a tolerance the antigen of interest. Therefore the vaccine composition may thus be useful for the treatment (i.e. the prophylactic treatment) of autoimmune diseases allergic diseases or graft rejection. Typically the antigen is selected from self-antigens or auto-antigens. Indeed, the activated immune cells that are directed against self or auto antigens can cause damage to the target organ or tissue or can damage other organs or tissues. The dysregulated immune cells secrete inflammatory cytokines that cause systemic inflammation or they can recognize self-antigens as foreign, thereby accelerating the immune response against self-antigens.

Antigens involved in autoimmune diseases, allergy, and graft rejection can be used in the compositions. For example, an antigen involved in any one or more of the following autoimmune diseases or disorders can be used in the present invention: diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia greata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis. Examples of antigens involved in autoimmune disease include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor.

Examples of antigens involved in allergy include pollen antigens such as Japanese cedar pollen antigens, rag Weed pollen antigens, rye grass pollen antigens, animal derived antigens such as dust mite antigens and feline antigens, histocompatiblity antigens, and penicillin and other therapeutic drugs. Examples of antigens involved in graft rejection include antigenic components of the graft to be transplanted into the graft recipient such as heart, lung, liver, pancreas, kidney, and neural graft components.

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: LPS with acylation defects activate human mDC to induce regulatory T cells. Human blood mDC were activated overnight either by medium or different LPS and co- cultured with allogeneic na^'ive CD4+ T cells. After 7 days, cells were incubated 6 h with PMA/Ionomycine in the presence of Brefeldin A. Foxp3 and CD25 expression was analysed in CD4+ T cell population. Experiments were performed on 4 different donors. Data for 2 representatives are shown.

EXAMPLE :

Material & methods

Ethics statement. Animal experimentation was conducted in strict accordance with good animal practice as defined by the French animal welfare bodies (Law 87-848 dated 19 October 1987 modified by Decree 2001-464 and Decree 2001-131 relative to European Convention, EEC Directive 86/609). All animal work was approved by the Direction Departmental des Services Veterinaires des Bouches du Rhone (authorization number 13.118). INSERM guidelines have been followed regarding animal experimentation (authorization No. 02875 for mouse experimentation).

Blood from healthy adult donors were collected at the Baylor Hospital Liver Transplant Clinic (Dallas, TX) after obtaining written informed consent. This study, including the consent form, was approved by the Institutional Review Board (IRB) of the Baylor Research Institute (BRI) (Dallas, TX). Any medical issue during blood collection from healthy donors was written and reported to the IRB at BRI. Lipopolysaccharides. The methods used in the extraction, purification and characterization of the LPS used in this study have been described previously (Lapaque et al, 2006). Briefly, Y. pestis KIM6, E. coli MLK3 and its lipid A mutants MLK53 htrB^~ (lauroyl- transferase), MLK 1067 msbB^~ (miristoyl-transferase) and MLK 986 htrB^'lmsbB^' were grown at the appropriate temperature, crude LPS obtained by the phenol-water method and then purified to remove traces of contaminant lipids and lipoproteins. The degree of lipid A acylation was determined by nano-electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) (Lapaque et al, 2006). For all experiments, LPS variants have been used at the concentration of 100 ng/ml.

