WO2012143516A1

WO2012143516A1 - Amplification of regulatory t cells by means of anti-thymocyte immunoglobulins with cytokine, inhibitor of tor protein kinase and/or differentiating agent

Info

Publication number: WO2012143516A1
Application number: PCT/EP2012/057281
Authority: WO
Inventors: Janine BERNAUD; Christian Bloy; Virginie MATHIAS-CHAPON; Dominique Rigal
Original assignee: Genzyme Polyclonals S.A.S.
Priority date: 2011-04-20
Filing date: 2012-04-20
Publication date: 2012-10-26
Also published as: FR2974369B1; FR2974369A1; AU2012245161A1

Abstract

The invention relates to an ex vivo method of preparing regulatory T cells from CD4+ T cells and anti-thymocyte immunoglobulins with cytokine, inhibitor of TOR protein kinase and/or differentiating agent, as well as the regulatory T cells that can be obtained by this method, a pharmaceutical composition containing them, and uses thereof for treating and/or preventing graft-versus-host disease, post-transplantation tissue or organ rejection, an autoimmune disease, an allergy, or diabetes. The invention also relates to a kit for amplifying regulatory T cells.

Description

Amplification of regulatory T cells by means of anti-thymocyte immunoglobulins with cytokine, inhibitor of TOR protein kinase and/or differentiating agent The invention relates to an ex vivo method of preparing regulatory T cells from CD4+

T cells and anti-thymocyte immunoglobulins with cytokine, inhibitor of TOR protein kinase and/or differentiating agent, as well as the regulatory T cells that can be obtained by said method, a pharmaceutical composition containing them, and uses thereof for treating and/or preventing graft-versus-host disease, post-transplantation tissue or organ rejection, an autoimmune disease, an allergy, or diabetes. The invention also relates to a kit for amplifying regulatory T cells.

The immune response that aims to ensure the integrity of an organism against aggressive factors, either external (microbiological agents or exogenous substances) or internal (cancer, autoimmunity, ageing) is governed by means that effect rejection or tolerance of a target called an antigen. These effectors are controlled by mechanisms of amplification (amplifying or "helper" cells) or of suppression (suppressor or regulatory cells).

There are several populations of regulatory T cells (Treg). However, one of them seems to have a major role in the arm of down-regulation of the immune response. This population of regulatory cells is characterized by specific surface and intracytoplasmic markers. Classically it is described as a population of CD4+CD25+Foxp3+ T cells and can express various supplementary markers such as CTLA4+, CD40L+, GITR+, GranzymeA+, Granzyme B+, ILT3+, ILT4+, CD109+, IL10+, CD127dim, CD62L+.

This population has two origins: one arises directly from intrathymic differentiation of CD4 cells into so-called "natural" regulatory T cells (nTreg) and the other is derived from the peripheral CD4 cells following stimulation of the effector system, producing "induced" regulatory CD4 cells (iTreg).

It is the latter that biologists wish to produce by inducing their differentiation and their expansion by various methods of stimulation. To date, the best induction is achieved with the combination of anti-CD3 (TCR) and anti-CD28 agonistic monoclonal antibodies. Unfortunately this TCR/CD28 signal is insufficient to induce the iTreg cells and this combination also induces effector cells and does not allow a totally pure population of iTreg cells to be obtained. Other factors are required for the induction of iTreg cells, such as solicitation of the gamma receptors of IL2 and TGF3. Now, the use of these signals is still insufficient to obtain a population of iTreg cells that is pure and effective.

Immunomanipulation of this regulatory branch with the cells supporting this activity represents a rational therapeutic means for treating autoimmune diseases, for preparing the organism to receive a tissue or organ transplant, or for treating situations of a runaway immune response or post-transplantation organ or tissue rejection, such as graft-versus-host disease (GvHD). In its primary conception the anti-lymphocyte serum (ALS) was intended to destroy the principal cells causing rejection of allogenic grafts. The objective was fully achieved, so that this product, developed in various ways, quickly became an important element in the pharmacopoeia for immunosuppression in organ or tissue transplantation. Thymoglobulin® (rabbit anti-human thymocyte immunoglobulins) has become the major product in this therapeutic class. It is produced by immunizing rabbits with human thymus, as described in international patent application WO 2002/026830. It is a preparation of IgG type polyclonal immunoglobulins. It was discovered some years ago that the lymphopenia- inducing effect of Thymoglobulin® does not represent the only mechanism of action. It has in fact been found that Thymoglobulin® also possesses the property of inducing the expansion of regulatory lymphocyte subpopulations (Feng et al., Blood, 1 1 1 (7), 3675-3683), opening up new therapeutic prospects.

The capacity of anti-thymocyte immunoglobulins (ATG) for inducing iTreg cells can be utilized for treating, notably, a severe pathology that arises following transplants of haematopoietic stem cells, namely graft-versus-host disease or GvHD.

GvHD is one of the major complications of allografts of haematopoietic stem cells and represents the first cause of mortality apart from relapse in familial grafts. It mainly affects the skin, alimentary canal and liver. Treatment of acute GvHD is based on adding, to the existing preventive immunosuppressant treatment (by calcineurin inhibitor or mycophenolate), corticotherapy (at 2 mg/kg/day), which is reduced once the response is obtained. There is no reference treatment beyond this line of treatment, nor in case of incomplete response or cortico-dependence.

Chronic graft-versus-host disease (chronic GvHD) takes the form of an auto-immune attack through deficiency of immune tolerance. The principal signs are cutaneous (even as far as scleroderma) and mucosal, affecting the digestive tract, sicca syndrome, and/or hepatic pulmonary effects or others. Moreover, in chronic GvHD, the first line of treatment of extensive forms (or IBMTR score > 3) is based on corticotherapy at 1 mg/kg/day whether or not combined with an immunosuppressant. In the case of incomplete response or of cortico- dependence, or beyond a first line of treatment, there is no reference treatment.

Reconstitution of regulatory T lymphocytes, evaluated by the CD4+ CD25+ Foxp3+ markers, is deficient for at least 6 to 12 months post-graft in humans, and the richness of the grafts of this cellular type, whether they are derived from bone marrow or from peripheral blood, is also very limited. The feasibility of the therapeutic use of regulatory T lymphocytes in humans is now established, based on regulatory T lymphocytes highly purified by flow cytometry, and then amplified. At present, there have not been any reports of undesirable effects in terms of tolerance. However, this type of procedure is very laborious and has low reproducibility. Now, in the blood of a healthy subject, the CD4+CD25+ cells represent almost all of the regulatory T cells and sorting just by the CD25+ marker is sufficient to obtain purification > 70% of these cells. Knowing that the objective is to obtain between 5.10⁵ and 1 .10⁶ cells per kg, it is possible to purify, based only on CD25 sorting, in GMP conditions, a lymphocyte population usable in therapeutics starting from cytapheresis of the donor. The quantity of non-regulatory T cells re-infused must not, however, exceed the threshold of 5.10⁵/kg, which is the maximum threshold permitted for sorting in haploidentical grafts (grafts in a situation of maximum risk of GvHD), which will limit the number of Treg cells re-infused and consequently will limit the efficacy of this cellular therapy.

That is why it is urgent to have ready availability of large amounts of purified iTreg cells for treating patients of this type.

The capacity of rabbit anti-human thymocyte immunoglobulins Thymoglobulin® (Genzyme, Cambridge, MA, USA) to induce CD4+CD25+Foxp3+ T cells from mononucleated cells of the peripheral blood (Feng et al., Blood, 2008, 1 1 1 (7), 3675-3683) or from CD25+ T cells of the peripheral blood (Sewgobind et al., Transplantation. 2010; 89(6):655-66) has been described. According to Feng et al. (Blood, 2008, 1 1 1 (7), 3675- 3683), horse anti-human thymocyte immunoglobulins ATGAM (Pharmacia & Upjohn, Kalamazoo, Ml, USA) would in contrast lead to a decrease in the absolute number and in the percentage of CD4+CD25+Foxp3+ T cells obtained from mononucleated cells of the peripheral blood. Moreover, maintaining CD4+CD25+Foxp3+ cells in culture in the presence of Thymoglobulin® leads progressively to apoptosis of the cells beyond 24 hours of culture (Feng et al., Blood, 2008, 1 1 1 (7), 3675-3683).

A certain number of obstacles have to be overcome before achieving expansion ex vivo of Treg cells for administering them to patients, as reported by Sakaguchi et al., Nat Rev Immunol. 2010, 10(7), 490-500. In fact, effector regulatory T cells are quick to enter apoptosis and they multiply with great difficulty, even in culture in the presence of high doses of IL2. Moreover, the use of high doses of IL2 can induce the production of pro-inflammatory cytokines by a non-negligible fraction of T cells. The development of a cocktail of cytokines and of chemical agents to permit expansion of pure functional regulatory T cells therefore seems desirable. The incorporation of rapamycin in such a cocktail has been suggested, so as to increase the purity with respect to Treg cells (Sakaguchi et al., Nat Rev Immunol. 2010, 10(7), 490-500).

However, to date, no amplification of iTreg cells after induction by anti-thymocyte immunoglobulins (ATG) has been reported.

