Regulatory t cells targeted by lymphotoxin alpha blocking agent and uses thereof

Abstract

To provide regulatory T cells and uses thereof.SOLUTION: Provided is a regulatory T cell which has been previously incubated with an effective amount of lymphotoxin alpha blocking agent. Also provided is a method of treating or preventing autoimmune disorders and inflammatory-associated cancers in a subject in need thereof, comprising the step of administering, to the subject, previously incubated regulatory T cells, together with a therapeutically effective amount of soluble lymphotoxin-β receptor.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method of treating or preventing autoimmune disorders and inflammation-associated cancers in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of regulatory T cells, wherein the population of regulatory T cells has been pre-incubated with an effective amount of a lymphotoxin alpha blocker.

[0002] 2. Background of the Invention CD4+CD25+Foxp3+ regulatory T cells (Tregs) constitute a subset of CD4+ T cells that play a key role in maintaining peripheral self-tolerance (1). This cell type has the unique ability to immunosuppress harmful autoreactive T cells that escape thymic negative selection, thereby preventing the development of inflammatory and autoimmune disorders. Foxp3+ Treg cells originate both from the thymus and from conversion of naive CD4+ T cells in the periphery, and are referred to as natural and inducible Tregs, respectively (2). During ontogeny, the development of natural Tregs is substantially delayed compared to that of conventional CD4+ T cells, as the first wave of Tregs arise during the perinatal period, whereas conventional CD4+ T cells appear earlier during the embryonic stage (3).

[0003] The importance of Foxp3+ Treg cells in controlling and maintaining the human immune system was demonstrated in scurvy mice that display a mutation in the Foxp3 gene resulting in a truncated, non-functional Foxp3 protein (4). These mice die at an early age because they are unable to generate thymus-derived Foxp3+ Tregs and therefore develop a fatal lymphoproliferative syndrome with multiorgan inflammation. Foxp3 was subsequently identified as a master regulator of Treg development, function, and homeostasis. Genetic mutations in the Foxp3 gene have also been identified in humans and are responsible for a severe autoimmune disease called immunoregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome (5). Foxp3+ Tregs suppress immune responses using several mechanisms (Workman et al., 2009). Four main mechanisms of action have been described: immunosuppressive cytokines (IL-10, TGF-β and IL-35), cytolysis of effector T cells and dendritic cells (granzymes B and A in mice and humans, respectively), metabolic disruption (CD39, CD73 and CD25) and modulation of antigen presentation in dendritic cells (CTLA-4 and LAG-3).

[0004] In mice, adoptive transfer of WT Tregs in inflammatory bowel disease (IBD) has been shown to prevent and cure established intestinal inflammation (6), type I diabetes (7), experimental autoimmune encephalomyelitis (EAE) (8), and asthma (9). Furthermore, Treg cells often promote tumorigenesis by attenuating tumor immune surveillance, but in contrast, they play an antitumor role in chronic inflammation-mediated cancers by suppressing inflammation, such as colitis-associated cancer (CAC) (10). In humans, Treg-based cell therapy is becoming a reality (11). For example, phase I clinical trials using human Tregs have been reported in patients suffering from type I diabetes (12), refractory Crohn's disease (13), or acute graft-versus-host disease (GVHD) during stem cell transplantation (14). However, there is still a need to develop novel therapies for autoimmune and inflammatory diseases.

[0005] Furthermore, the Treg adoptive transfer technique has a major limiting step: large numbers of cells are required for effective treatment in humans. Thus, there remains a need in the field of Treg cell therapy to reduce the number of cells required to effectively treat inflammatory and autoimmune disorders.

[0006] Summary of the Invention Foxp3 + CD4 + Regulatory T cells (Tregs) play a central role in peripheral tolerance due to their immunosuppressive and anti-inflammatory activities, thus crucially preventing the development of autoimmune and inflammatory disorders. + CD4 + Tregs express conventional CD4 as membrane-anchored LTα1β2 heterocomplexes. + It has been demonstrated that T cells express higher levels of lymphotoxin α (LTα). - / - Thymus and spleen Foxp3 from mice + CD4 + Treg (LTα - / - Tregs (Tregs) display a highly suppressive cell signature, suggesting that LTα negatively regulates the immunosuppressive function of this cell type. Interestingly, by limiting intestinal inflammation, LTα - / -Adoptive transfer of Tregs (AT) protects against dextran sulfate sodium (DSS)-induced colitis, cures inflammatory bowel disease (IBD), and attenuates the development of colitis-associated cancer (CAC). Importantly, by using mixed bone marrow chimeras, we found that specific LTα expression in hematopoietic cells negatively regulates the suppressive signature of Tregs. We identified that LTα negatively regulates the immunosuppressive properties of Tregs, and thus may be a valuable novel target in therapy to improve the suppressive activity of Tregs. We also found that LTα1β2 / LTβR interactions between Tregs and antigen-presenting cells (i.e., dendritic cells and thymic epithelial cells), respectively, regulate the immunosuppressive signature of Tregs. Finally, we found that AT of regulatory T cells pre-incubated with soluble lymphotoxin β receptor (LTβR) attenuates DSS-induced colitis. By incubating Tregs with soluble lymphotoxin β receptors, the number of cells injected in adoptive transfer can be reduced and a transfection or transduction step can be avoided, which represents a technical ease.

[0007] Detailed Description of the Invention A first aspect of the present invention relates to regulatory T cells pre-incubated with an effective amount of a lymphotoxin alpha blocking agent, which allows to enhance the suppressive activity of the regulatory T cells.

[0008] As used herein, the term "lymphotoxin alpha blocker" refers to an agent capable of blocking the binding of LTα ligands to LTβR, e.g., blocking the LTα1β2 / LTBR (predominant form) or LTα2β1 / LTβR interaction.

[0009] As used herein, the term "lymphotoxin alpha" or "LTα" (also known as tumor necrosis factor beta (TNF-β)) refers to a member of the tumor necrosis factor family. Lymphotoxin alpha is a cytokine secreted by lymphocytes. Lymphotoxin alpha (Uniprot reference: P01374 (Homo sapiens), P09225 (Mus musculus)) is encoded by the lymphotoxin alpha (LTA) gene (NCBI reference: Gene ID: 4049 (Homo sapiens), Gene ID: 16992 (Mus musculus)). The interaction of LTα with LTβ leads to the formation of a membrane-bound complex, LTα1β2 (predominant) or LTα2β1 / LTβR, which can interact with LTβR. The inventors show that blocking the LTα1β2 / LTβR interaction makes it possible to increase the suppressive activity of regulatory T cells.

[0010] In some embodiments, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0011] Thus, the present invention refers to regulatory T cells that have been pre-incubated with an effective amount of soluble lymphotoxin β receptor (LTβR).

[0012] As used herein, the term "lymphotoxin beta receptor (LTβR)" has its general meaning in the art and refers to a member of the tumor necrosis factor receptor (TNFR) family. LTβR (its Uniprot references: P36941 (Homo sapiens), P50284 (Mus musculus)) is a cell surface receptor for lymphotoxin that binds to lymphotoxin membrane-type LTα1β2 or LTα2β1 (a complex of lymphotoxin alpha and lymphotoxin beta) and is involved in apoptosis and cytokine release.

[0013] As defined herein, a "soluble LTβR" is a polypeptide that contains a lymphotoxin (LT)-binding fragment of the extracellular region of LTβR. For example, a soluble LTβR can contain all or a fragment of the extracellular domain of human LTβR. The extracellular domain of human LTβR consists of the amino acid sequence shown as SEQ ID NO:1.

[0014] [ka]

[0015] Soluble forms of lymphotoxin β receptors are known in the art and are disclosed in WO 97 / 03687. In some embodiments, the LTβR polypeptide is a full-length immature LTβR polypeptide from any species, such as any mammal, such as mouse, rat, or monkey. In preferred embodiments, the LTβR polypeptide is human.