Antibodies and reagents. The primary antibodies used for immunofluorecence microscopy were: mouse FK2 antibody (anti-mono- and polyubiquitinylated conjugates) (Enzo Life Science), affinity purified rabbit "Rivoli" antibody against murine I-A, NF-kB subunit p65/ReiA (Santa Cruz), CD 1 l c (Bolegend). Pam2CSK4 was purchased from InvivoGen to activate DC. Antibodies used for flow cytometry included APC-CD1 lc, FITC- CD40, FITC-CD80, PE-CD86, PE-IA-IE (MHC class II) (Pharmingen), as well as PB-CD8, A700-CD45.2, APC-CD44, PE-Cy7-CD25, APC-CD62L (BD Biosciences and eBiosciences). For intracellular labeling of cytokines, IL-12 (p40/p70)-PE and TNF-a PE monoclonal antibodies (Pharmingen) were used. The Aqua Dead Cell Stain (Invitrogen) was used to eliminate dead cells. Ovalbumine (OVA) was purchased from EndoGrade with purity>98% and endotoxin concentration <lEU/mg. SIINFEKL peptide was purchased from Schafer-N. Human mDC were sorted from PBMC of blood from healthy donors using lineage cocktail- FITC (BD Biosciences), CD123-PE (BD Biosciences), CDl lc-APC (Biolegend), HLA-DR- Quantum Red (Sigma). Human mDC were stained with CD86-PE, CD83-FITC, CD40-APC and HLA-DR-PB (eBiosciences or Biolegends). 7-AAD was used to exclude dead cells. For intracellular labelling IL13-APC, INF-y-PE-Cy7, IL-17-PE and Granzyme B-APC antibodies were used. Isotype matched controls were used appropriately. Alexa Fluor 647 conjugated phospho-specific antibodies were used for Phospho flow experiments on human IL-4 DC and were all from BD Biosciences. Akt(S478), Btk(Y557)/Itk(Y511), CREB(S 133)/ATF1(S63), ERK1/2(T202/Y204), IRF-7(S477/S479), Lck(Y505), NF-κΒ p65(S529), PLC-γΙ (Y783), PLC-y2 (Y759), p38 MAPK(T180/Y182), b-Catenin (S45), SHP-2(Y542), Src(Y418), SLP- 76(Y128), S6(S235/S236), STAT1(Y701), STAT1(S727), STAT3(Y705), STAT3(S727), STAT4(S693), STAT5(S694), STAT6(Y641), 4EBP1(T36/T45), Zap70(Y319)/Syk(Y352), JNK(T183/Y185). Mice and cells. C57B1/6 mice from Jackson Laboratory and OT-I, OT II TCR transgenic mice on C57B1/6 background were used. C57BL/6, Tlr4 ' and Tlr2 ' mice were maintained at the CIML animal house, France. Mouse bone marrow-derived DC (BMDC) and macrophages (BMDM) were prepared from 7-8 week-old female C57BL/6 mice as previously described (Lapaque et al, 2006).

Human DC. Human IL-4 monocyte-derived DC were generated from Ficoll-separated PBMC from healthy volunteers. Monocytes were enriched from the leukopheresis according to cellular density and size by elutriation as per manufacturer's recommendations. For DC generation, monocytes were resuspended in serum-free Cellgro DC culture supplemented with GM-CSF and IL-4. Blood myeloid DC (HLA-DR⁺CD1 lc⁺CD123Xin^~ ) were sorted from fresh PBMC using FACSAria (BD Biosciences). Na^'ive CD4⁺ and CD8⁺ T cells (CD45RA⁺CD45RO^~) (purity>99.2%) were purified by FACS-sorting.

Immunofluorescence microscopy. For immunofluorescence microscopy, stimulated BMDCs on coverslips were fixed in 3% paraformaldehyde at RT for 15 min, washed twice in PBS IX and processed for immunofluorescence labelling. To stain NF-κΒ, mouse BMDCs and BMDMs were permeabilized with PBS IX 1% saponin and then saturated with PBS IX 2% BSA. CD1 lc, NF-kB subunit p65/ReiA and MHC II were used as primary antibodies. After staining, samples were examined on a Zeiss LSM 510 laser scanning confocal microscope for image acquisition. Images were then assembled using Adobe Photoshop 7.0. Quantifications were done by counting at least 300 cells in 3 independent experiments. Flow cytometry. To analyse mouse BMDC maturation, cells were stimulated and stained with antibodies for classical activation markers. Appropriate isotype antibodies were used as controls. After staining, cells were washed with PBS 2% FCS, then PBS IX and fixed in 1.5% paraformaldehyde before analysis on a FACScalibur cytometer (Becton Dickinson). Cells were always gated on CDl lc for analysis and 100,000 CDl lc+ events were collected from each sample. For the intracellular staining of IL-12 and TNF-a in mouse BMDCs, BD Cytofix/Cytoperm and BD Perm/Wash buffers were used. At least 100.000 events were collected on FACSCanto II (BDBiosciences). For mouse CD4 and CD8 T cell assays, viable cells were analyzed for the decrease of CFSE (proliferation) and the expression of CD25, CD44 and CD62L (diluted in PBS IX EDTA 2mM). Human mDC activation was analyzed by checking the surface expression of maturation markers CD40, CD83, CD86, HLA-DR. Flow cytometry analysis was performed using the FlowJo software. Histograms were drawn from and median fluorescence intensity values were determined on gated populations. At least 100,000 events were collected on FACSCanto II (BDBiosciences) or FACSAria (BDBiosciences).

