WO2009037723A1

WO2009037723A1 - Method for the generation and expansion of gamma/delta t regulatory cells, cells thus obtained and their uses

Info

Publication number: WO2009037723A1
Application number: PCT/IT2008/000544
Authority: WO
Inventors: Rita Casetti; Chiara Agrati; Alessandra Sacchi; Federico Martini; Alessandra Rinaldi; Fabrizio Poccia
Original assignee: ISTITUTO NAZIONALE PER LE MALATTIE INFETTIVE
Current assignee: ISTITUTO NAZIONALE PER LE MALATTIE INFETTIVE
Priority date: 2007-08-10
Filing date: 2008-08-08
Publication date: 2009-03-26
Anticipated expiration: 2010-02-10
Also published as: ITRM20070437A1

Abstract

The invention relates to an in-vitro process for the generation and expansion of γδ T regulatory cells that express the transcription factor Foxp3 (γδ Treg cells), based on the culturing of peripheral blood mononuclear cells (PBMC) in the presence of cytokines, such as IL-2, IL- 15, growth factors and specific activator antigens. The invention also relates to the use of γδ Treg cells for the treatment of clinical frames that benefit from negative modulation of the immune response.

Description

Method for the generation and expansion of gamma/delta T regulatory cells, cells thus obtained and their uses

Field of the invention

The present invention relates to an in-υitro process for the generation and expansion of γδ T regulatory cells (γδ Treg cells) that express the transcription factor Foxp3 starting from a sample of peripheral blood mononuclear cells (PBMCs). The γδ Treg cells thus generated are used to induce a potent in-υitro inhibition of the proliferation of αβ T lymphocytes in man.

The regulatory capacity of the cells obtained by the method according to the invention can be exploited in the treatment of autoimmune diseases, for the prevention of transplant rejection, and, in general, in the treatment of clinical frames that benefit from negative modulation of the immune response.

Definitions

TCR: the T cell receptor (TCR) is a dimeric membrane protein of T lymphocytes that presents a portion of its own structure on the outer surface of these cells. TCR consists, in approximately 95% of lymphocytes, of a heterodimer comprising an α chain and a β chain (αβ T lymphocytes), whereas in the remaining portion of the lymphocyte population it consists of a heterodimer comprising a γ and a δ protein chain (γδ T lymphocytes). The function of the TCR consists in recognising antigens bound to MHC (Major Histocompatibility Complex) molecules on the cell surface. The TCR recognizes the antigen only in the context of MHC molecules.

Foxp3: Foxp3 is a nuclear transcription factor responsible for the suppression of the proliferation of αβ T lymphocytes. It acts by inhibiting the transcription of IL.-2 and increasing the expression of factors that suppress immune responses. Treg^: the T regulatory cells known until now the αβ receptor and are characterised as described below.

- nTreg cells: these are natural regulatory cells which originate in the thymus and constitutively express Foxp3. Their function is to inhibit the proliferation and production of cytokines by T lymphocytes in the course of the immune response once the pathogen has been eliminated. It has been demonstrated that they are responsible for the control of autoimmune diseases in that they inhibit the lymphocytes activated pathologically against autoantigens. In man, nTreg cells are represented by the T lymphocytes CD4/CD25^his^h, have a very low proliferative capacity in vitro and require the presence of exogenous IL- 2 to exert their suppressive activity. To perform their function they also need stimulation of the TCR and cell-to-cell contact (Paust S. and Cantor H. (2005) Immunol. Rev. 204, 195-207; Baecher-Allan C. and Hafler D.A. (2006) Immunol. Rev. 212, 203-216).

- iTreg cells: these are induced adaptive regulatory cells. These cells derive from the T lymphocytes CD4⁺/CD25" which, in the presence of IL-2, IL- 15 and TGF-β, differentiate into CD25⁺ regulatory cells that express Foxp3, secrete TGF-β and are capable of inhibiting the proliferation and production of cytokines of non-stimulated T lymphocytes with a mechanism independent of contact. These cells show a phenotype similar to the natural regulatory cells but the Foxp3 expression is transient and requires the continuous presence of IL-2 and TGF-β in the culture medium (Ramesh K., et al. Journal of Immunology (2007) 178: 7667-7677).