The present application therefore relates to a method of induction and of amplification of effector regulatory T cells from CD4+ T cells, by means of anti-thymocyte immunoglobulins. The regulatory T cells thus produced find application for treating and/or preventing GvHD, post-transplantation tissue or organ rejection, autoimmune diseases and allergies.

Method of preparation of regulatory T cells

The invention therefore relates to an ex vivo method of preparing regulatory T cells that comprises, or consists of, the steps consisting of:

a) cultivating CD4+ T cells in a culture medium containing anti-thymocyte immunoglobulins;

b) cultivating the cells obtained in step a) in a culture medium containing anti- thymocyte immunoglobulins and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent; and

c) collecting the CD4+CD25+Foxp3+ cells obtained in step b), the CD4+CD25+Foxp3+ cells being Treg cells. "Regulatory T cells" or "Treg" means CD4 T cells expressing at least the CD25 and

Foxp3 markers, i.e. CD4+CD25+Foxp3+ cells, in particular CD4+CD25++Foxp3+ cells, also called CD4+CD25^hlQhFoxp3+. The regulatory T cells can also present one or more of the following additional phenotypic markers of Treg: GITR, CTLA4, CD40L, GITR, Granzyme A, Granzyme B, ILT3, ILT4, CD109, IL10, CD127dim, CD62L, and CCR4. The Treg cells according to the invention can in particular be CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+ cells or

CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+ cells. They are more specifically induced regulatory T cells (iTreg).

Appropriate antibodies for detecting these various phenotypic markers are commonly available commercially to enable a person skilled in the art to determine whether a cell expresses one or other of the markers of regulatory T cells.

Advantageously, the method of preparing regulatory T cells according to the invention leads to functional Treg cells being obtained, i.e. cells capable of exerting immunosuppression.

The functional Treg cells express the GARP (glycoprotein-A repetitions predominant) marker, also called LRRC32 (leucine-rich repeat-containing protein 32), and the LAP (latency-associated peptide) marker, which is a molecule binding the GARP receptor. The expression of GARP can be detected principally at the intracellular level, but also at the membrane level. The functional Treg cells can therefore be identified by detecting the expression of the GARP/LRRC32 and/or LAP marker, for example by means of anti- GARP/LRRC32 or anti-LAP antibodies, as appropriate. The functional Treg cells can thus be for example CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+GARP+LAP+, or

CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ cells.

The functionality of the Treg cells can also be verified by any means known by a person skilled in the art, such as a test of inhibition of the allogenic reaction, also called mixed lymphocyte reaction. Such a test is described in example 1 of the present application. Briefly, so-called "responder" CD4 cells are mixed with so-called "stimulator" mononucleated homologous or allogenic cells, which have been irradiated so as to stop their proliferation, in the presence or absence of Treg cells that have been irradiated, and cultivated for, for example, 5 days of culture at 37°C and 5% C0₂. Tritiated thymidine is then added to the culture medium, and after, for example, a further 16 hours of culture, the level of incorporation of the tritiated thymidine by the cells is measured. A reduced amount of tritiated thymidine incorporated in the responder CD4 cells cultivated in the presence of Treg cells, relative to the mononucleated cells cultivated in the absence of Treg cells, is indicative of functional Treg cells, capable of exerting immunosuppression. The method of evaluating the function of Treg cells as described in the article of Feng et al. (Blood, 2008, 1 1 1 (7), 3675- 3683) can also be used. In the context of the method according to the invention, the CD4+ T cells, and therefore the regulatory T cells obtained from these cells, can be cells from a human or non- human mammalian subject, such as a primate (in particular humans, monkey), a rodent (in particular rat, mouse), a feline (in particular cat), a canine (in particular dog), an equine (in particular horse), a bovine (in particular cow), a sheep, or a pig.

The CD4+ T cells used in the context of the method according to the invention can come from various sources. They may be CD4+ T cells obtained from the peripheral blood of a healthy subject, of a subject intended to receive a tissue and/or organ graft, of a subject who has an autoimmune disease, or of a subject who has an allergy, i.e. for example a subject who has shown clinical signs of allergy (such as allergic rhinitis, allergic asthma, conjunctivitis, eczema). The CD4+ T cells can be obtained from the peripheral blood of a donor by any means known by a person skilled in the art, such as by cytapheresis, after administration of G-CSF or GM-CSF to the donor.

The CD4+ T cells can also be obtained from umbilical cord blood.

The CD4+ T cells can be purified from peripheral blood or from umbilical cord blood, from mononucleated cells isolated for example by Ficoll gradient centrifugation, and selection of the CD4+ cells from the mononucleated cells collected. Selection of the CD4+ cells can be positive selection carried out for example by means of magnetic beads covered with anti- CD4 monoclonal antibodies, as described in example 1 below, or by sorting by flow cytometry. Negative selection of the CD4+ cells, by removing the CD4- cells for example using cocktails of antibodies, gives the same results.

Preferably, after purification, the CD4+ T cells do not undergo a step of stimulation prior to being put in culture with the anti-thymocyte immunoglobulins according to step a). In other words, preferably in the method of preparing regulatory T cells according to the invention, purified CD4+ T cells are only maintained in a base culture medium, with or without serum, preferably for no more than 24 or 12 hours, prior to step a). Preferably, the anti-thymocyte immunoglobulins are directed against thymocytes of the same mammalian species as the species from which the CD4+ T cells are derived. In other words, and by way of illustration, the anti-thymocyte immunoglobulins will preferably be anti-human thymocyte immunoglobulins when the CD4+ T cells are human cells.

According to one embodiment, the anti-thymocyte immunoglobulins (ATG) are anti- human thymocyte immunoglobulins, in particular rabbit anti-human thymocyte immunoglobulins or horse anti-human thymocyte immunoglobulins. Contrary to the teaching of Feng et al. (Blood, 2008, 1 1 1 (7), 3675-3683), the inventors have in fact shown that Treg cells can be induced from CD4+ T cells by means of horse ATG. The ATGs are preferably the product Thymoglobulin® (Genzyme, Cambridge, MA, USA) or Lymphoglobulin® (Genzyme, Cambridge, MA, USA), both being commercially available.

The anti-human thymocyte immunoglobulins can be obtained by a method as described in international patent application WO 2002/026830. This method comprises or consists of:

(i) injecting a cellular preparation of human thymocytes into a specific pathogen free (SPF) animal;

(ii) collecting the human serum produced by the animal;

(iii) isolating the anti-human thymocyte immunoglobulins from the serum, without haemadsorption on erythrocytes, nor adsorption on human tissues, stroma or coarse extracts of these tissues.

The cellular preparation of human thymocytes used in step (i) can consist of cells of cell lines in culture, or can have been obtained by purification of fresh human thymocytes, preferably purified from fragments of thymus or optionally from suspensions of spleen, of tonsils, of lymph nodes, of thoracic trachea or of the peripheral blood. The fragments of human thymus can easily be taken during surgery, notably following cardiac surgery on children.

The SPF animal can in particular be a rabbit (Thymoglobulin®) or a horse (Lymphoglobulin®). In this method, the isolating step (iii) can employ ion-exchange chromatography and/or one or more precipitations. In particular, step (iii) employs ion-exchange chromatography on an anion exchange column (DEAE) at room temperature, followed by two successive steps of precipitation with sodium sulphate.

The immunoglobulins are not retained by the column and are quickly eluted from the column, which means they can be collected selectively. Chromatography on a specific affinity column, removing the antibodies that are still undesirable, can be applied in addition.

Steps of filtration/concentration/diafiltration can be employed for concentrating the preparation of immunoglobulins thus obtained.

According to another embodiment, the anti-thymocyte immunoglobulins are anti-non- human mammalian thymocyte immunoglobulins.

In steps a) and b), the culture media used comprise, or consist of, for step a) a "base culture medium" with ATG added, and for step b), a "base culture medium" with addition of ATG and said at least one cytokine and/or inhibitor of TOR protein kinase and/or a differentiating agent.

A suitable "base culture medium" for carrying out steps a) and b) can be a synthetic medium with or without serum, commonly available commercially, such as a serum-free medium of the RPMI or X-VIVO type (Lonza Walkersville, Inc.), or a medium with serum IMDM or DMEM. The presence of serum in the culture medium is not obligatory but improves the culture results. The base culture medium can classically contain antibiotics or have them added to prevent contamination during cell culture, and glutamine. A suitable base culture medium is typically an RPMI medium preferably with addition of 10% (vol/vol) of a pool of human serum AB, inactivated by heating, or of 10% (vol/vol) of fetal calf serum, optionally with further addition of 2 mM of glutamine and antibiotics such as 100 lU/ml of penicillin, and 100 μg/ml of streptomycin.

A base serum-free culture medium typically contains inorganic salts (such as Ca(N0₃)₂, MgS0₄, KCI, NaHC0₃, NaCI, NaHC0₃ and Na₂HP0₄), amino acids (in particular a mixture of the 20 natural amino acids), vitamins (such as D-biotin, choline chloride, folic acid, myo-inositol, niacinamide, p-aminobenzoic acid, D-pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B-12), or carbohydrate(s) (in particular glucose). A base serum-free culture medium can also contain transferrin, insulin, or albumin. A base serum-free culture medium also generally contains at least one antioxidant (for example reduced glutathione), at least one pH buffer (for example Hepes), and at least one pH indicator (for example phenol red).