Cytokine measurement. Murine IL-12 and TNF-a were quantified in culture supernatants of stimulated DC by sandwich enzyme-linked immunosorbent assays (ELISA) according to the manufacturer's protocol (Abcys). Human cytokine (IL-6, TNF-a, and IL- 12p40) were determined using the BeadLyte cytokine assay kit (Upstate, MA).

Immunoblotting. 30 μg of cell lysates were subjected to SDS-PAGE PAGE and, after transfer to nitrocellulose, the membrane was probed with a polyclonal antibody against phospho-S6 or S6 (Cell Signaling Technology) or an anti-actin antibody. Blots were subjected to enhanced chemiluminescence detection (ECL, PIERCE).

Quantitative RT-PCR. Total RNA was isolated with Trisol reagent, was reverse transcribed and analyzed by real-time quantitative PCR using the Power SYBR Green PCR Master Mix (Applied Biosystems). All reactions were performed in triplets. Data were acquired on a 7500 Fast Real-Time PCR system (Applied Biosystems) and were normalized to the expression of actin mRNA transcripts in individual samples. For a given real-time qRT- PCR sample, the RNA expression level was calculated from cycle threshold (Ct). In our analysis, given gene expression is shown as mean normalized expression (MNE) relative to the expression of β-actin.

In vitro antigen presentation assays. BMDC (5000 cells) were incubated overnight in 96-well culture plates either with media or OVA. T cells obtained from the lymph nodes and the spleen of OT-I and OT-II Rag-2^_/~ mice were purified with the T cell enrichment kit from Dynal following manufacturer's instructions. For CD4 and CD8 T cell proliferation assays, purified T cells were labeled with 10 μΜ carboxyfluorescein diacetate succinimidyl ester (CFSE from Invitrogen) for 10 min at 37°C. OT-II and OT-I cells (20000 cells) were added to BMDC that had been stimulated for 8 h with different LPS and then washed. The proliferation of OT-I and OT-II T cells was assessed after 3 days of co-culture by flow cytometry. The cells were washed and stained with anti-CD4 and anti-CD8 antibodies for identification. For CD4 and CD8 T cell activation assays, purified T cells were co-cultured with BMDC previously stimulated for 8 h with different LPS. After 3 days, the expression of surface markers such as CD25, CD44 and CD62L was analyzed by flow cytometry to study the cellular activation level. Co-culture of OT-II T cells with BMDC. CD4⁺ T cells were isolated from the spleen of OT-II Rag-2^~ mice using a CD4⁺ T cell isolation kit (Dynal; Invitrogen). Purity was determined by staining with CD4, CD5, and TCR Va2. A total of 3 x 10³ BMDC stimulated for 8 h with different LPS were co-cultured with 2 x 10⁴ OT-II Rag-2^~ T cells in the presence of ovalbumin, ovalbumin (257-264) peptide (0.06 μg/mL) and of TGF-β (1 ng/mL) as indicated. After 5 days of culture, the expression of Foxp3 and CD25 was evaluated.

Human CD4+ and CD8+ T cell responses. 5xl0³ blood mDC were co-cultured with CFSE-labeled allogeneic na^'ive CD4+ T and CD8+ T cells (l-2xl0⁵). The DC/T ratio was 1 : 1000 and 1 :20, respectively. Cell proliferation was tested by measuring CFSE-dilution on day 6. On day 7, the production of intracellular cytokines (INF-γ, IL-17, IL-13) and Granzyme B were analyzed after 6 h of T cell stimulation by PMA and Ionomycine, in the presence of Brefeldin A. Cells were stained for analysis by flow cytometry using different fluoro chrome-conjugated antibo dies . Phospho-flow analysis with fluorescent cell barcoding (FCB). Monocyte-derived