[0016] In some embodiments, the soluble LTβR comprises the amino acid sequence of SEQ ID NO:1.

[0017] As used herein, "polypeptide" and "protein" are used interchangeably and refer to any peptide linked chain of amino acids, regardless of length or post-translational modification. The LTβR, heterologous polypeptides or fusion proteins thereof used in any of the methods of the invention can contain or be human proteins or can be variants having up to 50 conservative amino acid substitutions.

[0018] As used herein, a "polypeptide fragment" refers to a segment of a polypeptide that is shorter than the full-length immature polypeptide. A "functional fragment" of a polypeptide has at least 10% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or 100%) of the activity of the mature polypeptide. Fragments of a polypeptide include terminal and internal deletion mutants of the polypeptide.

[0019] In some embodiments, the LTβR portion itself is soluble. In some embodiments, the LTβR is linked to a heterologous moiety that enhances its solubility, such as the Fc region of an immunoglobulin molecule. In some embodiments, the heterologous moiety can be covalently linked to the LTβR portion.

[0020] In some embodiments, soluble LTβR can be modified by the covalent attachment of a second polypeptide moiety, eg, a heterologous polypeptide (eg, to form a LTβR fusion protein) or a non-polypeptide moiety.

[0021] In some cases, such moieties can improve pharmacodynamic or pharmacokinetic parameters, such as solubility or half-life. LTβR fusion proteins can include all or part of an antibody constant region (e.g., Fc domain), transferrin, or albumin, such as human serum albumin (HSA) or bovine serum albumin (BSA). Fusion proteins can include a linker region between the LTβR sequence and the non-LTβR protein domain. In some embodiments, the soluble LTβR is modified by covalent attachment to a polymer, such as polyethylene glycol (PEG). Without being limited by any particular theory or mechanism, such soluble LTβR can act as a decoy receptor to reduce (block or neutralize) LTβR activity.

[0022] In some embodiments, the LTβR fusion protein has the LTβR extracellular ligand binding domain fused to an immunoglobulin constant heavy chain domain. More preferably, the LTβR ligand binding domain is fused to a human IgG Fc domain.

[0023] The term "Fc domain" of an antibody refers to the portion of the molecule comprising the hinge, CH2 and CH3 domains, but lacking the antigen binding site. The term is also meant to include equivalent regions of IgM or other antibody isotypes.

[0024] In some embodiments, the soluble LTβR is vaminercept. Vaminercept is a lymphotoxin beta receptor antagonist developed by Biogen Idec. Its CAS number is 909110-25-4 and its amino acid sequence consists of SEQ ID NO:2.

[0025] In some embodiments, the soluble LTβR protein comprises the amino acid sequence of SEQ ID NO:2.

[0026] [ka]

[0027] In some embodiments, the lymphotoxin alpha blocking agent is an anti-LTα blocking antibody.

[0028] As used herein, "antibody" includes both natural and non-natural antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, fully synthetic antibodies, single chain antibodies, and fragments thereof. Antibodies can be human or non-human antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0029] As used herein, "anti-LTα blocking antibody" refers to an anti-LTα antibody that prevents other antibodies or receptors from binding to LTα. Anti-LTα blocking antibodies interfere with the LTα1β2 / LTβR or LTα2β1 / LTβR interaction.

[0030] Examples of anti-LTα blocking antibodies are: [Table 1] Includes.

[0031] Antibodies are prepared according to conventional methods. Monoclonal antibodies can be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the present invention, mice or other suitable host animals are immunized with the antigen form of LTα at appropriate intervals (e.g., twice weekly, once weekly, twice monthly, or once monthly). The animals can be administered a final "booster" of antigen within one week of sacrifice. It is often desirable to use an immunological adjuvant during immunization. Suitable immunological adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in the art. Animals may be immunized subcutaneously, intraperitoneally, intramuscularly, intravenously, intranasally or by other routes. A given animal may be immunized with multiple forms of antigen by multiple routes.

[0032] Briefly, the antigen can be provided as a synthetic peptide corresponding to the antigenic region of interest in LTα. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph nodes or other organs of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form hybridomas. After fusion, the cells are placed in a medium that allows the growth of the hybridoma but not the fusion partner, using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). After culturing the hybridomas, the cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., antibodies that selectively bind to the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation and Western blotting. Other screening techniques are well known in the art. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigens, such as non-denaturing ELISA, flow cytometry and immunoprecipitation.

[0033] Importantly, as is well known in the art, the paratope, a small portion of an antibody molecule, is responsible for binding the antibody to its epitope (see generally Clark, WR (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). For example, the Fc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved or which has been produced without the pFc' region (called an F(ab')2 fragment) retains both antigen-binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved or which has been produced without the Fc region (called an Fab fragment) retains one antigen-binding site of the intact antibody molecule. Going further, an Fab fragment consists of a covalently bound antibody light chain and a portion of the antibody heavy chain, called Fd. The Fd fragment is the primary determinant of antibody specificity (one Fd fragment can be associated with up to 10 different light chains without altering antibody specificity) and retains epitope binding ability in isolation.

[0034] As is well known in the art, within the antigen-binding portion of an antibody there are complementarity determining regions (CDRs) that directly interact with the epitope of the antigen and framework regions (FRs) that maintain the tertiary structure of the paratope (see generally Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of an IgG immunoglobulin, there are four framework regions (FR1-FR4) separated by three complementarity determining regions (CDR1-CDRS). The CDRs, especially the m, and the CDRS regions, especially the heavy chain CDRS, are primarily responsible for antibody specificity.

[0035] It is now well established in the art that the non-CDR regions of a mammalian antibody can be replaced by analogous regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies, in which non-human CDRs are covalently linked to human FR and / or Fc / pFc' regions to produce a functional antibody.

[0036] The present invention provides compositions and methods that, in certain embodiments, include humanized forms of antibodies. As used herein, "humanized" describes an antibody in which some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in US 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are incorporated herein by reference. US 5,585,089 and 5,693,761 and WO 90 / 07861 also propose four possible criteria that can be used to design humanized antibodies. The first proposal was to use for the acceptor a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin being humanized or to use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of a human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor amino acid can be selected. The third proposal was that the donor amino acid rather than the acceptor amino acid can be selected at positions directly adjacent to the three CDRs in the humanized immunoglobulin chain. The fourth proposal was to use a donor amino acid that is present in a framework position where the amino acid is predicted to have a side chain atom within the 3A of the CDR in the three-dimensional model of the antibody and is predicted to be able to interact with the CDR. The above methods are only some examples of the methods that a person skilled in the art could utilize to prepare a humanized antibody. A person skilled in the art will be familiar with other methods for antibody humanization.

[0037] In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR regions are replaced with amino acids from a human immunoglobulin molecule, while some, most or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permitted as long as they do not abolish the ability of the antibody to bind to a given antigen. Suitable human immunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains the same antigen specificity as the original antibody. However, certain methods of humanization can be used to improve the binding affinity and / or specificity of the antibody using the method of "directed evolution", as described in Wu et al., / . Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

[0038] Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., US Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584 and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deficiency in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or part of the human germline immunoglobulin gene loci such that immunization of these animals will result in the production of fully human antibodies against the antigen of interest. After immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and therefore will not elicit a human anti-mouse antibody (KAMA) response when administered to humans.

[0039] In vitro methods for producing human antibodies also exist. These include phage display technology (US Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (US Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0040] Thus, as will be apparent to the skilled artisan, the present invention also provides F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions are replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.

[0041] The various antibody molecules and fragments can be derived from any of the commonly known immunoglobulin classes, including, but not limited to, IgA, secretory IgA, IgE, IgG, and IgM. IgG subclasses are also well known to those of skill in the art, including, but not limited to, human IgG1, IgG2, IgG3, and IgG4.

[0042] In another embodiment, the antibody of the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to a single heavy chain variable domain of an antibody of a type that can be found in camelid mammals, which naturally lack light chains. Such VHHs are also called "nanobodies". According to the invention, the sdAb can in particular be a llama sdAb. The term "VHH" refers to a single heavy chain with three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term "complementarity determining region" or "CDR" refers to hypervariable amino acid sequences that define the binding affinity and specificity of the VHH.