According to one embodiment, the culture medium in step a) does not contain any additive, notably cytokine, growth factor, or chemical agent, etc., other than the anti- thymocyte immunoglobulins and serum/antibiotic(s)/glutamine optionally present or added to the base culture medium. In other words, according to one embodiment, the only additives, in particular cytokines, growth factors, or chemical agents, present in the culture medium in step a) are the anti-thymocyte immunoglobulins and optionally serum and/or antibiotic(s) and/or glutamine. Thus, preferably the culture medium in step a) consists of water, inorganic salts, amino acids, vitamins, carbohydrates, anti-thymocyte immunoglobulins and optionally, or preferably in addition, serum, at least one antibiotic, and transferrin and/or insulin and/or albumin and/or at least one antioxidant and/or at least one pH buffer and/or at least one pH indicator. In particular the medium in step a) does not comprise added cytokines other than those contained in the serum optionally added.

Also, or alternatively, the culture medium in step b) need not contain any additive, notably cytokine, growth factor, or chemical agent, etc., other than the anti-thymocyte immunoglobulins and said at least one cytokine and/or inhibitor of TOR protein kinase and/or differentiating agent as described hereunder, as well as the serum/antibiotic(s)/glutamine optionally present or added to the base culture medium. In other words, according to one embodiment, the only additives, in particular cytokines, growth factors, or chemical agents, present in the culture medium in step b) are the anti-thymocyte immunoglobulins and said at least one cytokine and/or inhibitor of TOR protein kinase and/or differentiating agent as described hereunder, as well as optionally serum and/or antibiotic(s) and/or glutamine. Thus, preferably the culture medium in step b) consists of water, inorganic salts, amino acids, vitamins, carbohydrates, anti-thymocyte immunoglobulins, cytokine(s), at least one inhibitor of TOR protein kinase and/or at least one differentiating agent, and the medium is constituted optionally, or preferably in addition, of serum, at least one antibiotic, and transferrin and/or insulin and/or albumin and/or at least one antioxidant and/or at least one pH buffer and/or at least one pH indicator. In particular the medium in step b) does not contain cytokines other than i) one or more cytokine(s) selected from the group consisting of IL2, IL4, IL7, IL9, IL15 and IL21 , preferably from the group consisting of IL2 and IL15, and optionally ii) the cytokines contained in the serum, if serum is added.

The serum is generally added to the culture medium (when the base culture medium is a serum-free medium) at a rate of 5-15%, preferably 8-12%, in particular at about 10%, the percentage being expressed in volume of serum relative to the total volume of the final medium (i.e. with addition of serum). The serum is in general human serum AB, inactivated by heating, or fetal calf serum.

The culture steps a) and b) can be carried out conventionally at 37 ^<Ό and with 5%

C0₂. In step a) of the method according to the invention, the CD4+ T cells are cultivated in a culture medium containing the anti-thymocyte immunoglobulins (ATG) in conditions and for a sufficient time to induce Treg cells as defined above.

The CD4+ T cells can be seeded, for example, at a concentration from 0.1 .10^s to 5.10⁶ cells/ml_, in particular 0.5.10⁶ to 3.10⁶ cells/mL, or 1 .10⁶ to 2.10⁶ cells/mL.

For example, the CD4+ cells can be cultivated for 6 to 48 h or for 6 to 36 h, preferably for 8 to 48 h, more preferably for 8 to 36 h, for 12 to 36 h, for 20 to 28 h, for 23 to 25 h, or for 24 h.

Culture can be carried out in the presence of 1 to 200 μg/mL of ATG, in particular 10 to 150 g/mL, 20 to 100 g/mL, 30 to 80 g/mL, 40 to 70 g/mL, 50 to 60 μg mL, or in the presence of 50 μg mL of ATG, the ATG preferably being Thymoglobulin®. Culture can also be carried out in the presence of 1 to 200 μg/mL of ATG, in particular 30 to 180 g/mL, 50 to 150 g/mL, 80 to 150 g/mL, or 100 to 130 μg/mL of ATG, the ATG preferably being Lymphoglobulin®.

All combinations of these culture times and concentrations of ATG are possible.

The cells obtained at the end of step a) are then cultivated during step b) in a medium containing anti-thymocyte immunoglobulins and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent.

Prior to culture according to step b), the cells obtained from step a) can be washed and reseeded, for example at a concentration of 0.1 .10^s to 5.10⁶ cells/mL, in particular 0.5.10⁶ to 3.10⁶ cells/mL, or 1 .10⁶ to 2.10⁶ cells/mL. In particular, the cells obtained from step a) can be washed with a phosphate-buffered saline solution (for example PBS 1 X) prior to culture thereof according to step b).

The anti-thymocyte immunoglobulins are preferably of the same type as those used for culture step a), but not necessarily. They can be used at a concentration from 1 to 200 g/mL of ATG, in particular from 10 to 150 g/mL, 20 to 100 g/mL, 30 to 80 g/mL, 40 to 70 μg/mL, 50 to 60 μg mL, or in the presence of 50 μg mL of ATG, the ATG preferably being Thymoglobulin®. They can also be used at a concentration from 1 to 200 μg/mL of ATG, in particular 30 to 180 μg mL, 50 to 150 μg mL, 80 to 150 μg mL, or 100 to 130 μg/mL of ATG, the ATG preferably being Lymphoglobulin®. In particular, the anti-thymocyte immunoglobulins can be present in the culture media of steps a) and b) at one and the same concentration, but not necessarily.

An inhibitor of TOR ("Target Of Rapamvcin") protein kinase can be rapamycin (also called sirolimus, CAS number 53123-88-9) and/or an analogue thereof, namely temsirolimus (CAS number 162635-04-3) and everolimus (CAS number 159351 -69-6). The inhibitor of TOR protein kinase can be present in the culture medium at a total concentration from 5 to 25 ng/mL, in particular from 7.5 to 15 ng/mL, or from 9 to 1 1 ng/mL, for example 10 ng/mL.

A differentiating agent can be selected from retinoic acid and derivatives thereof, in particular tretinoin (or all-trans-retinoic acid), isotretinoin (or 13-cis-retinoic acid), or alitretinoin (9-cis-retinoic acid). The differentiating agent can typically be present in the culture medium at a concentration from 3 to 5 μg/mL.

The culture medium in step b) containing the anti-thymocyte immunoglobulins can contain at least one cytokine, for example two, three, or four cytokines, in particular at least one (for example two, three, or four) cytokine(s) binding the gamma chain of the receptor of IL2 (CD132) and optionally of TGF-β. In the context of the present application, "cytokine binding the gamma chain of the receptor of IL2" means IL2, IL4, IL7, IL9, IL15 or IL21 . The culture medium in step b) can in particular contain IL2 and/or IL15, more preferably IL2 and IL15.

The concentration of IL2 in the culture medium can be from 50 to 500 lU/mL, in particular from 100 to 400 lU/mL, or from 150 to 300 lU/mL, for example 180-220 lU/mL, in particular 200 lU/mL. The concentration of IL15 in the culture medium can be from 5 to 50 ng/mL, in particular from 5 to 30 ng/mL, or from 7.5 to 15 ng/mL, for example from 10 to 12 ng/mL, in particular 10 ng/mL. The concentration of TGF3 in the culture medium can be from 2 to 5 ng/mL. The concentration of IL4, IL7, IL9, or IL21 in the culture medium can be from 1 to 100 ng/mL, in particular from 5 to 50 ng/mL, from 5 to 30 ng/mL, or from 50 to 70 ng/mL.

The culture medium in step b) can in particular contain the anti-thymocyte immunoglobulins and

(i) at least one cytokine (in particular two, three, or four cytokines) and/or an inhibitor of TOR protein kinase;

(ii) at least one cytokine (in particular two, three, or four cytokines) binding the gamma chain of the receptor of IL2 and/or an inhibitor of TOR protein kinase;

(iii) at least one cytokine (in particular two, three, or four cytokines) and an inhibitor of TOR protein kinase;

(iv) at least one cytokine (in particular two, three, or four cytokines) binding the gamma chain of the receptor of IL2 and an inhibitor of TOR protein kinase; (v) two, three, or four cytokines binding the gamma chain of the receptor of IL2 including IL2 and/or IL15, and optionally or preferably in addition an inhibitor of TOR protein kinase; or

(vi) IL2, IL15 and at least one other cytokine, in particular at least one other cytokine selected from IL4, IL7, IL9 or IL21 , and an inhibitor of TOR protein kinase, in particular rapamycin. In particular, in the culture medium in step b), the additives added to the base culture medium can be limitatively those listed above, and optionally serum and/or antibiotic(s) and/or glutamine.

Thus, the medium containing anti-thymocyte immunoglobulins and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent, can in particular consist of said "base culture medium" with addition of:

- anti-thymocyte immunoglobulins, in particular Thymoglobulin® or Lymphoglobulin®;

- at least one cytokine binding the gamma chain of the receptor of IL2, in particular one, two, three, or four cytokines selected from the group consisting of IL2, IL4, IL7, IL9, IL15 and IL21 ; preferably IL2 and IL15;

- rapamycin and/or temsirolimus and/or everolimus, preferably rapamycin; and

- optionally serum and/or antibiotic(s) and/or glutamine

and the ATG, said at least one cytokine binding the gamma chain of the receptor of IL2, and rapamycin and/or temsirolimus and/or everolimus can be present in the culture medium in the ranges of concentrations stated above.