IL4 DC were generated as previously described. Briefly, human monocyte were enriched with human monocyte enrichment kit without CD 16 depletion (Stemcell Technologies, Canada) and suspended in CellGro DC medium (CellGenix, Germany) with GM-CSF and IL-4. On day 6, cells were washed and resuspended at 1 million/mL in RPMI supplemented with 2 mM L-Glutamine, 1 mM Sodium pyruvate, IX non essential amino acid, 50 μΜ b-ME, and 10 mM HEPES +10% FBS, and then cultured for 2 h in a C02 incubator. Cells were stimulated with different LPS (100 ng/ml) for 2, 5, 10, 30, 60, and 180 min. Equal amount of medium was used for stimulation control. All samples were immediately fixed by adding PFA (final 1.6%) for 10 min at RT. Fixed cells were centrifuged and washed once with PBS, and then permeabilized with ice-cold Methanol (500 μΐ/ΐ million cells) for 10 min at 4°C. Two dimension FCB was performed according to the previous report [11]. Pacific Blue-NHS and Alexa Fluor 488-NHS (Invitrogen, Carlsbad, CA) were added to each condition of cells at 0.02, 0.08, 0.32, 1.0, 3.0 μg/ml or 0.05, 0.2, 0.8, 3.0 μg/ml, respectively. Each sample has a unique combination of dyes with different concentrations. After 30 min on ice, barcoded cells were washed three times with PBS+0.5% BSA and combined into one tube. Combined barcorded cells were stained with Alexa Fluor 647 conjugated phospho-specific antibodies for 30 min at RT. Cells were washed two times with PBS+0.5% BSA. For purified anti-phospho- JNK antibody, cells were stained with secondary anti-rabbit DyLight 649 (Jackson Immunoresearch, West Grove, PA) for 30 min at RT and washed two times. Samples were immediately analyzed with FACS CantoII (BD Biosciences, San Jose, CA). Fold changes of phosphorylation were visualized as a Heatmap. The MFI of LPS-stimulated samples were normalized with medium- stimulated samples. Statistical analysis. All experiments were carried out at least 3 independent times and all the results correspond to the means ± standard errors. Statistical analysis was done using two-tailed unpaired Student's t test. Significance was defined when P values were <0.05.

Results:

Structural modifications of LPS affect cytokine secretion by DC

We used an array of LPS (Table I) differing in lipid A acylation to study their activation properties in mouse bone marrow-derived dendritic cells (BMDC) and bone marrow-derived macrophages (BMDM). In addition to the classical wild type hexa-acyl LPS purified from E. coli MLK strain, we used LPS from E. coli MLK mutants {msbB-, htrB- and msbB-lhtrB- double mutant) that produce mostly penta-acyl and tetra-acyl lipid A (Table I) or LPS purified from Y. pestis KIM grown at 37°C (mainly composed of tri- and tetra-acyl lipid A with small amounts of penta-acyl and hexa-acyl molecules, Table I). All LPS variants induced a BMDC maturation characterized by an up-regulation of the surface expression of major histocompatibility complex MHC-II and co-stimulatory molecules (CD40, CD86) However, significant lower levels of secreted TNF-a and IL-12 were detected in DC stimulated by tetra-acyl LPS purified from E. coli MLK {msbB-lhtrB-) double mutants or LPS purified from Y. pestis compared to DC stimulated with wild type E. coli hexa-acyl LPS. Moreover, the LPS variants did not induce any IFN-a secretion not shown). Similarly, in BMDM activated by tetra-acyl LPS, TNF-a secretion was strongly decreased compared to BMDM incubated with hexa-acyl LPS as previously observed in macrophage cell lines [8,9,10].

We then tested the ability of tetra-acyl LPS (referred as purified either from E. coli MLK msbB-lhtrB- double mutant or Y. pestis grown at 37°C) to induce human blood myeloid DC (mDC) activation. Hexa-acyl and tetra-acyl LPS induced a similar up-regulation of classical cell surface activation markers (HLA-DR, CD40, CD86, and CD83). However, mDC treated with tetra-acyl LPS secreted lower levels of IL-12, IL-6 and TNF-a than those stimulated by hexa-acyl LPS. Tetra-acyl LPS from Y. pestis, which contains small amounts of hexa-acyl LPS had a stronger capacity to trigger IL-12, IL-6 and TNF-a secretion (p<0.01) than LPS purified from E. coli (msbB-, htrB-) double mutant (devoid of hexa-acyl LPS) (Table I). Together, our data show that structural modifications of LPS induce an intermediate phenotype of maturation in mouse and human DC characterized by high levels of MHC-II and co-stimulatory molecule expression, but low levels of pro -inflammatory cytokine secretion.

Tetra-acyl LPS induce a TLR4-dependent DC activation

LPS recognition by host cells is mediated through the To 11- like receptor 4 (TLR4/MD2/CD14) receptor complex [12]. To determine the contribution of TLR4 in the cell activation induced by LPS with acylation defects, BMDC derived from Tlr4 , Tlr2^~ and wild type mice were treated with the LPS variants. No activation was observed in Tlr4 mice-derived BMDC stimulated either by hexa-acyl or tetra-acyl LPS (p<0.001), as measured by the secretion of TNF-a. In addition, TLR2 was not implicated in DC activation induced by the different LPS, showing that LPS preparations were not contaminated by lipoproteins.