[0043] The VHHs of the invention can be readily prepared by one of ordinary skill in the art using routine experimentation. VHH variants and modified forms thereof can be produced by any technique known in the art, for example, in vitro maturation.

[0044] VHHs or sdAbs are usually generated by PCR cloning of V-domain repertoires from blood, lymph node or spleen cDNA obtained from immunized animals into phage display vectors, such as pHEN2. Antigen-specific VHHs are typically selected by panning phage libraries on immobilized antigens, such as antigens coated on the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinity for their antigens than VHHs derived from animals that have received some immunization. The high affinity of VHHs from immune libraries results from the natural selection of mutant VHHs during clonal expansion of B cells in lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site-directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH and low antigen concentration). Camelid-derived VHHs are easily expressed in and purified from the E. coli periplasm at levels much higher than the corresponding domains of conventional antibodies. VHHs generally exhibit high solubility and stability and can be easily produced in yeast, plants and mammalian cells. For example, the "Hamers Patents" describe methods and techniques for generating VHHs against any desired target (see, for example, US Pat. Nos. 5,800,988; 5,874,541 and 6,015,695). The "Hamers patents" describe, inter alia, the production of VHHs in bacterial hosts such as E. coli (see, e.g., US 6,765,087) and lower eukaryotic hosts such as molds (e.g., Aspergillus or Trichoderma) or yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see, e.g., US 6,838,254).

[0045] As used herein, the term "regulatory T cells" or "Treg" refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune and inflammatory diseases. These cells generally suppress or downregulate the induction and proliferation of effector T cells and modulate antigen-presenting cell function. Tregs are cells capable of suppressive activity (i.e., inhibiting the proliferation of conventional T cells) either through cell-to-cell contact or through the release of immunosuppressive cytokines.

[0046] Another object of the invention relates to a population of regulatory T cells according to the invention.

[0047] As used herein, the term "population" refers to a population of cells, where a majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have a particular characteristic of a cell of interest and express a marker of interest.

[0048] Another object of the present invention is an ex vivo method for stimulating regulatory T cell immunosuppressive activity, comprising: i) obtaining a biological sample from a subject; ii) isolating regulatory T cells from said sample; and iii) expanding regulatory T cells in vitro; and iv) incubating the isolated regulatory T cells with an effective amount of a lymphotoxin alpha blocking agent, such as a soluble lymphotoxin β receptor (LTβR), to block the LTα1β2 / LTβR or LTα2β1 / LTβR interaction.

[0049] As used herein, the term "subject" refers to mammals, such as rodents, cats, dogs, and primates. Preferably, the subject of the present invention is a human.

[0050] As used herein, the term "biological sample" refers to any bodily fluid or tissue. In one embodiment, the biological sample is a blood sample.

[0051] As used herein, the term "regulatory T cell immunosuppressive activity" is well known in the art and refers to the ability of Tregs to suppress or downregulate the induction and proliferation of effector T cells. As used herein, the term "stimulation of regulatory T cell immunosuppressive activity" refers to enhancement of regulatory T cell immunosuppressive activity.

[0052] As used herein, "isolate" refers to removing a cell or cell population from its native environment. As used herein, "isolated" refers to a cell or cell population that has been removed, isolated, purified or separated from its native environment (e.g., a blood sample) and is at least about 75%, 80%, 85%, and preferably about 90%, 95%, 96%, 97%, 98%, 99% free from other cells with which it is naturally present.

[0053] According to the method of the present invention, the regulatory T cells of the present invention are isolated from a sample. Any technique known to those skilled in the art can be used. In one embodiment, the regulatory T cells are CD8 + and CD19 + CD4 by cell depletion + After pre-enrichment of T cells, they were isolated by cell sorting. The purity of the selected regulatory T cells was >97%.

[0054] A further object of the present invention relates to a pre-incubated regulatory T cell of the invention, characterized in that it expresses a TCR or a chimeric antigen receptor which recognizes / binds to a self-antigen.

[0055] As used herein, the term "TCR" has its general meaning in the art and refers to a molecule found on the surface of a T cell that is responsible for recognizing antigens bound to MHC molecules. In particular, a "T cell characterized as expressing a TCR" means that a T cell has been genetically engineered to express said TCR, for example by transfecting or transducing said T cell in vitro or ex vivo with a nucleic acid molecule encoding said TCR.

[0056] As used herein, the term "chimeric antigen receptor" or "CAR" has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide that contains an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. In the context of the present invention, the antigen-binding domain of the antibody recognizes / binds to an autoantigen.

[0057] As used herein, the terms "recognize" or "bind" mean that a TCR or chimeric antigen receptor has affinity for an antigen.

[0058] As used herein, the term "autoantigen" refers to an endogenous antigen or its active fragments that are recognized by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins including cell surface receptors. Examples of autoantigens include, but are not limited to, preproinsulin (PPI), glutamic acid decarboxylase (GAD), insulinoma-associated protein 2 (IA-2), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), zinc transporter 8 (ZnT8) and chromogranin A for T1D; myeloperoxidase and proteinase 3 for granulomatosis with polyangiitis; myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) in multiple sclerosis; gliadin in celiac disease.

[0059] Another object relates to a population of regulatory T cells of the invention that express a TCR or chimeric antigen receptor that recognizes / binds to a self-antigen.

[0060] In one embodiment, the regulatory T cells of the present invention are transfected or transduced in vitro or ex vivo with a vector encoding a chimeric antigen receptor or an antigen-specific TCR to express a chimeric antigen receptor or an antigen-specific TCR that recognizes / binds to an autoantigen. Therefore, another object of the present invention relates to a method for producing a regulatory T cell of the present invention expressing a chimeric antigen receptor or an antigen-specific TCR that recognizes / binds to an autoantigen, the method comprising the step of transfecting or transducing the regulatory T cells of the present invention in vitro or ex vivo with a vector encoding a chimeric antigen receptor or an antigen-specific TCR.

[0061] The term "transduction" or "transducing" refers to the viral transfer of genetic material and its expression in a recipient cell.

[0062] As used herein, the terms "transfection" or "transfecting" refer to the process of introducing DNA (e.g., an assembled DNA expression vector) into a cell, thereby allowing cell transformation.

[0063] As used herein, the term "vector" refers to a nucleic acid molecule that allows the insertion of foreign nucleic acid without destroying the ability of the vector to replicate and / or integrate in a host cell.

[0064] The regulatory T cell populations of the present invention (populations of regulatory T cells that have been pre-incubated with an effective amount of a lymphotoxin alpha blocking agent, e.g., a soluble lymphotoxin beta receptor (LTβR), and that may or may not express a TCR or chimeric antigen receptor that recognizes / binds to an autoantigen) are particularly suitable for therapeutic use.

[0065] A further object of the present invention relates to a population of regulatory T cells, expressing or not expressing TCRs or chimeric antigen receptors that recognize / bind to self-antigens, pre-incubated with an effective amount of lymphotoxin alpha blocker, for use in adoptive cell therapy.

[0066] As used herein, the term "adoptive cell therapy" refers to cell-based immunotherapy involving the infusion of autologous or allogeneic lymphocytes, which may or may not be genetically modified.