- TrI cells: these are regulatory cells induced following repeated in-υitro stimulations of human CD4⁺ lymphocytes in the presence of IL- 10. TrI cells inhibit the T cell-mediated response to pathogens, alloantigens and cancer cells. TrI cells have a very low proliferative ability and their suppressive capacity is mediated by soluble factors, particularly IL-10. They do not express Foxp3. - Th3 cells: these cells constitute the only subgroup of regulatory cells that are induced by antigens introduced into the oral cavity with food. Their suppressive ability is not based on direct cell contact but is mediated by TGF-β. They do not express Foxp3.

- CD8⁺CD28 cells": these cells suppress the immune response in a manner dependent upon contact between cells, interacting directly with APCs (Antigen Presenting Cells) and rendering the latter tolerogenic. APCs are defined as tolerogenic when, as a result of lack of expression of particular surface proteins, their interaction with the cells of the immune system induces a block of their proliferation instead of activation, as normally occurs.

αβ T lymphocytes: These express the αβ heterodimer of the TCR on their surface. They comprise CD4 and CD8 T lymphocytes. They take part in specific immunity and expand as a result of contacting the specific peptide antigen, presented in the context of MHC molecules by specialised cells. They can exhibit both direct cytostatic activity (CD8⁺ T lymphocytes) on the target cells (for example, cells containing a pathogenic agent or tumour cells) and a support activity (CD4 helper cells) or inhibitory activity (CD4 Treg cells) in the specific immune response.

γδ lymphocytes: these are T lymphocytes that express the γδ heterodimer of the TCR. Unlike the αβ T lymphocytes, they recognise non-peptide antigens via a mechanism independent of presentation by MHC molecules (Bukowsky J.F. J. Immunol. (1995) 154: 998; Morita C. T. Immunity. (1995) 3: 495). In man, two populations of γδ T lymphocytes are present:

- γδ T lymphocytes with the Vγ9Vδ2 receptor, which represent the majority population in peripheral blood;

- γδ T lymphocytes with the Vδl receptor, which represent the majority population in the mucosae and have only a very limited presence in peripheral blood. Vγ9Vδ2 T lymphocytes are involved in the immune response against intracellular pathogens and haematological diseases (Poccia F. et al. Immunol. Today, 1998, 19:253; Poccia et al. Curr. MoI. Med. 2001, 1: 137; Ferrarini M. et al. Trends Immunol. 2002, 23: 14).

Background art

The homeostasis of the immune system depends on the equilibrium between the response to an invading pathogen and the tolerance of the immune system to autoantigens.

This equilibrium is achieved by rigid selection at the thymus level, by means of which the T lymphocytes with TCR specific for self antigens are eliminated, whereas all the others, capable of recognising foreign antigens, migrate into the lymph nodes at peripheral level, where the contact with the possible pathogen may take place. Not all the autoreactive T lymphocytes, however, are eliminated in this phase, and those that escape this selection enter the bloodstream. At the peripheral level these potentially autoreactive clones are inhibited thanks to the presence of αβ CD4⁺ regulatory cells. The CD4⁺ regulatory cells, in turn, also have the function of suppressing the immune response to a pathogen, when the latter has been eliminated, avoiding the possibility of tissue damage due to an excessive immune response.

In autoimmune diseases, αβ T lymphocytes reactive against autoantigens damage the tissues and organs, causing major clinical damage, as in the case of type I diabetes or autoimmune thyroiditis. For the reasons indicated above, both CD4 and CD8 T lymphocytes which have escaped the negative selection of the autoreactive clones at thymus level are capable of exerting a potentially pathogenic autoreactive response.

T regulatory lymphocytes (T regulatory cells, Treg) play a very significant role in the induction and maintenance of tolerance on the part of the other T lymphocytes. They are responsible, in fact, for the limitation of the immune response and for the inhibition of induction of autoimmunity (Sakaguchi, S. (2004) Ann. Rev. Immunol. 22, 531).

Treg cells have so far been identified as T lymphocytes that express the αβ heterodimer of the TCR and the molecules CD4+CD25^hie^h (Paust S. and Cantor H. (2005) Immunol. Rev. 204, 195-207; Baecher-Allan C. and Hafler D.A. (2006) Immunol. Rev. 212, 203-216). From recent studies it has emerged that these Treg cells specifically express the nuclear transcription factor Foxp3. It would appear that Foxp3 is the main gene controlling the development and function of Treg cells (nTreg and iTreg) and to date the protein is considered the best molecule to identify these cells (L.A. Schubert, et al. J. Biol. Chem. (2001) 276, 137672-37679; R. Khattri, et al, Nat. Immunol. (2003) 4, 337; Stephens G.L. et al., J. Immunology (2007) 178, 6901). Other types of T cells that are capable of inhibiting the proliferation of αβ T cells, such as the type 1 regulatory cells (TrI) that produce IL-10 and the Th3 cells that produce TGF-β, never express the protein Foxp3.