The measurement of DC viability following treatment with different LPS showed that both hexa-acyl and tetra-acyl LPS induce a very low percentage of dead cells (0.93%). We next tried to understand if the decrease of pro -inflammatory cytokine secretion in BMDC activated by tetra-acyl LPS was related to a defect in signal transduction. It has been shown that NF-KB translocation is a key event in LPS-induced TLR4 signalling [13]. Under unstimulated conditions, NF-κΒ is kept in the cytosol as an inactive form. Under hexa-acyl LPS stimulation NF-κΒ is translocated into the nucleus where it can bind to several gene promoters [13,14]. After 15 and 30 min of cell stimulation, tetra-acyl LPS induced a significant (p<0.01) stronger NF-κΒ translocation than hexa-acyl LPS. Similar results were observed in macrophages.

Since the activation of the mammalian target of rapamycin (mTOR) pathway has been implicated in DC maturation [16], we then analyzed the phosphorylation of the ribosomal protein S6, one of downstream elements of the TLR4 pathway. Compared to hexa-acyl LPS, tetra-acyl LPS induced a stronger S6 phosphorylation at 30 min post-cell activation No difference for S6 phosphorylation was observed at later time points either by hexa-acyl or tetra-acyl LPS. These data show for the first time that LPS with acylation defects induce an early and strong activation of the TLR4-dependent signalling pathway in mouse DC and macrophages.

We extended this study to human monocyte-derived IL-4 DC by using the phospho- flow technology. Fluorescent cell barcoding (FCB) was applied to analyze many conditions simultaneously, using a collection of several anti-phosphorylated proteins [11]. All LPS variants LPS were equally able to increase the phosphorylation levels of several signaling molecules including MAPKs (ERK, p38, JNK), Akt-mTOR pathway molecules (Akt, 4EBP1, S6), and some transcription factors (CREB, NFkB p65). Thus, LPS purified from E. coli MLK (msbB-, htrB-) double mutant and Y. pestis were able to trigger TLR4-dependent signalling in human DC, in agreement with data obtained on mouse BMDC.

Altogether these data show that LPS with acylation defects act as agonists to the TLR4 pathway and efficiently induce signal transduction in mouse and human DC.

Tetra-acyl LPS induce an early synthesis of pro-inflammatory cytokines followed by their proteasome-dependent degradation

We then investigated whether the decrease of pro-inflammatory cytokine secretion in BMDC activated by tetra-acyl LPS was due to a defect in cytokine synthesis (transcription/translation). BMDC were activated with different LPS and quantitative RT- PCR used to analyse gene expression. In BMDC treated by tetra-acyl LPS an earlier and stronger transcription of tnf-a, p35 and p40 genes was observed compared to BMDC treated by hexa-acyl LPS. Therefore, the decrease of pro-inflammatory cytokine secretion observed in Figure 4B cannot be attributed to transcriptional defects.