[0067] The population of Tregs of the present invention can be utilized in methods and compositions for adoptive cell therapy according to known techniques or modifications thereof that will be apparent to one of skill in the art based on this disclosure. See, for example, US 2003 / 0170238 to Gruenberg et al. See also US 4,690,915 to Rosenberg. In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing and concentrating them in a therapeutically effective amount in a medium and container system (a "pharmacologically acceptable" carrier) suitable for administration. A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition depends on the age and weight of the recipient and the severity of the condition being targeted. Classically, the number of Tregs infused is about 1-3×10 6(Adair et al. Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision. Front Immunol 2017). However, when using the Tregs of the present invention, the dose can be reduced due to their increased immunosuppressive activity. In one embodiment, the dose can be reduced by at least 50%. In one embodiment, the dose can be reduced by 75%. In one embodiment, standard cell therapy doses can be used. These cell amounts are approximately 10 3 / kg, preferably 5 x 10 3 The number of pieces per kg can be reduced by about 10 7 pcs / kg, preferably 10 8 The number of cells can be as many as 10 or 20 cells / kg. The number of cells will depend on the end use for which the composition is intended, as will the type of cells contained therein. Clinically relevant numbers of immune cells can be distributed over multiple infusions that cumulatively equal or exceed the total desired amount of cells.

[0068] For purposes of the present invention, the regulatory T cells of the invention used in adoptive cell therapy can be isolated from the subject ("autologous cells") or from another individual ("allogeneic cells").

[0069] As used herein, "allogeneic cells" refer to cells isolated from one subject (the donor) and injected into another subject (the recipient or host).

[0070] As used herein, "autologous cells" refer to cells that are isolated and infused back into the same subject (recipient or host).

[0071] In one embodiment, the regulatory T cells of the invention used in adoptive cell therapy can be derived from stem cells.

[0072] As used herein, the term "stem cell" refers to a cell in an undifferentiated or partially differentiated state that has the property of self-renewal and has the developmental potential to differentiate into multiple cell types, without any particular connotation as to developmental potential (i.e., totipotency, pluripotency, multipotency, etc.).

[0073] In certain embodiments, the regulatory T cells of the present invention used in adoptive cell therapy can be derived from induced pluripotent stem cells.

[0074] As used herein, the terms "iPSC" and "induced pluripotent stem cell" are used interchangeably and refer to pluripotent stem cells that are artificially derived (e.g., by induced or complete reversal) from non-pluripotent cells, typically adult somatic cells, for example, by inducing the forced expression of one or more genes.

[0075] In certain embodiments, the regulatory T cells of the invention used in adoptive cell therapy can be derived from embryonic stem cells.

[0076] As used herein, the term "embryonic stem cells" refers to naturally occurring pluripotent stem cells of the inner cell mass of embryonic blastocysts (see, e.g., US Pat. Nos. 5,843,780; 6,200,806; 7,029,913; 7,584,479, which are incorporated herein by reference). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (nuclear transfer) (see, e.g., US Pat. Nos. 5,945,577; 5,994,619; 6,235,970, which are incorporated herein by reference). Embryonic stem cells are pluripotent and give rise to all derivatives of the three primary germ layers, ectoderm, endoderm, and mesoderm, during development. That is, they can develop into each of the more than 200 cell types of an adult when given sufficient and necessary stimulation for that particular cell type. They do not contribute to the extraembryonic membranes or the placenta, i.e., they are not totipotent.

[0077] In one embodiment, the regulatory T cells of the invention used in adoptive cell therapy can be derived from the conversion of conventional CD4+ T cells.

[0078] A further object of the present invention relates to a method for treating or preventing an autoimmune disorder, comprising administering to a patient in need thereof an amount of a lymphotoxin alpha blocking agent effective to stimulate regulatory T cell immunosuppressive activity. In particular, the present invention relates to a method for treating or preventing an autoimmune disorder or inflammation-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of regulatory T cells, wherein the population of regulatory T cells has been pre-incubated with an effective amount of a lymphotoxin alpha blocking agent. The incubation with the lymphotoxin alpha blocking agent allows for an increase in the suppressive activity of the regulatory T cells.

[0079] In one embodiment, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0080] In one embodiment, regulatory T cells express a TCR or chimeric antigen receptor that recognizes / binds to an autoantigen.

[0081] In some embodiments, regulatory T cells do not express a TCR or chimeric antigen receptor that recognizes / binds to a self-antigen.

[0082] Thus, the present invention refers to a method for treating or preventing an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of regulatory T cells pre-incubated with an effective amount of soluble LTβR and / or a population of regulatory T cells pre-incubated with an effective amount of soluble LTβR and expressing a TCR or chimeric antigen receptor that recognizes / binds to an autoantigen.

[0083] As used herein, the term "autoimmune disease" refers to the presence of an autoimmune response (an immune response directed against self or self-antigens) in a subject. Autoimmune diseases include diseases caused by a breakdown in self-tolerance, such that the adaptive immune system, in cooperation with cells of the innate immune system, responds to self-antigens and mediates cell and tissue damage. In some embodiments, an autoimmune disease is characterized as being at least in part the result of a humoral and / or cellular immune response. Examples of autoimmune diseases include acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune inflammatory bowel disease ... Immune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuropathy, Behçet's disease, bullous pemphigoid, autoimmune cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Kogans syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, Granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis,Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignoconjunctivitis, linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), Mooren's ulcer, Muscha-Haverman disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), autoimmune neutropenia, Ophthalmic cicatricial pemphigoid, Optic neuritis, Relapsing rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Paley-Romberg syndrome, Parsonage-Turner syndrome, Parsplanitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Autoimmune polyglandular syndromes types I, II and III, Polymyalgia rheumatica, Polymyositis , post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, total These include, but are not limited to, stiff-body syndrome, subacute bacterial endocarditis (SBE), Sacca syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Trosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular dermatosis, vitiligo, Waldenstrom's macroglobulinemia (WM), and Wegener's granulomatosis [granulomatosis with polyangiitis (GPA)]. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiple sclerosis, scleroderma, Addison's disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren's syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS),Selected from the group consisting of Waldenstrom's macroglobulinemia (WM), chronic inflammatory demyelinating polyneuropathy (CIDP), Hashimoto's encephalopathy (HE), Hashimoto's thyroiditis, Graves' disease, and Wegener's granulomatosis [granulomatosis with polyangiitis (GPA)].

[0084] In one embodiment, the autoimmune disease is inflammatory bowel disease.

[0085] In one embodiment, the autoimmune disease is multiple sclerosis or type 1 diabetes.

[0086] A further object of the present invention relates to a method of treating or preventing inflammation-associated cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of regulatory T cells, wherein the population of regulatory T cells has been pre-incubated with an effective amount of a lymphotoxin alpha blocker.

[0087] In some embodiments, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0088] As used herein, the term "inflammation-associated cancer" refers to any cancer in which inflammation is considered to be at least one of the pathogenic mechanisms involved in the initiation and development of the cancer. Examples of inflammation-associated cancer include, but are not limited to, colitis-associated cancer, gastric adenocarcinoma, bladder cancer, liver cancer, rectal cancer, bile duct cancer, colon cancer, colorectal cancer, gallbladder cancer, hepatocellular carcinoma, ovarian cancer, cervical cancer, skin cancer, esophageal cancer, bladder cancer, mesothelioma, lung cancer, oral squamous cell carcinoma, pancreatic cancer, vulvar squamous cell carcinoma, salivary gland cancer, lung cancer, and MALT lymphoma.

[0089] In one embodiment, the inflammation-associated cancer is colitis-associated cancer, which is a subtype of colon cancer.

[0090] A further object of the present invention relates to a method for treating or preventing allergy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of regulatory T cells, wherein the population of regulatory T cells has been pre-incubated with an effective amount of a lymphotoxin alpha blocker.