Culture according to step b) can be carried out for a minimum of 1 day and up to 14 days (for example from D1 to D15, if step a) lasts 24 hours), in particular up to 1 1 days (for example from D1 to D13, if step a) lasts 24 hours), up to 9 days (for example from D1 to D10, if step a) lasts 24 hours), up to 7 days (for example from D1 to D8, if step a) lasts 24 hours), or up to 4 days (for example from D1 to D5, if step a) lasts 24 hours). The minimum duration of culture step b) can be 3 days, 5 days or 7 days, for example. Thus, the culture time in step b) can be for example from 3 to 1 1 days, or from 5 to 9 days.

During culture according to step b), the cultivated cells can, at least once, be washed and then diluted for reseeding at a concentration from 0.1 .10^s to 5.10⁶ cells/mL, in particular 0.5.10⁶ to 3.10⁶ cells/mL, or 1 .10⁶ to 2.10⁶ cells/mL. This washing/reseeding can be applied for example after 1 to 4 days, in particular after 3 to 4 days of culture according to step b), notably when the total culture time according to step b) is from 7 to 14 days. Washing can be carried out with phosphate-buffered saline solution (for example PBS 1 X). According to a first embodiment, the method according to the invention comprises the steps consisting of:

a) cultivating CD4+ T cells in a culture medium containing anti-thymocyte immunoglobulins for 6 to 48 h;

a1 ) washing the cells obtained in step a);

b) cultivating the washed cells from step a1 ) in a culture medium containing anti- thymocyte immunoglobulins and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent, for 1 to 4 days, in particular for 3 to 4 days; b1 ) washing the cells obtained in step b);

b2) cultivating the washed cells from step b1 ) in the culture medium defined in step b), i.e. a fresh culture medium containing anti-thymocyte immunoglobulins, and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent, for a maximum of 6 or 1 0 days, in particular for 2 to 6 days or for 5 to 1 0 days, in particular for 2 to 5 days;

c) collecting the CD4+CD25+Foxp3+ cells obtained in step b2), the CD4+CD25+Foxp3+ cells being Treg cells.

Preferably, in this embodiment, the duration of step b2) is at most 1 0 days when step b) took 1 to 2 day(s). Preferably, the duration of step b2) is at most 6 days when step b) took 3 to 4 day(s).

According to a second embodiment, the method of preparing regulatory T cells according to the invention comprises, or consists of, the steps of:

a) cultivating, for 6 to 48 h, in particular for 8 to 36 h, for 20 to 28 h, or in particular for 23 to 25 hours, in particular about 24 hours, CD4+ T cells in a culture medium containing the anti-thymocyte immunoglobulins (ATG), in particular Thymoglobulin® or Lymphoglobulin®, at a concentration from 1 to 200 g/mL, and in particular from 10 to 125 g/mL, from 20 to 1 00 μg/mL, from 30 to 80 g/mL, from 40 to 70 g/mL, or from 50 to 60 μg mL, preferably 50 μg mL (notably for Thymoglobulin®), or in particular from 30 to 180 g/mL, 50 to 150 g/mL, 80 to 1 50 g/mL, or 100 to 130 yg/mL (notably for Lymphoglobulin®);

a1 ) optionally or preferably washing the cells obtained in step a);

b) cultivating, for 1 to 14 days, in particular for 3 to 1 1 days, or 5 to 9 days, the cells obtained from step a), or from step a1 ) if applicable, in a culture medium containing

- said anti-thymocyte immunoglobulins (ATG), in particular Thymoglobulin® or Lymphoglobulin®, at a concentration from 1 to 200 g/mL, and in particular from 10 to 125 μg/mL, from 20 to 1 00 g/mL, from 30 to 80 g/mL, from 40 to 70 g/mL, or from 50 to 60 μg mL, preferably 50 μg mL (notably for Thymoglobulin®), or in particular from 30 to 1 80 g/mL, 50 to 1 50 g/mL, 80 to 150 g/mL, or 100 to 130 yg/mL (notably for Lymphoglobulin®) ;

- IL2 at a concentration from 50 to 500 lU/mL, in particular from 1 00 to 400 lU/mL, or from 1 50 to 300 lU/mL, for example 1 80-220 lU/mL, in particular 200 lU/mL;

- IL15 at a concentration from 5 to 50 ng/mL, in particular from 5 to 30 ng/mL, or from 7.5 to 1 5 ng/mL, for example from 1 0 to 12 ng/mL, in particular 1 0 ng/mL; and

- rapamycin, or an analogue thereof, at a concentration from 5 to 25 ng/mL, in particular from 7.5 to 15 ng/mL, or from 9 to 1 1 ng/mL, for example 1 0 ng/mL; and

c) collecting the CD4+CD25+Foxp3+ cells obtained in step b), the CD4+CD25+Foxp3+ cells being Treg cells. According to this second embodiment, culture step b) can be performed by b1 ) culture of the cells obtained from step a), or from step a1 ) if applicable, in said culture medium defined in step b) for 1 to 4 days, in particular for 3 to 4 days; then b2) washing the cells; b3) culture of the washed cells in said culture medium of step b1 ) for at most 6 or 10 days, in particular for 2 to 6 days or for 5 to 10 days, in particular for 2 to 5 days.

According to a third embodiment, the method of preparing regulatory T cells according to the invention comprises, or consists of, the steps of:

a) cultivating, for 20 to 28 hours, preferably for 23 to 25 hours, in particular about 24 hours, CD4+ T cells in a culture medium containing the anti-thymocyte immunoglobulins (ATG), in particular Thymoglobulin® at a concentration from 50 to 60 μg mL or Lymphoglobulin® at a concentration from 80 to 150 g/mL;

a1 ) optionally or preferably washing the cells obtained in step a);

b) cultivating, for 5 to 9 days, in particular for 9 days, the cells obtained from step a) or a1 ), if applicable, in a culture medium containing said anti-thymocyte immunoglobulins (ATG), in particular Thymoglobulin® at a concentration from 50 to 60 μg mL or Lymphoglobulin® at a concentration from 80 to 150 g/mL; and IL2 at a concentration of 180-220 lU/mL, in particular 200 lU/mL; IL15 at a concentration from 7.5 to 15 ng/mL, in particular 10 ng/mL; and rapamycin at a concentration from 9 to 1 1 ng/mL, for example 10 ng/mL; and

c) collecting the CD4+CD25+Foxp3+ cells obtained in step b), the

CD4+CD25+Foxp3+ cells being Treg cells.

According to a fourth embodiment, the method of preparing regulatory T cells according to the invention comprises, or consists of, the steps of

a) cultivating CD4+ T cells for 20 to 28 hours in a culture medium containing the anti- thymocyte immunoglobulins at a concentration from 50 to 60 μg mL;

a1 ) washing the cells obtained in step a);

b) cultivating the washed cells from step a1 ) for 1 to 4 days in a culture medium containing said anti-thymocyte immunoglobulins at a concentration from 50 to 60 μg mL, and IL2 at a concentration of 180-220 lU/mL, IL15 at a concentration from 7.5 to 15 ng/mL, and rapamycin at a concentration from 9 to 1 1 ng/mL;

b1 ) washing the cells obtained in step b);

b2) cultivating the washed cells from step b1 ) in the culture medium defined in step b) for 2 to 5 days;

According to these embodiments of the method according to the invention, step b) can consist of b1 a) cultivating, for 1 to 4 days, in particular 3 to 4 days, more particularly for 4 days, the cells obtained from step a) in a culture medium containing said anti-thymocyte immunoglobulins (ATG), in particular Thymoglobulin® at a concentration from 50 to 60 μg mL or Lymphoglobulin® at a concentration from 80 to 150 g/mL; and IL2 at a concentration of 180-220 lU/mL, in particular 200 lU/mL; IL15 at a concentration from 7.5 to 15 ng/mL, in particular 10 ng/mL; and rapamycin at a concentration from 9 to 1 1 ng/mL, for example 10 ng/mL;

bi b) washing the cells and reseeding them, for example at a concentration from 0.1 .10^s to 5.10⁶ cells/mL, in particular 0.5.10⁶ to 3.10⁶ cells/mL, or 1 .10⁶ to 2.10⁶ cells/mL, in a culture medium containing said anti-thymocyte immunoglobulins (ATG), in particular Thymoglobulin® at a concentration from 50 to 60 μg mL or Lymphoglobulin® at a concentration from 80 to 150 g/mL; and IL2 at a concentration of 180-220 lU/mL, in particular 200 lU/mL; IL15 at a concentration from 7.5 to 15 ng/mL, in particular 10 ng/mL; and rapamycin at a concentration from 9 to 1 1 ng/mL, for example 10 ng/mL; and

b2) cultivating the cells reseeded in step bi b) for 2 to 5 days, in particular for 5 days, in the culture medium defined in step b1 a).