We next investigated whether the defect in cytokine secretion by DC stimulated with tetra-acyl LPS was due to a change in protein translation. BMDC were incubated with the different LPS in the presence of brefeldin A to block the secretion of newly synthesized cytokines. Intracellular levels of IL-12 and TNF-a were analysed by flow cytometry. LPS with acylation defects induced significant higher TNF-a and IL-12 synthesis at 2 h and 4 h post-stimulation compared to hexa-acyl LPS. However, at 8 h post-stimulation, the level of intracellular cytokines was lower in DC treated with tetra-acyl LPS than in DC treated by hexa-acyl LPS. It has been shown that glucose or energy deprivation, calcium homeostasis perturbation or elevated synthesis of secretory proteins induce an alteration of the Endoplasmic Reticulum (ER) homeostasis [15,16]. This leads to the disruption of protein folding, the accumulation of unfolded proteins and ER stress response or unfolded protein response (UPR) to restore ER normal function. One of the major components of UPR is the degradation of misfolded proteins by the proteasome (ER associated degradation, ERAD) [15,16]. We therefore determined if the decrease of cytokine secretion observed in DC activated by tetra-acyl LPS could be due to a proteasome-mediated degradation of newly synthesized cytokines. Epoxomycine or Mgl32 proteasome inhibitors were used in BMDC treated by the different LPS for 8 h and intracellular the IL-12 expression was analysed. As expected, in the absence of proteasome inhibitors the level of intracellular IL-12 expression was lower in tetra-acyl LPS-treated DC than in hexa-acyl LPS-treated DC. However, in the presence of proteasome inhibitors DC treated with tetra-acyl LPS levels of intracellular IL-12 were similar to those expressed by DC treated with hexa-acyl LPS. We then studied the ubiquitinylation of proteins following DC activation by different LPS. It has been shown that upon inflammatory stimulation, DC accumulate newly synthesized ubiquitinylated proteins in large cytosolic structures. These DC aggresome-like induced structures (DALIS) are transient and require continuous protein synthesis [16]. Mouse DC treated with LPS variants underwent maturation and displayed MHC II surface localization as well as DALIS formation. However, after 4h of tetra-acyl LPS treatment, the percentage of DALIS- containing cells was significantly higher as compared to cell stimulated by hexa-acyl LPS. At 24 h, the number of DALIS decreased, consistent with the transient DALIS expression previously demonstrated in the process of DC maturation (not shown) [16]. These data strongly suggest that tetra-acyl LPS induce a degradation of IL-12 by the proteasome machinery in DC. It is therefore tempting to hypothesize that LPS with acylation defects could induce an ER stress in DC activating the proteasome machinery. This will lead to the down-regulation of cytokine intracellular levels and consequently to a decrease of their secretion. LPS with acylation defects induce antigen-specific CD8 an d CD4⁺ T cell responses

We next studied the antigen presentation capacity of tetra-acyl LPS-treated DC and their ability to promote T cell responses. We used transgenic mice that express either a TCR specific for the MHC class-I restricted OVA (OT-I Rag-2^~ ) or a TCR specific for the MHC class-II restricted OVA (OT-II Rag-2^~ ). BMDC incubated in either medium alone or medium containing ovalbumin (OVA) were activated by different LPS and co-cultured with OTI (CD8⁺) and OTII (CD4⁺) T cells for 3 days. Basal level of T cell responses was determined. BMDC incubated with LPS alone or OVA alone could not induce any T cell response (data not shown). However, BMDC incubated with OVA and activated by different LPS efficiently induced antigen-specific CD8 and CD4 T cell responses. DC activated by tetra-acyl LPS induced a higher OTI and OTII T cell proliferation than cells treated by hexa- acyl LPS. DC stimulated by tetra-acyl and hexa-acyl LPS were able to trigger T cell activation characterized by a CD25 up-regulation and a CD62L down-regulation. However hexa-acyl LPS-treated BMDC led to a higher down-regulation of CD62L by OT II T cells than those treated with tetra-acyl LPS. Altogether, these data show that BMDC induced by LPS with acylation defects are able to efficiently promote antigen presentation and induce CD8⁺ and CD4⁺ T cell responses.

We then investigated the functional properties of human DC stimulated with LPS variants. Human blood myeloid DC (mDC) activated by the different LPS were able to induce the proliferation of allogeneic naive CD4⁺ and CD8⁺ T cells, although to a lower level for E. coli tetra-acyl LPS compared to other LPS. Tetra-acyl LPS from Y. pestis, which contains small amounts of hexa-acyl LPS had a stronger capacity to trigger T cell responses than LPS purified from E. coli (msbB-, htrB-) double mutant (devoid of hexa-acyl LPS) (Table I). These results show that tetra-acyl LPS-treated DC are able to promote CD4⁺ and CD8⁺ T cell responses both in mouse and human models.

We then characterized the effector T cells induced by LPS-treated mDC. Cells were stimulated with PMA/Ionomycin and stained for intracellular IFN-γ (T_RI response), IL-13 (T_H2 response) and IL-17 (T_Hi7 response). mDC stimulated either by hexa- or tetra-acyl LPS polarized allogeneic naive CD4⁺ T cells into IFN-y-expressing T_RI cells. CD4⁺ T cells co- cultured with either hexa-acyl LPS-activated mDC or tetra-acyl-activated mDC did not express IL-13 or IL-17. mDC stimulated by tetra-acyl LPS were also able to induce IFN-γ and Granzyme B synthesis in CD8⁺ T cells. However, we observed lower levels of IFN-γ and Granzyme B production with LPS purified from E. coli MLK {msbB-, htrB-) double mutant compared to other LPS .

These data indicate that DC activated by either hexa-acyl or tetra-acyl LPS induce T_RI responses and activate CD8⁺ T cells.