[0091] In some embodiments, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0092] As used herein, the term "allergy" generally refers to an inappropriate immune response characterized by inflammation, including, but not limited to, food allergies, respiratory allergies, and other allergies that cause or may cause a systemic response, such as Quincke's edema and anaphylaxis. The term encompasses allergies, allergic diseases, hypersensitivity-related diseases, or respiratory diseases associated with airway inflammation, such as asthma or allergic rhinitis. In some embodiments, the methods of the present invention are effective in preventing, treating, or alleviating one or more symptoms associated with anaphylaxis, drug hypersensitivity, skin allergies, eczema, allergic rhinitis, urticaria, atopic dermatitis, dry eye disease, allergic contact allergy, food hypersensitivity, allergic conjunctivitis, insect venom allergy, bronchial asthma, allergic asthma, intrinsic asthma, occupational asthma, atopic asthma, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD). Hypersensitivity-related diseases or disorders that can be treated by the method of the present invention include, but are not limited to, anaphylaxis, drug reactions, skin allergies, eczema, allergic rhinitis, urticaria, atopic dermatitis, dry eye disease (or other diseases called keratitis sicca (KCS) (also called xerophthalmia)), allergic contact allergies, food allergies, allergic conjunctivitis, insect venom allergies, and respiratory diseases associated with airway inflammation, such as IgE-mediated asthma and non-IgE-mediated asthma. Respiratory diseases associated with airway inflammation include, but are not limited to, rhinitis, allergic rhinitis, bronchial asthma, allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, occupational asthma, atopic asthma, exercise-induced asthma, cough-induced asthma, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD).

[0093] A further object of the present invention relates to a method for treating or preventing an immune response to an exogenously administered molecule in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of regulatory T cells, wherein the population of regulatory T cells has been pre-incubated with an effective amount of a lymphotoxin alpha blocker.

[0094] In some embodiments, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0095] Non-limiting examples of this type include immune responses to replacement therapy for genetic deficiencies such as hemophilia A, hemophilia B, congenital deficiencies of other clotting factors such as factor II, prothrombin and fibrinogen, primary immune deficiencies (e.g., severe combined immunodeficiency, X-linked agammaglobulinemia, IgA deficiency), primary hormone deficiencies such as growth hormone deficiency and leptin deficiency, congenital enzyme and metabolic disorders such as carbohydrate metabolism disorders (e.g., sucrose-isomaltase deficiency, glycogen storage diseases), amino acid metabolism disorders (e.g., phenylketonuria, maple syrup urine disease, glutaric acidemia type 1), urea cycle disorders (e.g., carbamoyl phosphate synthase I deficiency), organic acid metabolism disorders (e.g., erythrocyte sedimentation syndrome ... Disorders of fatty acid oxidation and mitochondrial metabolism (e.g., medium-chain acyl-coenzyme A dehydrogenase deficiency), porphyrin metabolism disorders (e.g., porphyrias), purine or pyrimidine metabolism disorders (e.g., Lesch-Nyhan syndrome), steroid metabolism disorders (e.g., lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia), mitochondrial dysfunction disorders (e.g., Kearns-Sayre syndrome), peroxisome dysfunction (e.g., Zellweger syndrome), lysosomal storage disorders (e.g., Gaucher disease, Niemann-Pick disease).

[0096] A further object of the present invention relates to a method for treating or preventing an immune response against transplanted tissue or cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of regulatory T cells, wherein the population of regulatory T cells has been pre-incubated with an effective amount of a lymphotoxin alpha blocker.

[0097] In some embodiments, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0098] As used herein, the term "transplant" refers to an organ and / or tissue and / or cells that can be obtained from a first organism (or donor) and transplanted into a second organism (or recipient). Typically, a subject can be transplanted with a graft selected from the group consisting of heart, kidney, lung, liver, pancreas, pancreatic islets, brain tissue, stomach, large intestine, small intestine, cornea, skin, trachea, bone, bone marrow, muscle, or bladder. The method of the present invention is also particularly suitable for preventing or suppressing immune responses associated with the rejection of donor tissue, cell, graft, or organ transplant by the recipient subject. Graft-related diseases or disorders include graft-versus-host disease (GVHD), such as associated with bone marrow transplantation, and immune disorders resulting in or associated with the rejection of organ, tissue, or cell graft transplants (e.g., tissue or cell allografts or xenografts), including grafts such as skin, muscle, neurons, pancreatic islets, organs, liver parenchymal cells, etc. Thus, the methods of the invention are useful for preventing host-versus-graft disease (HVGD) and graft-versus-host disease (GVHD). The chimeric construct can be administered to the subject before, during, and / or after transplantation (e.g., at least one day before transplantation, at least one day after transplantation, and / or during the transplantation procedure itself). In some embodiments, the chimeric construct can be administered to the subject periodically before and / or after transplantation.

[0099] As used herein, the term "subject" refers to mammals, such as rodents, cats, dogs, and primates. Preferably, the subject of the present invention is a human.

[0100] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease, preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving the quality of life and / or prolonging survival. The term "treatment" includes prophylactic treatment. As used herein, the term "preventing" or "prevention" refers to a reduction in the risk of acquiring or developing a given condition.

[0101] As used herein, the term "administer" or "administration" refers to the act of injecting or in any manner physically delivering a substance present outside the body into a subject, for example, by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When a disease or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or a symptom thereof. When a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or a symptom thereof.

[0102] A "therapeutically effective amount" is determined using techniques routinely employed by those skilled in the art to provide an "improved therapeutic outcome". However, it will be understood that the daily dosing regimen of the compositions of the present invention will generally be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound utilized; the specific composition utilized; the age, weight, general health, sex, and diet of the subject; the duration of administration, route of administration, and excretion rate of the specific compound utilized; duration of treatment; drugs used in combination, as well as factors well known in the medical field. According to the present invention, the regulatory T cells can be incubated for a wide range of times, from 5 to 120 minutes, over a wide range of 1 to 500 μg / ml soluble lymphotoxin β receptor (LTβR). To this end, regulatory T cells can be incubated with 1, 2, 5, 10, 30, 50, 100, 150, 200 or 500 μg / ml for 5, 10, 30, 60, 90 or 120 minutes.

[0103] In some embodiments, regulatory T cells are incubated with 50 μg / ml soluble lymphotoxin β receptor (LTβR) for 30 minutes.

[0104] Although the innate immune system is well known to be involved in tissue healing processes, the adaptive immune system has recently emerged as a key player. T cells, particularly regulatory T cells (Tregs), have been shown to promote repair and regeneration of various organ systems (15). Thus, a further object of the present invention relates to a population of regulatory T cells, expressing or not expressing TCRs or chimeric antigen receptors that recognize / bind to self-antigens, pre-incubated with an effective amount of lymphotoxin alpha blocker, for use in regenerative therapy.

[0105] As used herein, the term "regenerative therapy" refers to the process of regenerating human or animal cells, tissues, or organs to restore or establish normal function. Regenerative medicine has the potential to heal or replace tissues and organs damaged by aging, disease, or trauma, and to correct congenital defects. This field holds the promise of engineering damaged tissues and organs by stimulating the body's own repair mechanisms to functionally heal previously irreparable tissues or organs.

[0106] Regenerative therapies include, but are not limited to, tissue healing processes, such as skin regeneration, and organ regeneration, such as myocardial regeneration, liver regeneration, kidney regeneration, pancreatic regeneration, lung regeneration, hair follicle regeneration, heart regeneration; bladder regeneration; muscle regeneration (e.g., skeletal muscle regeneration and cardiac regeneration), bone regeneration, and central nervous system regeneration.

[0107] In some embodiments, the regenerative therapy is muscle regeneration, skin regeneration, bone regeneration, or central nervous system regeneration.

[0108] As used, the term "organ or tissue regeneration" refers to reinitiating the proliferative activity of differentiated resident cells in an organ or tissue to replace their massive loss of cells following organ injury.

[0109] As used herein, the term "organ damage" has its general meaning in the art and refers to any type of reversible or irreversible damage to the cells of an organ. Organ damage may be caused by any condition. Exemplary organ damage includes ablative therapies (such as those required to prepare a patient for bone marrow transplantation (BMT)), complications associated with HIV / AIDS, the aging process, malnutrition, and radiation poisoning. The term also includes age-related involution, i.e., the gradual shrinkage of an organ with age. Organ damage, e.g., thymus damage, may be caused by, for example, irradiation as a medical procedure. Examples of diseases that are partially treated with radiation include, but are not limited to, cancers such as multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, acute myeloid leukemia, neuroblastoma, ovarian cancer, germ cell tumors, acute lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndromes, myeloproliferative disorders, chronic lymphocytic leukemia, juvenile chronic myeloid leukemia, and other diseases such as autoimmune disorders, amyloidosis, aplastic anemia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, Blackfan-Diamond anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, and inborn errors of metabolism.