The mechanism of action is different for the various subtypes of Treg Foxp3⁺. In particular, the inhibitory activity of nTreg Foxp3⁺ cells is based on a mechanism independent of cytokines, i.e. it does not involve the action of cytokines with a suppressive function, such as IL-4, IL-10 and TGF-β 1, but needs cell-to-cell contact. Although it is still not clear how the suppression occurs, it has been hypothesised (Toda A. and Piccirillo CA. , J. Leukoc. Biol. (2006) 80) that one of the possible mechanisms bywhich Treg cells inhibit the effector cells is the one in which Treg cells negatively modulate the function of APCs. Many studies indicate, in fact, that one of the mechanisms on the basis of which CD4 regulatory cells inhibit the effector response is mediated by their influence on the maturation of dendritic cells, rendering them tolerogenic and thus no longer capable of activating an adaptive immune response. This effect is demonstrated by the reduced expression of co-stimulatory molecules by the dendritic cells, essential for proper stimulation of the effector cells. Other studies (Thornton A.M., Shevach E.M. J. Exp. Med. (1998) 188: 287; Gondek D.C. et al. J. of Immunology (2005) 174: 1783), on the other hand, have demonstrated that the Treg suppression is probably based on different mechanisms again. Currently, we have no detailed knowledge of the functional mechanism used by Treg cells to inhibit the response of CD4 or CD8 effector cells either in vitro or in vivo, though various authors have done a great deal of work in the field (Thornton A.M., Shevach E.M. J. Exp. Med. (1998) 188: 287; Thornton A.M. et al. J. Immunol (2004) 172: 6519; Jonuleit H. et al. J. Exp. Med. (2002) 196: 255).

Two hypotheses have been formulated as to the origin of autoimmune diseases. According to the first one, such diseases depend upon a reduced number or reduced function of regulatory cells. The second hypothesis postulates that autoimmune diseases are the consequence of the development of pathogenic T cells resistant to regulation. As regards diabetes, for example, the regulatory cells control the progression of the disease by inhibiting the activation of effector cells and their function: nevertheless, effector cells resistant to regulation may accumulate over time, giving rise to onset of the disease (Tang Q. and Bluestone JA. Immunological Review (2006) 212: 217). Furthermore, in-vitro experiments have demonstrated that Treg cells inhibit the proliferation of other T cells, blocking the production of IL- 2. In-υivo studies suggest lastly that the regulatory cells control various aspects of the immune response including proliferation, production of cytokines, migration towards target tissues and the effector functions of the cells present in the tissue concerned.

Any organ within the cardiocirculatory system, the digestive tract, the visual system, the endocrine system, the skin, joints, kidneys and lungs, the muscular system and the nervous system can be affected by an autoimmune disease (NIH Publication No.02 4858, (2002) "Questions and answers about autoimmunity").

As a result, the treatment of autoimmune diseases depends considerably on the type of organ affected, on the severity of the disease and on its symptoms. The general clinical approach to this class of diseases aims at reducing the symptoms (which may include the prescription of analgesic drugs or extend even to the removal of the part affected), at preserving the functions of the organ affected (for example, by using anti-inflammatory drugs such as corticosteroids or hormone replacement therapies) and at reducing the general immune response (for example, by means of therapy with immunosuppressors such as cyclosporin and cyclophosphamide). These treatments, however, are often of limited efficacy and/or induce adverse side effects.

It is therefore clear that, in actual fact, no real cure has yet been found for autoimmune diseases.

Another important function of CD4 regulatory cells is that of being potent inhibitors of effector functions following organ transplantation. Recognition of alloantigens by CD4 T lymphocytes leads to the expansion not only of antigen-specific effector cells with pathogenic effects but also of antigen-specific CD4 regulatory cells, which can inhibit transplant rejection in an antigen-specific manner. Studies are in progress to apply this tolerogenic capacity to organ transplantation, particularly when a reduced tissue compatibility would lead for sure to rejection. (Boschiero L. et al., Transplantation Proceedings 2007; 39:2013-2017).