All the particular characteristics disclosed with respect to the method according to the invention can of course be combined with these embodiments. The cumulative total culture time, for steps a) and b), can be from 2 to 15 days, in particular from 4 to 12 days, or from 7 to 10 days. In fact, prolongation of the total culture time beyond 15 days does not contribute to increasing the level of amplification of the Treg cells produced by the method according to the invention, because beyond 15 days, the Treg cells seem to become sensitive again to the anti-thymocyte immunoglobulins and gradually enter apoptosis.

Step c) of harvesting the cells can be applied for example by collecting all of the cells obtained at the end of step b), or b2) if applicable, and optionally, or preferably, then selecting the Treg cells. According to one embodiment, in step c), the CD4+CD25+Foxp3+ cells are harvested in the form of a cell population comprising CD4+CD25+Foxp3+ cells.

The Treg cells can for example be selected by performing sorting by flow cytometry or using beads, in particular magnetic beads, covered for example with anti-CD25 and/or anti-FoxP3 and/or anti-LAP and/or anti-GARP antibodies.

The Treg cells collected are in particular functional Treg cells, in particular CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells. The method according to the invention can include one or more supplementary steps, after step c), consisting for example of washing and/or purifying and/or concentrating the Treg cells, and/or freezing the Treg cells, and/or irradiating the Treg cells, and/or suspending the Treg cells in a pharmaceutically acceptable vehicle.

Preferably, the method according to the invention comprises, after step c), at least one supplementary step consisting of preparing a pharmaceutical composition comprising the Treg cells, in particular the functional Treg cells.

The Treg cells, and notably the functional Treg cells, can be irradiated to stop their proliferation while preserving their functional properties.

The method of preparation according to the invention makes it possible to obtain: cultures or cell populations containing from 50 to 80%, notably from 60 to 80%, or from 70 to 80% of CD4+/CD25+/FoxP3+ T cells, in particular CD4+CD25+Foxp3+GAPtP+LAP+ functional Treg cells, in particular CD4+CD25++Foxp3+ GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells; and

- an amplification factor of at least 20, 40, 60, 80, or 100 and in particular an amplification factor from 20 to 120, from 40 to 120, from 60 to 120, or from 80 to 120, of the total number of CD4+/CD25+/FoxP3+ cells, in particular of CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular of CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells. The amplification factor is calculated relative to the number of CD4+/CD25+/FoxP3+ cells, or CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells present among the CD4+ T cells cultured in step a), as appropriate.

Thus, the method of preparation according to the invention makes it possible to obtain, at the end of step b), a population of cells which comprises:

a) for example from about 15.10⁶ to 20.10⁶ CD4+ T cells, 3.10⁶, preferably 6.10⁶,

8.10⁶, 10.10^s, 12.10^s, 15.10^s, 17.10^s, 20.10^s, 22.10^s or 24.10^s CD4+/CD25+/FoxP3+ T cells, in particular CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+ GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells; and/or b) from 50 to 80%, notably from 60 to 80%, or from 70 to 80% of

CD4+/CD25+/FoxP3+ T cells, in particular CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells.

The method of preparation according to the invention also makes it possible to obtain, always for example starting from about 15.10^s to 20.10⁶ CD4+ T cells, 3.10⁶, preferably 6.10⁶, 8.10⁶, 10.10^s, 12.10^s, 15.10^s, 17.10^s, 20.10^s, 22.10^s or 24.10^s of these functional Treg cells. The total number of Treg cells, in particular of functional Treg cells that can be obtained is proportional to the number of CD4+ T cells put in culture. Thus, if about 30.10^s to 40.10^s CD4+ T cells were put in culture, then 6.10^s, 12.10^s, 16.10^s, 20.10^s, 24.10^s, 30.10^s, 34.10^s, 40.10^s, 44.10^s or 48.10^s of these functional Treg cells can be obtained.

These results were obtained in particular starting from CD4+ cells of the peripheral blood and after 1 day of induction culture with anti-thymocyte immunoglobulins, and 9 days of culture in the presence of anti-thymocyte immunoglobulins, IL2, IL15 and rapamycin.

Regulatory T cells and therapeutic applications

The invention also relates to the regulatory T cells, in particular the functional Treg cells, obtained, or that can be obtained, by the method of preparation according to the invention, and the therapeutic applications thereof. The Treg cells in fact constitute a cellular therapy product for the application of specific immunotherapies.

The regulatory T cells obtained, or that can be obtained, by the method of preparation according to the invention are present in a cell population comprising predominantly or essentially Treg cells, in particular functional Treg cells.

The invention therefore relates in particular to a population of cells that comprises predominantly or essentially Treg cells, in particular functional Treg cells, said cell population comprising, or consisting of:

a) for example starting from about 15.10^s to 20.10^s CD4+ T cells initially put in culture, at least 3.10^s, preferably 6.10^s, 8.10^s, 10.10^s, 12.10^s, 15.10^s, 17.10^s, 20.10^s, 22.10^s or 24.10^s CD4+/CD25+/FoxP3+ T cells, in particular CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells.

The total number of Treg cells, in particular of functional Treg cells that can be obtained being proportional to the number of CD4+ T cells put in culture. Since, from umbilical cord blood, generally from about 30.10^s to about 70.10^S CD4+ T cells can be purified; from a blood pouch generally from about 140.10^s to about 210.10^s CD4+ T cells can be purified; and from a peripheral blood stem cell donation, generally from about 100.10^s to about 500.10^s CD4+ T cells can be purified, this means that the method of the invention makes it possible to generate a cell population comprising or consisting of 100.10^s to 250.10^s CD4+/CD25+/FoxP3+ T cells, in particular at least 100.10⁶, preferably at least 120.10⁶, 140.10⁶, 160.10⁶, 180.10⁶, 200.10⁶, 210.10⁶, 220.10⁶, 230.10⁶, 240.10⁶ or 250.10⁶ CD4+/CD25+/FoxP3+ T cells, in particular CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells;

and/or

b) from 50 to 80%, notably from 60 to 80%, or from 70 to 80% of CD4+/CD25+/FoxP3+ T cells, in particular of CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular of CD4+CD25++Foxp3+GARP+LAP+ T cells, or

CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells.

The population of cells preferably consists of syngeneic cells. The population of cells is also preferably autologous to the subject intended to receive cell therapy.

Thus, the invention relates to the regulatory T cells according to the invention, and in particular said population of cells that comprises predominantly or essentially Treg cells, in particular functional Treg cells, for use for treating and/or preventing graft-versus-host disease, rejection of a tissue or organ transplant, an autoimmune disease or an allergy.

The invention also relates to a method of treating and/or preventing graft-versus-host disease, rejection of a tissue or organ transplant, an autoimmune disease, or an allergy, said method comprising the administration of regulatory T cells according to the invention, and in particular said population of cells that comprises predominantly or essentially Treg cells, in particular functional Treg cells, to a subject in need thereof.

The regulatory T cells according to the invention, and in particular said population of cells that comprises predominantly or essentially Treg cells, in particular functional Treg cells are preferably purified with a view to therapeutic use. Thus, said population of cells that comprises predominantly or essentially Treg cells, in particular functional Treg cells, comprises, or is preferably purified and consists of 80 to 100%, notably from 85 to 100%, 90 to 100%, 95 to 100% of CD4+/CD25+/FoxP3+ T cells, in particular of CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular of CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells.

In the context of the invention, the term "treat" or "treatment" means suppressing, alleviating or preventing the progression of a disorder or preventing the appearance of such a disorder or of one or more symptoms connected with said disorder.

The regulatory T cells constitute an immunosuppressant treatment for induction of tolerance with respect to transplanted organ(s) or tissue(s), or for preventing rejection of transplanted organ(s) or tissue(s), in particular during an autologous graft, an allograft (between two different subjects belonging to one and the same biological species, or homologous graft) or a xenograft (between two subjects belonging to two different but closely related biological species). The tissue and/or organ transplanted, also called graft, can in particular be bone marrow, kidney, liver, lung, heart, heart-lung block, pancreas, heart valve, cornea, or hand and part of the face.

"Graft-versus-host disease" or "GvHD" is a complication of allografts of bone marrow, of stem cells from peripheral blood or from umbilical cord blood, frequently observed when the donor and the recipient are incompatible and the recipient is profoundly immunodepressed and the graft contains immunocompetent cells such as T lymphocytes. A distinction is made between acute graft-versus-host disease, which generally occurs between 2 and 4 weeks after transplantation, and can last up to about 3 months, and chronic graft-versus-host disease, which generally appears after 100 days post-transplantation.

The regulatory T cells can also be useful for treating and/or preventing an autoimmune disease. "Autoimmune disease" denotes non-exhaustively lupus erythematosus, disseminated lupus erythematosus, rheumatoid arthritis, systemic scleroderma, multiple sclerosis, autoimmune haemolytic anaemias, autoimmune thrombopenia, polymyositides, dermatomyositides, vesicular pemphigus or pemphigus vulgaris, psoriasis, type 1 diabetes, Berger disease, Basedow disease, Hashimoto thyroiditis, primary myxoedema, coeliac disease, Crohn's disease, herpetiform dermatitis, myasthenia, haemorrhagic rectocolitis, primary biliary cirrhosis, primary sclerosing cholangitis, Biermer anaemia, CREST syndrome, acquired epidermolysis bullosa, Lambert-Eaton myasthenic syndrome, polymyositis, Goujerot-Sjogren syndrome, Guillain-Barre syndrome, optical neuritis, psoriasis, rheumatoid arthritis, bone-marrow aplasia, Reiter syndrome, primary biliary cirrhosis, antiphospholipid antibody syndrome, opsoclonus-myoclonus syndrome, temporal arteritis, acute disseminated encephalomyelitis, Goodpasture syndrome, Wegener granulomatosis, Churg-Strauss syndrome, sarcoidosis, nephrotic syndrome and La Peyronie disease.