[0110] In some embodiments, organ damage may also result from ischemia. Ischemia can be reversible or persistent (i.e., permanent) ischemia. Persistent ischemia is characterized by an organ being inadequately supplied with blood and therefore hypoxic even in a resting subject.

[0111] In some embodiments, organ damage may be caused by any form of chemical or physical agent, such as a drug, environmental toxin, or any other agent that contacts the subject and causes damage to the organ or tissue, directly or indirectly. It also includes damage caused by successful therapeutic treatment of the subject, such as a treatment that results in the induction of cellular apoptosis in the organ (e.g., chemotherapy).

[0112] Thus, the present invention relates to a population of regulatory T cells, expressing or not expressing a TCR or chimeric antigen receptor that recognizes / binds to an autoantigen, pre-incubated with an effective amount of a lymphotoxin alpha blocker, for use in regenerating tissues or organs.

[0113] In some embodiments, the organ or tissue is skeletal muscle, cardiac muscle, bone, skin, or the central nervous system.

[0114] Thus, the present invention relates to a population of regulatory T cells, expressing or not expressing TCRs or chimeric antigen receptors that recognize / bind to self-antigens, pre-incubated with an effective amount of a lymphotoxin alpha blocker, for use in regenerating skeletal muscle, cardiac muscle, bone, skin or the central nervous system.

[0115] According to the present invention, the population of regulatory T cells of the present invention is administered to a subject in the form of a pharmaceutical composition.

[0116] Thus, a further object of the present invention relates to a pharmaceutical composition comprising a population of regulatory T cells pre-incubated with an effective amount of a lymphotoxin alpha blocking agent and / or a population of regulatory T cells expressing a TCR or chimeric antigen receptor that recognizes / binds to an autoantigen and that has been pre-incubated with an effective amount of a lymphotoxin alpha blocking agent.

[0117] In some embodiments, the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

[0118] Thus, the present invention relates to a pharmaceutical composition comprising a population of regulatory T cells pre-incubated with an effective amount of soluble LTβR and / or a population of regulatory T cells pre-incubated with an effective amount of soluble LTβR and expressing a TCR or chimeric antigen receptor that recognizes / binds to an autoantigen.

[0119] Typically, the population of Tregs can be combined with a pharma- ceutically acceptable excipient and, optionally, a sustained-release matrix, such as a biodegradable polymer, to form a therapeutic composition. "Pharmaceutically" or "pharma- ceutically acceptable" refers to molecular entities and compositions that do not produce adverse allergic or other untoward reactions when administered appropriately to a mammal, particularly a human. A pharma- ceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary. In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient can be administered to animals and humans alone or in combination with another active ingredient, in unit dosage form, in admixture with a conventional pharmaceutical support. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms.

[0120] In one embodiment, the Treg population of the present invention is administered by a parenteral route. In a preferred embodiment, the Treg population of the present invention is administered by an intravenous route.

[0121] Typically, the pharmaceutical composition contains a pharma- ceutically acceptable medium for injection preparations. These can be, in particular, isotonic and sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts) or dry, in particular lyophilized compositions. These can be made up into injection solutions by the addition of sterile water or saline, as the case may be. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of injection solutions or suspensions. In all cases, the form must be sterile and fluid to the extent that it can be easily injected. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions of the invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. The population of Tregs can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein). Acid addition salts are formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be obtained from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of suspensions, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition. Sterile injectable solutions are prepared by incorporating the required amount of active compound in a suitable solvent, optionally with some of the other ingredients listed above, followed by filtration sterilization. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation are vacuum drying and freeze-drying techniques that produce a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution. The preparation of more or highly concentrated solutions for direct injection is also contemplated. In this case, the use of DMSO as a solvent is expected to result in very rapid penetration and deliver high concentrations of active agent to small tumor areas. Depending on the formulation, the solution will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulation is easily administered in a variety of dosage forms, such as the types of injection solutions described above, but drug release capsules and the like can also be utilized. For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be utilized will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the dosage appropriate for the individual subject.

[0122] The present invention will be further illustrated by the following figures and examples, which should not be construed in any way as limiting the scope of the invention. [Brief description of the drawings]

[0123] [Figure 1] Figure 1. Adoptive transfer of LTα- / - Tregs protects against the severity of DSS-induced colitis. (A) Representative flow cytometry profiles of Foxp3 expression in purified splenic CD4+CD25+ cells from WT and LTα- / - mice. (B) Experimental setup: Colitis was induced in WT mice injected 2 days prior with 2×105 WT or LTα- / - Tregs by administration of 2% DSS in drinking water for 7 days, followed by water alone until day 11. Colon inflammation and CD4+ T cell priming in mesenteric lymph nodes were analyzed on days 11 and 4 of the protocol, respectively. (C) Weight loss relative to the initial weight on day 0 in WT mice injected with 2×105 WT or LTα- / - Tregs. Data are from three independent experiments with 4 mice per group. (D) Disease activity index (DAI) was monitored during the course of DSS-induced colitis. (E) Histogram showing colon histological scores in both groups of mice. [Diagram 2] Figure 2. Adoptive transfer of WT Tregs preincubated with soluble LTβR-Fc fusion protein attenuates DSS-induced colitis. (A) Experimental setup: Colitis was induced in WT mice injected 2 days previously with either 2x105 WT Tregs preincubated or not with LTβR-Fc protein or with LTα- / -Tregs by administration of 2% DSS in drinking water for 7 days followed by water alone until day 11. Body weight was monitored daily and colon length was measured on day 11 of the protocol. (B) Weight loss relative to initial body weight on day 0 in WT mice injected with either WT Tregs preincubated or not with soluble LTβR-Fc protein or with LTα- / -Tregs. Data are from one independent experiment with 3-9 mice per group. (C) Histograms show colon lengths observed at the end of the experimental protocol. [Diagram 3]Figure 3. The suppressive signature of Treg cells is controlled by the LTα1β2 / LTβR axis. (A) Expression levels of Il10, Ebi3, Tgfb1, Ifn-γ, Gzmb and Fasl mRNA were measured by qPCR in purified CD45.2 WT and LTα- / - Tregs from donor groups. (B) Purified WT Foxp3+CD4+ Tregs, with or without pre-incubation with soluble LTβR-Fc fusion protein and co-cultured with purified CD11c+ dendritic cells for 24 h, were analyzed for expression levels of Klrg1, Il10, Ebi3, Tgfb1, Gzmb and Fasl by qPCR.

[0124] Working Example Materials and Methods mouse All mice - CD45.1 WT, CD45.1 × CD45.2 WT, CD45.2 WT and CD45.2 LTα - / - mice - were on a pure C57BL / 6 background and maintained under specific pathogen-free conditions at CIML (France). Standard food and water were provided ad libitum. Males and females were used at 6-8 weeks of age. All procedures involving animals were performed in accordance with institutional and ethical guidelines.

[0125] Treg cell isolation Splenic Treg cells were isolated by scratching the spleen through a 70 μm mesh. Splenic red blood cells were lysed with lysis buffer (eBioscience). Prior to cell sorting, CD4+ T cells were pre-enriched via a depletion program by depleting CD8+ and CD19+ cells using anti-CD8α (clone 53.6.7) and anti-CD19 (clone 1D3) biotinylated antibodies with anti-biotin microbeads by AutoMACS (Miltenyi Biotech). CD4+CD25+ Tregs were sorted using a FACSAriaIII cell sorter (BD).

[0126] DSS-induced colitis experiment Two days before induction of colitis, WT recipient mice were transfected with 2.10 IgG1-derived LTα- / - mice selected from WT or LTα- / - mice. 5 CD4+CD25+ splenic Tregs were injected intravenously. Induction of colitis was assessed by administering 2% DSS (Alfa Aesar) in drinking water for 7 days followed by water only until sacrifice on day 11. Body weight, rectal bleeding and stool consistency were monitored daily after DSS administration and used to determine the DAI.