γδ T lymphocytes also play an important role in regulating the early inflammatory response to microbial infections and thus in preventing excessive tissue damage (Fu Y. -X. et al. J. Immunol. (1994) 153: 3101; King D. P. et al. J. Immunol. (1999) 162: 5033; Egan P.J. et al. J. Exp. Med. (2000) 191: 2145; Skeen M.J. et al. Infect. Immun. (2001) 69: 7213; Skeen M.J. et al. J. Leukoc. Biol. (2004) 76: 104; Tagawa T. et al. J. Immunol. (2004) 173: 5156). Experimental evidence suggests that γδ T lymphocytes perform their regulatory function by influencing the chemotaxis and function of effector cells that play a key role in inflammation, such as neutrophils (Fu Y.-X. et al. J Immunol. (1994) 153: 3101; D'Souza CD. et al. J. Immunol. (1997) 158: 1217), macrophages (Egan P.J. et al. J. Exp. Med. (2000) 191: 2145; Tagawa T. et al. J Immunol. (2004) 173: 5156), and NK cells (Li B. et al. J. Immunol. (1998) 161 1558; Vincent M.S. et al. J. Exp. Med. (1996) 184: 2109).

Since the cells that express the γδ receptor recognise non-protein antigens in a manner not restricted to MHC very early in the course of an infection, they are considered part of the first line of defence of the host. Moreover, the resemblance of the pathogenic antigens recognised by the γδ T lymphocytes to the molecules expressed by the host's damaged cells suggests that the responses of the γδ T lymphocytes are induced more by non-specific molecules of damaged cells than by the excessive presence of microbial antigens or foreign antigens (Hayday A.C. Ann. Rev. Immunol. (2000) 18: 975). The early activation of γδ T lymphocytes may lead to the production of IFNγ, essential in the activation of macrophages and NK cells, which are important in the early antibacterial protection before the specific response of the αβ T cells is activated (see Hayday A. C. above)

In man, the substantial expansion of γδ T lymphocytes during infections by L. monocytogenes, Mycobacterium tuberculosis, Brucella melitensis, and Ehrlichia chaffeensis emphasises the great importance that these cells have in the immune response of the host (Spada F.M. et al. J. Exp. Med. (2000) 191: 937).

Methods are known for expanding γδ T cells in vitro starting from PBMCs by stimulation with phosphorylated compounds of bacterial origin containing nucleotides or by means of isoprenoid pyrophosphates such as isopentenyl pyrophosphate (IPP) in the presence of cytokines, such as IL-2 and IL-15 (WO 03/070921), for the purposes of obtaining a large number of γδ T cells that can be used in the course of infectious diseases and tumours utilising the known effector functions, for example, the cytotoxicity and the adaptive response induction function of αβ cells by means of cytokines, for stimulating and thus increasing the immune defences of the subject treated. Nevertheless, though many studies have been conducted on the functions of γδ T cells, no evidence has been found among these cells for the possible existence of a subpopulation of γδ T cells with a function comparable to that of the αβ Treg cells or which can be characterised by expression of the Foxp3 transcription factor and having the function of suppressing the response of other activated T lymphocytes.

Summary of the invention

An in-υitro method has now been found for generating and expanding γδ T cells that express the Foxp3 transcription factor with a regulatory function and here resides the present invention. These cells, designated γδ Treg cells, possess the ability to inhibit the replication of αβ. cells.

The method is based on the stimulation of lymphocytes with a specific antigen for Vγ9Vδ2 T lymphocytes such as isopentenyl pyrophosphate (IPP), in the presence of cytokines, such as interleukin-2 (IL-2) and interleukin-15 (IL- 15) and a growth factor such as Transforming Growth Factor β-1 (TGF-βl). The combination of these reagents induces the significant expression of the Foxp3 transcription factor on γδ T lymphocytes after three days' culture.

One subject of the invention is therefore a process for the production of γδ Treg cells that comprises:

(i) culturing peripheral blood mononuclear cells, possibly thawed, in the presence of a synthetic activator of γδ, lymphocytes, and in the presence of cytokines and growth factors in conditions such as to ensure the proliferation of γδ Treg cells; and

(ii) recovery of the cells thus obtained. The peripheral blood mononuclear cells should preferably derive from cytapheresis. The cells used are preferably human and may come from frozen biological samples.

Another object of the invention resides in that the cell cultures obtained with the process according to the invention, characterised by the fact that they contain, in the context of the γδ T cell population, at least 30% of γδ Treg cells expressing the protein Foxp3.

Anadditional object of the invention resides in the subpopulation of Vγ9Vδ2 T regulatory cells with the characteristics described here below.

A further object of the invention resides in pharmaceutical compositions characterised in that they contain a population of γδ T cells in which at least 30% consist of γδ T regulatory cells expressing Foxp3. Preferably, the compositions additionally contain a pharmaceutically acceptable vehicle such as a stabilising agent, for example, human serum albumin.