In contrast to murine BMDC, tetra-acyl LPS activate human mDC to induce T_reg cells

DC with MHC II^high, co-stimulation^high, pro -inflammatory cytokines ^low phenotype are referred in the literature as semi-mature. It has been shown that these cells are able to trigger the differentiation of regulatory T cells (T_reg) [17]. We thus evaluated whether mouse BMDC activated by tetra-acyl LPS displaying a semi-mature phenotype were capable of generating T_reg cells characterized by the expression of the transcriptional factor Foxp3 and a high CD25 expression at their cell surface. When maintained on a Rag-2^~ background, transgenic mice that express a TCR specific for I-A^b-OVA complexes (OT-II Rag-2^~ mice) contain only conventional (Foxp3 ) CD4⁺ T cells in their periphery, a situation that facilitates the measurement of their conversion into T_reg cells [18]. Such conversion requires I-A^b+ DC and the presence of the OVA-derived peptide specifically recognized by OT-II CD4⁺ T cells. It also depends on the secretion by the antigen-presenting DC of TGF-β [18]. Accordingly, BMDC stimulated with different LPS variants were incubated with OT-II Rag-2^~ T cells in the presence of the OVA or OVA₂₅₇-264 peptide (0.06 μg/mL), with or without TGF-β. We could observe that OVA and peptide-pulsed BMDC were both capable of inducing the activation of OT-II Rag-2^~ CD4⁺ T cells as measured by CD25 expression. However, DC stimulation either by tetra-acyl or hexa-acyl LPS did not trigger T_reg responses in mouse BMDC. The addition of exogenous TGF-β to the culture did not confer to LPS-activated DC the ability to generate T_reg cells.

We then studied the capacity of human mDC activated by tetra-acyl LPS to induce T_reg cells. Human DC activated by LPS variants were co-cultured with allogeneic naive CD4⁺ T cells and T_reg population was analysed by flow cytometry (Figure 1). We could observe that mDC activated by tetra-acyl LPS induced a higher T_reg population characterized by the expression of Foxp3 and a high CD25 expression at the cell surface (Figure 1).

Discussion:

The innate immune system possesses various mechanisms to detect and facilitate host responses to microbial components such as LPS [19]. It has been described that each change in chemical composition of LPS causes a dramatic decrease of its activity down to a complete loss of endotoxicity [6]. Different cell types, mainly human and mouse monocytes/macrophages have been used to study LPS structural requirements for its immuno stimulatory properties. However, to determine the endotoxic activity of enterobacterial LPS, previous studies have mainly concentrated on cytokine production. Consequently, a decrease in IL-8, IL-6 and TNF-a secretion by cells stimulated with LPS harboring acylation defects has been considered as a lack of immuno genicity or a defect of pro-inflammatory signaling [9,10,20]. In contrast, we show here that LPS with acylation defects efficiently induce a potent activation of TLR4-dependent signaling in mouse and human DC that leads to a strong cytokine synthesis, which in turn triggers the activation of the proteasome machinery. The consequence is the degradation of intracellular proinflammatory cytokines and consequently the decrease of their secretion. This hypothesis corroborates previous results, which showed a decrease of cytokine secretion in tetra-acyl LPS-treated macrophages [8,9,10,20].

The difference in the activation potential of LPS variants in terms of cytokine secretion could affect the output of the DC immune response. DC activated by tetra-acyl LPS triggered CD4⁺ T and CD8⁺ T cell responses both in mouse and human DC. However, human DC activated by LPS with acylation defects displayed a semi-mature phenotype and induced T_reg responses. There could be several mechanisms by which tetra-acyl LPS interact with human DC to elicit distinct types of T_H responses. Functional differences between the different subsets of human myeloid DC could be one possible explanation. Two main populations of circulating DC termed myeloid (mDC) and plasmacytoid (pDC) were identified in the blood of healthy donors. Additional distinctions can be made within the mDC subset with CDlc⁺CD141^" mDCl, CDlc^"CD141⁺ mDC2 and CD16⁺ mDC [21]. It has been shown that mDC l and mDC2 differ for the expression of surface markers, cytokine production profile and the differentiation of T_H responses. When co-cultured with purified human peripheral blood cells, mDC l produce IL-12 and favor T_RI differentiation, while mDC2 produce high levels of IL-10 and direct the differentiation of T_R2- Moreover, the identification of numerous phenotypic and functional differences among pulmonary mDCl and mDC2 suggests a possible preferential role for mDC2 in regulating immunity and disease pathogenesis in the respiratory tract distinct from that of mDCl . Distinct roles in host immunity for each human DC were previously shown [21,22,23,24]. For instance, the human CDlc^"CD141⁺ mDC2 subset is the functional equivalent of mouse CD8a⁺ DC, capable of cross presentation of exogenous antigens. Regarding their capacity to secrete IL-10, mDC2 might also induce T_reg populations.