[0127] In vitro co-culture assay 2.10 for co-culture assays 3 Cell sorting total CD11c hi Dendritic cells were pre-incubated with soluble LTβR-Fc recombinant protein (2 μg / ml; R&D systems) for 1 h or not. 4 Purified CD4 + CD25 + The cells were co-cultured with Tregs for 24 hours at 37°C.

[0128] Treg cell incubation with soluble LTβR-Fc protein in a DSS-induced colitis model 2.105 CD4+CD25+ splenic Tregs purified from CD45.2 WT mice were pre-incubated or not with soluble LTβR-Fc fusion protein (50 μg / ml; R&D Systems) for 30 min in culture medium (RPMI ThermoFisher containing 10% FBS (Sigma Aldrich), 2 mM L-glutamine (ThermoFisher), 1 mM sodium pyruvate (ThermoFisher) and 2×10-5 M 2-mercaptoethanol (ThermoFisher)). Tregs were then adoptively transferred intravenously into CD45.2 WT recipients. 2.105 CD4+CD25+ splenic Tregs from LTα- / - mice were used as controls. Two days later, colitis was induced by administering 2% DSS (Alfa Aesar) in drinking water for 7 days, followed by water only until sacrifice on day 11. Body weight was monitored daily after DSS administration.

[0129] Flow cytometry Anti-CD4 (RM4.5) antibody was obtained from BD. For intracellular staining with Foxp3 antibody, cells were fixed, permeabilized, and stained with Foxp3 staining kit according to the manufacturer's instructions (eBioscience). Stained cells were analyzed by FACSCanto II (BD) and data were analyzed using FlowJo software.

[0130] Quantitative RT-PCR Total RNA was isolated using TRIzol (Invitrogen) and cDNA was synthesized using random oligo-dT primers and Superscript II reverse transcriptase (Invitrogen). qPCR was performed using SYBR Premix Ex Taq master mix (Takara) on an ABI 7500 fast real-time PCR system (Applied Biosystem). Results were normalized to actin mRNA.

[0131] statistical analysis Statistical significance was assessed using unpaired Student's t-test or Mann-Whitney test using GraphPad Prism 6 software. Two-way Anova test with Bonferroni correction was used for the analysis of tumor growth, body weight loss and DAI. * , P < 0.05; ** , P < 0.01; *** , P < 0.001; **** , P<0.0001. Normal distribution of data was assessed using the D'Agostino-Pearson omnibus normality test. Error bars represent the mean ± SEM.

[0132] result LT Alpha - / - Adoptive transfer of Tregs protects against ulcerative colitis LT Alpha - / - Considering that Tregs highly express several genes involved in Treg suppressive function (data not shown), LTα - / - We assessed whether adoptive transfer of Tregs would show therapeutic benefit for protecting against dextran sulfate sodium (DSS)-induced colitis. - / - Purified Foxp3 from mouse + 2.10, which mainly contains Tregs 5 CD4 + CD25 + The cells (Fig. 1A) were injected into WT recipient mice 2 days before induction of colitis with 2% DSS (Fig. 1B). - / - We observed that mice infused with Tregs lost significantly less weight than those infused with WT Tregs (Fig. 1C). Moreover, the disease activity index (DAI), which combines stool consistency, rectal bleeding, and weight loss, was substantially less significant in these mice (Fig. 1D). In accordance with weight loss and DAI, these mice had less damage to the colonic epithelium at the end of the experiment (data not shown) and a lower histological score for colitis (Fig. 1E). We also found that LTα - / -We observed a decrease in the expression of proinflammatory cytokines, such as Il6, Ifnγ, Tnf-α, Il17A, Il1α, and Il33, as well as chemokines involved in immune cell recruitment, such as Ccl2 and Cxcl12, in the colon of Treg-transferred mice (data not shown). Furthermore, we analyzed the nature of colon-infiltrating immune cells by flow cytometry. Neutrophils, macrophages, dendritic cells, B cells, and CD4 + The number of T cells is - / - In mice transferred with Tregs, there was a dramatic decrease in WT Tregs compared with mice transferred with CD3 + and B220 + A reduction in infiltration of Th1 and Th17 effector CD4 + The number of T cells was also decreased (data not shown). As a result, the Treg / Th1 and Treg / Th17 ratios were significantly decreased in the LTα - / - Tregs were elevated in the colon of transferred mice (data not shown). 5 Pieces to 1.10 5 1, then 0.5.10 5 By reducing it to LTα - / - The potential for Tregs to protect against colitis was evaluated. 5 LTα - / - 2.10 Tregs are characterized by reduced weight loss 5 It was observed that 0.5.10 Tregs showed better protection than WT Tregs (data not shown). 5 LTα - / - Tregs are 2.10 5 The protective effect of LTα Tregs is the same as that of WT Tregs. - / - We show that Tregs are approximately 4-fold more suppressive in vivo than their WT counterparts.

[0133] Next, LTα - / - Adoptive transfer of Tregs increased CD4+ T cells in mesenteric lymph nodes 5 days after DSS administration. +We further determined whether T cell priming was inhibited. - / - It was found that mice infused with Tregs already exhibited longer colon lengths and reduced colon weight / length ratios at this time point, indicating attenuated colonic inflammation (data not shown). Surprisingly, Th1 and Th17 effector CD4 + T cell numbers were substantially reduced in the mesenteric lymph nodes of these mice (data not shown). - / - Tregs inhibit naive CD4 + In conclusion, these data suggest that LTα inhibits the conversion of T cells into effector T cells. - / - Adoptive transfer of Tregs suppresses colonic inflammation and pathogenic CD4 in mesenteric lymph nodes + Attenuating T cell priming has been shown to protect against the development of ulcerative colitis.

[0134] Adoptive transfer of WT Tregs preincubated with soluble LTβR-Fc fusion protein attenuates DSS-induced colitis We next assessed whether adoptive transfer of Tregs preincubated with soluble LTβR-Fc fusion protein would show a therapeutic benefit in protecting against dextran sulfate sodium (DSS)-induced colitis. - / - or Foxp3 purified from WT mice + 2.10, which mainly contains Tregs 5 CD4 + CD25 + Cells (Fig. 1A) were preincubated with or without soluble LTβR-Fc fusion protein in culture medium for 30 min. Tregs were injected into WT recipient mice 2 days before inducing colitis with 2% DSS (Fig. 2A). In mice injected with Tregs preincubated with LTβR-Fc, LTα - / -We observed that mice injected with LTβR-Fc lost significantly less weight than mice injected with WT Tregs (Fig. 2B). Furthermore, the colons of mice injected with Tregs preincubated with soluble LTβR-Fc were significantly longer than those of mice injected with WT Tregs. - / - similar to the length of the injected mice (Figure 2C).

[0135] In conclusion, these data demonstrate that adoptive transfer of WT Tregs preincubated with soluble LTβR-Fc enhances the upregulation of LTα - / - It has been shown that it protects against the development of ulcerative colitis as effectively as Treg.

[0136] Expression of LTα in hematopoietic cells and the LTα1β2 / LTβR axis negatively regulate the suppressive signature of Treg cells LT Alpha - / - Because mice exhibit a disorganized thymic and splenic microenvironment, LTα - / - We analyzed the contribution of non-hematopoietic stromal cells to the highly immunosuppressive phenotype of Tregs. To this end, lethally irradiated CD45.2 WT or LTα - / - Recipient mice were translocated to CD45.1 congenic mice (WT CD45.1:WT and WT CD45.1:LTα, respectively). - / - We generated bone marrow (BM) chimeras reconstituted with WT BM cells from donor mice (Fig. 1b). Six weeks after BM transplantation, the CD4 + CD25 + Treg cells were sorted from the spleen and analyzed for expression of several genes associated with Treg effector function (data not shown). + Tregs were observed in both groups of mice (data not shown). Furthermore, the expression of some genes, such as Klrg1, Tgfb, Gzmb, and Fasl, was similar in both groups of mice. This suggests that non-hematopoietic cells express LTα - / - These results suggest that Tregs are not involved in the highly suppressive signature of Tregs observed in mice (data not shown).