The regulatory T cells can also be useful for treating and/or preventing an allergy, in particular an allergy associated with a deficiency of regulatory T cells. Allergy is a humoral response triggered in response to an allergen that is associated with secretion of IgE by the plasmocytes. An allergy may be associated with clinical manifestations such as eczema, asthma, allergic rhinitis, atopic dermatitis, conjunctivitis, and in the most severe cases anaphylactic shock. The allergens triggering allergies can be, non-exhaustively, pollen allergens (from trees, grasses, etc.), mite allergens (from house dust or storage), insect allergens (from hymenoptera, cockroaches, etc.), animal allergens (from dog, cat, horse, rat, mouse, etc.), mould allergens and food allergens. The Treg cells, and in particular said population of cells that comprises predominantly or essentially Treg cells, can be formulated in the form of a pharmaceutical composition comprising the Treg cells and a pharmaceutically acceptable vehicle. The Treg cells can in particular have been frozen and are then reconstituted before use in the form of a suspension in a pharmaceutically acceptable vehicle. The Treg cells can also have been irradiated, which stops their proliferation while maintaining their functional properties. Optionally, the Treg cells can be irradiated before or after freezing.

These pharmaceutical compositions also form part of the invention. In particular, a pharmaceutical composition according to the invention can be obtained by the method according to the invention, when the latter comprises at least one supplementary step consisting of preparing a pharmaceutical composition comprising CD4+CD25+Foxp3+ T cells, in particular CD4+CD25+Foxp3+GARP+LAP+ T cells.

"Pharmaceutically acceptable vehicle" means a vehicle suitable for use in contact with human or animal cells, without inducing toxicity, irritation, or undue allergic response. Non-limiting examples of pharmaceutically acceptable vehicles notably include physiological solution, i.e. having the same osmolarity as blood, and which can be a solution of doubly- distilled water containing 0.9 g/L of NaCI, or Ringer solution or Ringer lactate solution. The Treg cells can be suspended for example in a total volume of physiological solution of 10, 50, 100, 150, 200, 250, 300, 400, or 500 ml_.

The pharmaceutical composition preferably comprises a therapeutically effective amount of regulatory T cells, i.e. a sufficient amount for treating and/or preventing the disease in question. The amount of regulatory T cells and of the composition according to the present invention as well as the frequency of administration can be determined by clinical studies, by the doctor or by the pharmacist. The "therapeutically active" dose specific to each subject may depend on a number of factors such as the nature and severity of the disease to be treated, the composition used, the subject's age, weight, general state of health, sex and diet, the method of administration, the duration of treatment (in monodose or in several doses), and on the medicinal products used in combination and on other factors that are well known by medical specialists.

The pharmaceutical composition according to the invention can comprise a minimum of 0.5.10⁶ Treg cells/mL, in particular a minimum of 1 .10⁶ Treg cells/mL, or 2.10⁶ Treg cells/mL, or 2.5.10⁶ Treg cells/mL. The pharmaceutical composition preferably contains at least 100.10⁶, preferably at least 120.10⁶, 140.10⁶, 160.10⁶, 180.10⁶, 200.10⁶, 210.10⁶, 220.10⁶, 230.10⁶, 240.10⁶ or 250.10⁶ Treg cells. The pharmaceutical composition may preferably comprise or consist of 100.10⁶ to 250.10⁶ Treg cells. They are in particular functional Treg cells, notably CD4+CD25+Foxp3+GARP+LAP+ T cells, in particular CD4+CD25++Foxp3+GARP+LAP+ T cells, or CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells or CD4+CD25++Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells.

The regulatory T cells or the pharmaceutical composition according to the invention can be administered to a subject in need thereof by the intravenous, intraperitoneal, intramuscular, or topical route, or in a lymph node, notably by infusion or injection.

The regulatory T cells can be for heterologous use, i.e. the Treg cells can have been obtained from CD4+ cells from a subject other than the subject to be treated.

Preferably, the regulatory T cells are for autologous use, i.e. they are autologous cells from the subject to be treated. The "subject" can be a human or non-human mammal, as described above.

The regulatory T cells can be used in combination, for simultaneous or sequential administration, with another immunosuppressant treatment, such as ciclosporin A, tacrolimus, an inhibitor of TOR protein kinase as defined in the present application, mycophenolic acid, a glucocorticoid, methotrexate, cyclophosphamide (Endoxan), azathioprine, sulphasalazine, an anti-TNF antibody, an anti CD-20 antibody (for example Mabthera).

Kit

The invention also relates to a kit for amplifying regulatory T cells comprising, preferably exclusively:

- anti-thymocyte immunoglobulins; and

- one or more means for purifying CD4+ cells and/or

- at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent; and/or

- one or more markers for selection of

CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ cells.

Preferably, the kit according to the invention comprises, preferably exclusively:

- anti-thymocyte immunoglobulins; and

- two cytokines selected from the group consisting of IL2, IL4, IL7, IL9, IL15 and IL21 ; and

- and an inhibitor of TOR protein kinase; and

- optionally one or more means for purifying CD4+ cells and/or one or more markers for selection of CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ cells.

Said anti-thymocyte immunoglobulins, at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent are as defined above in the section "Method of preparation of regulatory T cells". Preferably said at least one or said at least two cytokines are IL2 and IL15. Preferably the kit according to the invention does not comprise any cytokine other than IL2 and IL15.

The kit includes in particular anti-thymocyte immunoglobulins and at least one cytokine and an inhibitor of TOR protein kinase. It can further comprise one or more means for purifying CD4+ cells.

It can also comprise one or more antibodies selected from the group consisting of anti-CD4, anti-CD25, anti-Foxp3, anti-GITR, anti-CTLA4, anti-CD62L, anti-CCR4, anti-GARP and anti-LAP antibodies, preferably anti-CD25, anti-Foxp3, and anti-GARP and/or anti-LAP antibodies.

Means for purifying CD4+ cells are well known by a person skilled in the art and include for example means for positive selection such as sorting of CD4+ cells by flow cytometry or beads, notably magnetic, covered with anti-CD4 antibodies, or means for negative selection such as beads covered with antibodies permitting capture of the CD4- cells.

The invention will be further illustrated by the following examples and figures. FIGURES

Fig. 1 depicts inhibition of the allogenic response of responder CD4 cells incubated in the presence of regulatory T cells induced by anti-thymocyte immunoglobulins (CD4 ATG) and mononucleated allogenic cells, relative to the responder CD4 cells alone (CD4). The regulatory cells were added in a ratio relative to the responder cells of 1/20, 1 /10, 1 /4, 1/2, 0.75/1 , 1/10, 2/1 and 5/1 . The controls used consist of responder CD4+ cells in the presence of mononucleated allogenic cells (T Pos), responder CD4+ cells cultivated in the presence of CD4 irradiated autologous cells (T auto) and responder CD4+ cells alone in culture (T Neg).

Fig. 2 shows that culture volume has no effect on the expression profile of the phenotypic markers of the CD25/Foxp3, GITR, CTLA4, CD62L, and CCR4 Treg cells and on the annexin/propidium iodide markers of apoptosis. EXAMPLES

Example 1 : Materials and methods

Purification of human CD4+ cells

Peripheral blood is obtained from blood donors in good health taken from the Lyons blood bank. The mononucleated cells (MNCs) are isolated by centrifuging whole blood diluted to half in PBS deposited on a Ficoll gradient of density 1 .077. The MNCs are then incubated with magnetic beads covered with an anti-CD4 monoclonal antibody. A magnet is applied to the tube to retain the CD4 cells, while the cells that are not retained are withdrawn (positive selection with the kit from Milteny Biotech GmbH, Germany). At the end of the procedure, the purity of the CD4 cells evaluated in flow cytometry is above 95%.

Cell culture

The purified CD4 cells are incubated at a rate of 2.10⁶ cells/ml in a culture medium of the RPMI type with 10% of a pool of human serum AB inactivated by heating, with addition of 2 mM of glutamine, 100 lU/ml of penicillin, and 100 μg/ml of streptomycin.

In this culture medium, the effect of adding different inducers of cellular differentiation on amplification of Treg cells was tested with the following range of concentrations:

- Thymoglobulin® from 1 to 150 μg/ml

- IL2 from 50 to 500 lll/mL

- IL10 from 10 to 100 ng/mL

- IL15 from 5 to 50 ng/mL

- TGF3 from 2 to 5 ng/mL

- retinoic acid from 3 to 5 μg mL

- rapamycin from 10 to 22.5 ng/mL

- conditioned medium (mixed lymphocyte culture supernatant) from 1 to 20%.