T_reg are key players in the immune regulation, particularly in tolerance. This cell population plays a crucial role in suppressing immune responses to self-antigens and in preventing autoimmune diseases [25,26]. Evidence is emerging that T_reg can control immune responses to pathogens. They are beneficial to the host through limiting the immunopathology associated with anti-pathogen immune responses and enabling the development of immune memory. However, pathogens can exploit T_reg to subvert the protective immune responses of the host in order to survive and establish a chronic infection [27,28]. Microbes have evolved strategies for programming DC to induce T_reg in order to maintain immune homeostasis that controls unbridled host immunity [4,27]. For example, filamentous hemagglutinin (FHA) from the bacteria Bordetella pertusis induces DC to provide IL-10 and prime T_reg. Moreover, Yersinia pestis is known to activate DC by means of the dimer of TLR2 and TLR6 to induce

There is growing evidence that the induction of tolerance is not restricted to immature DC. Within the tolerogenic pool of DC, a third population is proposed, called semi-mature [17]. This new subset or developmental stage of DC is distinguished as mature by their surface marker analysis (MHC II^hlgh and co-stimulation ^hlgh). However, semi-mature DC do not release high level of pro -inflammatory cytokines, such as IL-Ιβ, IL-6, TNF-a or IL-12p40 or IL-12p70. IL-10 production by semi-mature DC has been described, but it is not an absolute requirement for T_reg differentiation [17]. Inducers of DC semi-maturation can be lactobacilli from the gut flora [30], intranasally applied OVA [31], apoptotic cells [32], Bordetella pertussis FHA [33] or TNF-a [34]. Here we show that, structural modifications of LPS are able to induce semi-mature human and mouse DC characterized by MHC-II^hlgh, co- stimulation¹¹¹⁸¹¹, pro -inflammatory cytokines ^low phenotype. In the human model, these semi- mature DC induce high levels of T_reg cells.

In conclusion, we describe a new mechanism which regulates the pro -inflammatory cytokine decrease in cells activated by LPS with acylation defects. We propose that cell stimulation by tetra-acyl LPS trigger the activation of the proteasome machinery. This leads to the degradation of intracellular pro-inflammatory cytokine levels and consequently to a decrease of their secretion. Our results provide new insights into the understanding of early steps of endotoxin action and suggest that structural modifications of LPS could represent an important strategy for pathogens to subvert adaptive immunity by T_reg cell induction in order to survive.

Bacterial strain (relevant genetic Proportions of lipid A species features) ^a (molecular mass)

E.coli MLK3 >90% hexaacyl (1823.3 Da);

traces of penta and tetraacyl.

E.coli MLK53 (htrB-) rough-LPS; pentaacyl lipid A

deficient in C12 oxyacyl of 3- OH-C14 acyl at GlcN C2' (1615.1

Da) E.coli MLK 1067 (msbB-) rough-LPS; > 90% pentaacyl

(1587.0 Da); tetraacyl traces

E.coli MLK986 (msbB-, htrB-) rough-LPS; 29% pentaacyl

(1643.0 Da); 54% tetraacyl (1404.8 Da); and 17% triacyl (1178.6 Da)

Y. pestis KIM rough-LPS, 9% hexaacyl (1797.2

Da); 10% pentaacyl; 40% tetraacyl (1404.8 Da); 7% arabinosamine- tetraacyl (1535.9

Da); 30% triacyl (1178.6 Da)

a All are rough-type LPSs

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method for obtaining a population of regulatory T cells, said method comprising the steps of (i) coculturing a population of dendritic cells with a population of na^'ive CD4+ T cells in a culture medium having an amount of at least one tetraacylated lipopolysaccharide (LPS), and (ii) isolating the population of regulatory T cells as obtained in step i).

2. A culture medium having an amount of at least one tetraacylated lipopolysaccharide (LPS).

3. A tetraacylated lipopolysaccharide (LPS) according to the present invention for use in a method for inducing in a subject in need thereof high level of T regulatory cells