[0137] Lethally irradiated CD45.1 × CD45.2 WT recipient mice were then transfected with either WT CD45.1 and WT CD45.2 (WT donor group) or WT CD45.1 and LTα - / - CD45.2(LTα - / - We determined the contribution of each of the hematopoietic compartments by generating mixed bone marrow chimeras reconstituted with BM cells (50:50) from 1 donor group (data not shown). After 6 weeks, LTα was significantly higher in the spleen than in the control group compared to that derived from WT CD45.2 BM cells. - / - CD45.2 CD4 derived from BM cells + Foxp3 + An increase in the frequency and number of Tregs was found (data not shown). - / - CD45.2 Tregs showed enhanced expression of Il10, Ebi3, Tgfb1, Ifng, Gzmb, and Fasl genes compared with WT CD45.2 Tregs (Figure 3A). These data indicate that expression of LTα in hematopoietic cells negatively regulates the immunosuppressive signature of Treg cells.

[0138] Since it was observed that Tregs express LTα as a membrane-anchored LTα1β2 heterocomplex (data not shown), we assessed the contribution of the LTα1β2 / LTβR axis in controlling the inhibitory signature of Tregs. In particular, we analyzed whether blocking the LTα1β2 / LTβR interaction between Tregs and dendritic cells would affect the inhibitory signature of Treg cells. To this end, we analyzed purified WT CD4+ cells with or without preincubation with soluble LTβR-Fc fusion protein. + CD25 + Tregs were isolated using purified CD11c +Tregs were co-cultured with dendritic cells. Interestingly, Tregs pre-incubated with LTβR-Fc upregulated the expression of several genes related to Treg suppressive function, such as Klrg1, Il10, Ebi3, Tgfb1, Gzmb, and Fasl, compared with non-pretreated Tregs (Figure 3B). Thus, these data indicate that the LTα1β2 / LTβR interaction between Tregs and dendritic cells negatively regulates the suppressive signature of Tregs.

[0139] Expression of LTα is conserved in peripheral blood-derived human Tregs We next assessed whether LTα expression was conserved in human Tregs derived from peripheral blood of female and male healthy donors. + CD4 + Classically, Tregs are CD4 + CD25 + CD127 lo Intracellular LTα protein (data not shown) and cell surface LTα1β2 heterocomplexes (data not shown) were virtually detected by flow cytometry in Tregs from all donors analyzed, indicating that this expression is conserved from mouse to human.

[0140] Discussion Several studies have identified a number of molecules involved in the positive regulation of Treg cell development and function. In contrast, few reports have described signals that negatively regulate Treg function. Here, by analyzing distinct T cell populations endowed with regulatory properties, we find that Foxp3+ Tregs express substantially LTα as a membrane-anchored LTα1β2 heterocomplex. LTβR - / - As in mice, LTα - / - Mice express CD4 in the thymus + Foxp3 + It does not show any obvious defect in Treg cell development. On the other hand, the gene signature related to suppressive function is - / -Both LTα and LTβR expression in Tregs and dendritic cells were significantly increased in IL-1 and IL-2, indicating that LTα, and more precisely, the LTα1β2 / LTβR interaction between Tregs and dendritic cells, negatively regulates their immunosuppressive signature.

[0141] Interestingly, LTα - / - We have previously demonstrated that adoptive transfer of Tregs protects against DSS-induced colitis (Figure 1) and treats IBD more efficiently than WT Tregs, as compared to mice injected with LTα - / - In mice transferred with Tregs, this was reflected by reduced weight loss, increased colon length, and reduced histological scores. - / - We observed that Tregs substantially reduced colon inflammation and infiltration of inflammatory immune cells. In the DSS-induced colitis model, LTα - / - It was found that the transfer of Tregs reduced the priming and / or proliferation of Th1 and Th17 pathogenic cells in the mesenteric lymph nodes. Importantly, the ratios of Tregs / Th1 and Tregs / Th17 were elevated in the colon in both DSS-induced colitis and IBD models, suggesting that Tregs can also exert their suppressive effects locally in this tissue. The ability to suppress colonic inflammation may be due to the LTα - / - Adoptive transfer of Tregs also attenuates the development of CAC, which is known to be driven by chronic inflammation. Altogether, these data suggest that LTα - / - It has been shown that Tregs exhibit a higher ability to treat colitis and protect against both colitis and CAC development. It has also been shown that Tregs and dendritic cells, particularly Sirpα + We also found that LTα1β2 / LTβR interaction between conventional and plasmacytoid dendritic cells negatively regulates the suppressive signature of Treg cells, suggesting that direct cell contact with antigen-presenting cells regulates Treg suppressive activity.

[0142] Herein, adoptive transfer of WT Tregs pre-incubated with soluble LTβR-Fc resulted in the upregulation of LTα - / - We show that WT Tregs are protected from the development of ulcerative colitis as effectively as soluble LTβR-Fc (Figures 2B-C). Furthermore, WT Tregs pre-incubated with soluble LTαR-Fc were protected from the development of ulcerative colitis as effectively as soluble LTβR-Fc (Figures 2B-C). - / - These results suggest that LTβR-Fc-induced IBD is a potent inhibitory factor for Tregs, and that LTαR-Fc-induced IBD is a potent inhibitory factor for Tregs. - / - It is also expected to attenuate the development of CAC as effectively as adoptive transfer of Tregs.

[0143] References Throughout this application various references are included to describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. [Table 2]

Claims

1. pre-incubated with an effective amount of a lymphotoxin alpha blocker, Regulatory T cells.

2. The regulatory T cell of claim 1 , wherein the lymphotoxin alpha blocker is a soluble lymphotoxin beta receptor (LTβR).

3. A population of regulatory T cells according to claim 1 or 2.

4. The regulatory T cell according to claim 1 , which expresses a TCR or a chimeric antigen receptor that recognizes / binds to an autoantigen.

5. The population of regulatory T cells according to claim 4.

6. transfecting or transducing regulatory T cells in vitro or ex vivo with a vector encoding a chimeric antigen receptor or TCR; A method for producing the regulatory T cells according to claim 4.

7. 10. The population of regulatory T cells according to claim 3 and / or 5 for use in adoptive cell therapy in a subject in need thereof.

8. A population of regulatory T cells according to claim 3 and / or 5. Pharmaceutical compositions.

9. 1. An ex vivo method for stimulating regulatory T cell immunosuppressive activity, comprising: i. obtaining a biological sample from a subject; ii. isolating regulatory T cells from the sample; iii. Expanding regulatory T cells in vitro; iv. incubating the isolated regulatory T cells with an effective amount of a lymphotoxin alpha blocking agent to block LTα1β2 / LTβR or LTα2β1 / LTβR interaction; method.

10. The method of claim 9 , wherein the biological sample is a blood sample.

11. 1. A method of treating or preventing an autoimmune disorder in a subject in need thereof, comprising: Administering a therapeutically effective amount of a population of regulatory T cells according to claim 3 or / and 5 to a subject. method.

12. 2. The method of claim 1, wherein the autoimmune disorder is inflammatory bowel disease.

13. 1. A method for treating or preventing inflammation-associated cancer or allergy in a subject in need thereof, comprising: Administering a therapeutically effective amount of the population of regulatory T cells according to claim 3 to a subject. method.

14. 2. The method of claim 1, wherein the inflammation-associated cancer is colitis-associated cancer.

15. 1. A method for treating or preventing an immune response to an exogenously administered molecule or to a transplanted tissue or cell in a subject in need thereof, comprising: Administering a therapeutically effective amount of the population of regulatory T cells according to claim 3 to a subject. method.

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