Thus, in the culture medium of the RPMI type described above, the inducers of differentiation and proliferation of the CD4 cells were tested according to the following scheme:

DO) Start of culture of CD4+ at 2.10⁶ cell/mL and stimulation by Thymoglobulin® at 50 μg mL;

D1 ) Washing and resumption of culture at 1 .10⁶ cells/mL then restimulation by Thymoglobulin® at 50 μg mL + inducer(s) of differentiation;

D5) Washing and resumption of culture at 1 .10⁶ cells/mL then restimulation by Thymoglobulin® at 50 μg mL + inducer(s) of differentiation

The best combination for amplification of Treg cells was Thymoglobulin® 50 μg mL + IL2 200 IU/mL + IL15 10 ng/ml + rapamycin at 10 ng/mL.

Thus, in the culture medium of the RPMI type described above, a typical culture protocol is carried out according to the following scheme:

D1 ) Washing and resumption of culture at 1 .10⁶ cells/mL then restimulation by Thymoglobulin® at 50 μg mL + IL2 200 lU/mL + IL15 10 ng/ml+ rapamycin at 10 ng/mL;

D5) Washing and resumption of culture at 1 .10⁶ cells/mL then restimulation by Thymoglobulin® at 50 μg mL + IL2 200 lU/mL + IL15 10 ng/ml+ rapamycin at 10 ng/mL; Analysis of cells by flow cytometryThe cells cultivated in the presence or absence of anti-thymocyte immunoglobulins (also called ATG) are first labelled with a fluorescein- labelled anti-CD4 antibody (anti-CD4-FITC) and a phycoerythrin-labelled anti-CD25 (anti- CD25-PE) for 30 min. The cells are then washed and resuspended in 1 ml of fixing/permeabilizing buffer (Cold Fix/Perm Buffer from eBioscience) for 45 min at 4^<C. The cells are again washed twice with permeabilizing buffer (eBioscience) and blocked with a PBS buffer containing 2% of rat serum for 15 min.

An allophycocyanin-labelled anti-Foxp3 antibody (PCH101 ) is added to the cells for 30 min at 4^<C in the dark. The cells are once again washed twice before being analysed in flow cytometry with a FACSCalibur from Becton Dickinson.

For surface labelling of the other cellular markers, the following antibodies were used: anti CD62L-FITC, CD49d-FITC, CTLA4-PE, CD127-PE, GITR-PE, from Beckman and GARP from Alexis Biochemicals and LAP-PE, and CCR4-PE from R&D.

The analysis of events was performed using the CellQuest software from Becton

Dickinson.

Apoptosis and viability of the cells were measured using the Annexin-V-Fluos kit from Roche, according to the manufacturer's recommendations, followed by analysis by flow cytometry.

Mixed lymphocyte reaction (MLR)

The so-called responder MNC cells from a blood donor "X", at 10⁵ cells/well, are put in the presence of stimulating MNCs at 10⁵ cells/cell, from a donor "Y", irradiated at 40 Gy, to induce an allogenic reaction. The cells are incubated in 200 μΙ of a culture medium of the RPMI type with 10% of a pool of human serum AB inactivated by heating plus 2 mM of glutamine, plus 100 lU/ml of penicillin, plus 100 μg/ml of streptomycin in a round-bottomed 96-well plate. Irradiated CD4+CD25+Foxp3+ Treg cells are added to this reaction for determining their capacity for immunosuppression of the allogenic reaction in a ratio of regulatory cells to responder cells of 1/20, 1 /10, 1/4, 1 /2, 0.75/1 , 1/10, 2/1 and 5/1 . The control consists of irradiated T CD4 responder cells added in the same proportions or ratio as Treg cells..

After 5 days of culture at 37°C and 5% C0₂, 1 .0 pCi/well of tritiated thymidine is added. The cells are collected 16 hours later and the incorporation of the thymidine is measured with a gamma counter. All the measurements are taken in triplicate. The incorporation of tritiated thymidine reflects the level of proliferation of the responder cells, and therefore the level of development of the allogenic reaction. Example 2: Production and amplification of Treg cells by culture of CD4 cells in the presence of Thymoglobulin®

Cells cultivated in the presence of anti-thymocyte immunoglobulins (ATGs) alone, and in particular CD4+CD25+Foxp3+ cells induced on D1 by the product Thymoglobulin®, enter apoptosis if they are maintained in the presence of ATGs alone. Conversely, CD4+CD25+Foxp3+ cells induced by ATG, if they are cultivated in the presence of a differentiating agent without ATG, are dedifferentiated to a CD4+ phenotype.

Culture of CD4+CD25+Foxp3+ cells induced in a medium containing ATG and one or more differentiating factors is therefore necessary to achieve maintenance, and if possible amplification, of these cells.

The effect of different inducers of cellular differentiation was tested on D5, in the context of the culture protocol defined in example 1 . The results are shown in Table 1 .

Table 1 : Effect of inducers of cellular differentiation on maintenance/amplification on

These results show that anti-thymocyte immunoglobulins alone, IL10 and the conditioned medium were not suitable for maintaining CD4+CD25+Foxp3+ cells induced on D1 by anti-thymocyte immunoglobulins. TGF-β and retinoic acid, on their own, lead to satisfactory maintenance of CD4+CD25+Foxp3+ cells.

The most effective combination for amplification of CD4+CD25+Foxp3+ Treg cells was Thymoglobulin® + IL2, IL15, and rapamycin.

Starting from CD4+ cells having less than 3% of CD4+CD25++Foxp3+ cells, after 10 days of culture, up to about 80% of the cells had become CD4+CD25++Foxp3+ Treg cells. The expansion factor of the Treg cells is 120 in the 10-day cultures in the presence of IL2, IL15, Rapamycin and Thymoglobulin® (Table 2). Table 2: Production of CD4+CD25++Foxp3+ cells induced by Thymoglobulin®

Moreover, analysis of these cells shows that they also possess the other surface markers CTLA4, GITR, CD127dim, CD62L and CCR4 present on induced Treg cells.

To date, the commonest markers for the identification and characterization of Treg cells are the molecules: CD4, CD25, CD127 and FoxP3. The transcription factor FoxP3 is necessary but insufficient for the development and function of T-Reg lymphocytes.

A new molecule also regulates the expression of FoxP3: this molecule GARP (glycoprotein-A repetitions predominant) also called LRRC32 (leucine-rich repeat-containing protein 32) is a membrane receptor binding the molecule LAP (latency-associated peptide). They act by maintaining strong expression of FoxP3 and are markers of functional Treg cells.

Activated CD4s expressing the molecule GARP are directed against regulatory CD4s strongly expressing FoxP3 to induce active and functional Treg cells. We demonstrated that the GARP marker is also strongly expressed intracytoplasmically, and much more moderately in the membrane, as well as the LAP molecule, after induction of Treg cells with the mixture Thymoglobulin®+IL2+IL15+rapamycin.

These cells are therefore

CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ T cells.

Other experiments with culture of CD4+ cells in the presence of IL2, IL15, rapamyci and Thymoglobulin®, in the conditions of example 1 , led to the following results (Table 3):

Table 3: Production of CD4+CD25+Foxp3+ cells induced by Thymoglobulin®

Example 3: Effect of the sequence of induction by the anti-thymocyte immunoglobulins (ATG) alone and then culture of the induced cells in a medium containing ATG and one or more differentiating factors

CD4+ cells were cultivated in RPMI medium with addition of 10% of a pool of human serum AB inactivated by heating, 2 mM of glutamine, 100 lU/ml of penicillin, and 100 μg/ml of streptomycin and optionally Thymoglobulin® 50 μg ml, IL2 200 lU/ml, rapamycin (Rapa) 10 ng/ml and TGF-β 5 ng/ml, according to the information given in Table 4 below.

Table 4: Effect of stimulation of CD4+ cells by Thymoglobulin® for 24h

These results show that without stimulation by Thymoglobulin®, there is no induction of Treg cells. When all reagents are added at the same time right at the start of culture, good induction of Treg cells is obtained but it is also accompanied by strong induction of apoptosis. When the CD4+ cells are stimulated for 24h by Thymoglobulin®, and are then cultivated without restimulation by Thymoglobulin®, more than 50% of the Treg cells are lost (the number of CD4+ CD25+ FoxP3- cells is increased). The most favourable sequence for amplification of Treg cells is therefore an initial step of stimulation with Thymoglobulin®, then culture in the presence of cytokines and/or differentiation inducer and/or inhibitor of TOR protein kinase and Thymoglobulin®.

Example 4: Treg cells induced by Thymoglobulin® inhibit the allogenic reaction

(MLR)

When a mixture is prepared from responder cells and CD4+CD25++Foxp3+ Treg cells induced by Thymoglobulin® (CD4 ATG), we observe dose-dependent inhibition of MLR depending on the quantity of Treg cells (CD4 ATG) added to the reaction. Inhibition of 80% is obtained for a ratio of responder cells to CD4 ATG of 1 /5 (Fig. 1 ). Example 5: Treg cells can be induced by Thymoglobulin® from umbilical cord blood

Transplants of cells derived from umbilical cord blood are commonly used for treating haemopathies. It was verified that the protocol described in example 1 is able to produce Treg cells from naive CD4s from umbilical cord blood.

Example 6: Treg cells induced by Thymoglobulin® can be produced in large volume in vials of 2, 5, 25, and 75 ml without changes in their properties

To verify that we could produce these Treg cells in large quantities, for immunotherapy testing, we produced these cells in vials from 2 to 75 ml, after seeding at a rate of 2.10⁶ CD4+ cells per ml and 10 days of culture, with change of medium and reseeding at 1 .10⁶ cells per ml on D1 and D5. It is found that the culture volume does not alter the quality of the cells produced (Fig. 2).

Moreover, the Treg cells induced by Thymoglobulin® can be frozen and thawed without altering their functional properties.

Example 7: Comparison of the capacity of Thymoglobulin® to induce Treg cells with that of another preparation of anti-human thymocyte immunoglobulins

The capacity of the two products Thymoglobulin® and Lymphoglobulin® (horse anti- human thymocyte immunoglobulins, Genzyme) to induce CD4+CD25+Foxp3+ regulatory T cells, in identical culture conditions (Thymoglobulin® or Lymphoglobulin® at 0, 10, 50 or 100 μg mL in RPMI medium with 10% of a pool of human serum AB inactivated by heating, with addition of 2 mM of glutamine, 100 lU/ml of penicillin, and 100 μg/ml of streptomycin) was compared after 24 hours of culture. The results obtained are shown in Table 5.

Table 5: Comparison of Thymoglobulin® and Lymphoglobulin®

Concentration ^g/mL) % of CD4 CD25+Foxp3+ cells

Control 0 1

Thymoglobulin® 10 10

50 52

100 80

Lymphoglobulin® 10 2

50 8

100 30

Claims

1 . Ex vivo method of preparing regulatory T cells (Treg), said method comprising the steps consisting of:

b) cultivating the cells obtained in step a) in a culture medium containing anti- thymocyte immunoglobulins and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent for 1 to 14 days; and

c) harvesting the CD4+CD25+Foxp3+ T cells obtained, the CD4+CD25+Foxp3+ T cells being Treg cells.

2. Method according to Claim 1 , which comprises the steps consisting of:

a1 ) washing the cells obtained in step a);

b) cultivating the washed cells from step a1 ) in a culture medium containing anti- thymocyte immunoglobulins and at least one cytokine and/or one inhibitor of TOR protein kinase and/or one differentiating agent for 1 to 4 days;

b1 ) washing the cells obtained in step b);

b2) cultivating the washed cells from step b1 ) in the culture medium defined in step b), for a maximum of 6 or 10 days;

3. Method according to Claim 1 or 2, in which the CD4+ T cells are cultivated in step a) for 12 to 36 h.

4. Method according to Claim 2 or 3, in which, in step b), the washed cells from step a1 ) are cultivated for 3 to 4 days and, in step b2), the washed cells from step b1 ) are cultivated for 2 to 6 days.

5. Method according to any one of Claims 1 to 4, in which, in steps a) and b), said anti-thymocyte immunoglobulins are rabbit or horse anti-human thymocyte immunoglobulins.

6. Method according to any one of Claims 1 to 5, in which, in steps a) and b), said anti-thymocyte immunoglobulins are rabbit anti-human thymocyte immunoglobulins Thymoglobulin® or horse anti-human thymocyte immunoglobulins Lymphoglobulin®.

7. Method according to any one of Claims 1 to 6, in which said anti-thymocyte immunoglobulins are present at a concentration from 1 to 200 μg/mL.

8. Method according to any one of Claims 1 to 7, in which the culture medium in step b) contains anti-thymocyte immunoglobulins, at least two cytokines and one inhibitor of TOR protein kinase.

9. Method according to any one of Claims 1 to 8, in which said at least one cytokine is selected, or said at least two cytokines are selected, from the group consisting of IL2, IL4, IL7, IL9, IL15 and IL21 .

10. Method according to any one of Claims 1 to 9, in which said culture medium in step b) contains anti-thymocyte immunoglobulins, at least the two cytokines IL2 and IL15 and one inhibitor of TOR protein kinase.

1 1 . Method according to any one of Claims 1 to 10, in which said inhibitor of TOR protein kinase is rapamycin.

12. Method according to any one of Claims 1 to 1 1 , in which said culture medium in step b) contains 5 to 25 ng/mL of inhibitor of TOR protein kinase.

13. Method according to any one of Claims 1 to 12, in which said culture medium in step b) contains 50 to 500 lU/mL of IL2 and/or 5 to 50 ng/mL of IL15.

14. Method according to any one of Claims 1 to 13, in which said culture medium in step a) is a base serum-free culture medium, with addition of only said anti-thymocyte immunoglobulins and optionally serum and/or antibiotic(s) and/or glutamine.

15. Method according to any one of Claims 1 to 14, in which said culture medium in step b) is a base serum-free culture medium with addition of only said anti-thymocyte immunoglobulins and said at least one cytokine and/or said inhibitor of TOR protein kinase and/or said differentiating agent, and optionally serum and/or antibiotic(s) and/or glutamine.

16. Method according to any one of Claims 1 to 5, which comprises the steps of: a) cultivating, for 20 to 28 hours, CD4+ T cells in a culture medium containing anti- thymocyte immunoglobulins at a concentration from 50 to 60 μg mL;

a1 ) washing the cells obtained in step a);

b) cultivating, for 1 to 4 days, the washed cells from step a1 ) in a culture medium containing said anti-thymocyte immunoglobulins at a concentration from 50 to 60 μg mL, and IL2 at a concentration of 180-220 lU/mL, IL15 at a concentration from 7.5 to 15 ng/mL, and rapamycin at a concentration from 9 to 1 1 ng/mL;

b1 ) washing the cells obtained in step b);

c) harvesting the CD4+CD25+Foxp3+ T cells obtained in step b2), the CD4+CD25+Foxp3+ T cells being Treg cells.

17. Method according to any one of Claims 1 to 16, in which the CD4+CD25+Foxp3+

T cells are collected in the form of a cell population comprising CD4+CD25+Foxp3+ cells.

18. Method according to any one of Claims 1 to 17, in which the CD4+CD25+Foxp3+ T cells are collected in the form of a cell population comprising 50 to 80% of CD4+CD25+Foxp3+ T cells.

19. Method according to any one of Claims 1 to 18, in which the total number of CD4+CD25+Foxp3+ regulatory T cells obtained was amplified by a factor of 20 to 120 relative to the total number of CD4+CD25+Foxp3+ T cells present among the CD4+ T cells cultured in step a).

20. Method according to any one of Claims 1 to 19, in which the CD4+CD25+Foxp3+ T cells are CD4+CD25+Foxp3+ GARP+LAP+ T cells.

21 . Method according to any one of Claims 1 to 20, in which the CD4+ T cells are

CD4+ T cells from a subject intended to receive a tissue or organ transplant, or from a subject with an autoimmune disease or an allergy.

22. Method according to any one of Claims 1 to 19, in which the CD4+ T cells are CD4+ T cells derived from umbilical cord blood.

23. Method according to any one of Claims 1 to 22, which comprises, after step c), one or more supplementary steps consisting of washing and/or purifying and/or concentrating, and/or freezing the regulatory T cells, and/or suspending the regulatory T cells in a pharmaceutically acceptable vehicle.

24. Method according to any one of Claims 1 to 23, which comprises, after step c), at least one supplementary step consisting of preparing a pharmaceutical composition comprising the CD4+CD25+Foxp3+ T cells.

25. Cell population comprising CD4+CD25+Foxp3+ regulatory T cells that can be obtained by the method according to any one of Claims 1 to 24.

26. Cell population according to Claim 26 comprising 100.10⁶ CD4+CD25+Foxp3+GARP+LAP+ T cells.

27. Cell population according to Claim 25 or 26 which comprises from 80 to 100% of CD4+CD25+Foxp3+GARP+LAP+ cells.

28. Pharmaceutical composition comprising a cell population according to any one of Claims 25 to 27 and a pharmaceutically acceptable vehicle.

29. Pharmaceutical composition that can be obtained by the method according to Claim 24.

30. Pharmaceutical composition according to Claim 28 or 29, comprising 100.10⁶

CD4+CD25+Foxp3+GAPtP+LAP+ regulatory T cells.

31 . Cell population according to any one of Claims 24 to 27 or pharmaceutical composition according to any one of Claims 28 to 30 for use for treating and/or preventing graft-versus-host disease, post-transplantation tissue or organ rejection, an autoimmune disease, a type 1 diabetes or an allergy in a subject.

32. Cell population or pharmaceutical composition for use according to Claim 31 , the regulatory T cells being autologous or heterologous cells of the subject to be treated.

33. Cell population or pharmaceutical composition for use according to Claim 31 or 32, the regulatory T cells being used in combination, for simultaneous or sequential administration, with another immunosuppressant treatment.

34. Kit for amplification of regulatory T cells comprising:

- anti-thymocyte immunoglobulins; and

- one inhibitor of TOR protein kinase; and

- optionally one or more means for purifying CD4+ cells and/or one or more selection markers for CD4+CD25+Foxp3+GITR+CTLA4+CD62L+CCR4+GARP+LAP+ cells.

35. Kit according to Claim 34, not comprising any cytokine other than IL2 and IL15.