Yet a further object of the present invention resides in the use of the subpopulation of said γδ T regulatory cells expressing Foxp3 for pharmaceutical, therapeutic, experimental, cosmetic and research purposes, with particular reference to clinical frames that benefit from negative modulation of the immune response, such as, for example, autoimmune diseases and the prevention of transplant rejection.

Further objects of the invention will be evident from the description provided here below.

Brief description of the figures

Figure 1. Induction of γδ Treg cells.

Figure 2. Phenotypic characterisation of γδ Treg cells. Figure 3. Inhibition of proliferation of αβ cells by γδ Treg cells.

Detailed description of the invention

The in-vitro method according to the invention makes it possible to generate, starting from a biological sample, generally a sample of peripheral blood mononuclear cells (PBMCs), a particular subpopulation of γδ T cells, i.e. Vγ9Vδ2 cells, with regulatory activity which, as well as expressing the Foxp3 nuclear transcription factor, present the following additional characteristics: a. they express the following surface markers: CD25, CTLA-4, CD 127, CD45RO, CD69, and CD27; b. they exert a regulatory capacity in that they inhibit the proliferation of αβ T lymphocytes; c. the inhibition they exert on the proliferation of αβ lymphocytes does not require cell-to-cell contact but is mediated by one or more soluble factors, in that it is also exerted when the two cell populations (γδ Treg cells and stimulated PBMCs) are separated by a porous membrane that does not allow the passage of cells but only of the soluble factors produced by them (experiment not shown); d. they are capable of influencing the differentiation and maturation of dendritic cells, modulating the expression of a number of molecules typical of differentiated and/or mature dendritic cells (CD80, CD86) (experiment not shown).

The γδ Treg cells that are obtained with the method according to the invention are CD4 and CD8 negative cells since they derive from γδ T lymphocytes that normally do not express these markers. Furthermore, the cells according to the invention are all positive for the proteins CD25 and CTLA-4, which are typical markers of activated leukocytes. The γδ Treg cells that are obtained by means of the method according to the invention are also all positive for the activation marker CD 127. CD 127 is a protein normally expressed on all human T cells and one whose presence is regulated negatively as a result of activation of T lymphocytes. In αβ Treg cells, which by definition express Foxp3, the presence of the protein CD 127 would appear to remain constantly suppressed precisely as a result of the Foxp3 action (Weihong L. et al. J Exp Med (2006) 203; 1701). The presence of CD 127 on the γδ Treg cells obtained by the method according to the invention is therefore a further feture that differentiates them from the Treg cells studied up to date.

A fundamental characteristic of γδ Treg cells that differentiates them from CD4 regulatory cells is also the fact that the suppressive function of γδ Treg cells is not antigen-specific, unlike other cells, which in contrast exert their action only following recognition of a specific antigen in the context of the major histocompatibility complex. The advantage might be that a more extensive suppressive activity is obtained, and one applicable regardless of the specificity of the immune response that has to be inhibited. The lack of antigen specificity is the main advantage of γδ cells over the corresponding αβ cells.

The method according to the invention for the preparation of a composition of cells including γδ Treg cells expressing Foxp3 comprises the following steps:

(i) culture a biological preparation, preferably of blood cells, typically cells deriving from cytapheresis, in the presence of a synthetic activator compound of T lymphocytes, and in the presence of cytokines, typically IL-2 and IL- 15, and of a growth factor, typically TGF-beta, for a time period sufficient to obtain a γδ T cell population containing at least 30% of γδ Treg cells expressing Foxp3;

(ii) recover the cells thus obtained, for example, by cytofluorimetric separation, e.g. with a FACS-Vantage, FACS-Aria Flow Cytometer (Becton Dickinson) or the like, or by means of immunomagnetic methods (Molday RS, Yen SP, Rembaum A. Nature (1977) 268:437).- The biological preparation of stage (i) is generally a sample of lymphocytes obtained, for example, by purification of PBMCs starting from a normal sample of peripheral blood or by cytapheresis, also thawed. The PBMCs from peripheral blood are preferably isolated by means of the Ficoll-Hypaque technique according to methods with which experts in the field are familiar. In particular, the blood is stratified on a density gradient (Ficoll-Hypaque) and, by means of centrifuging, the mononuclear cells form a ring (buffy coat) which is easily collected and used.

The lymphocytes present in the buffy coat are cultured in suitable medium selected from those available on the market, for example, RPMI 1640 complete medium containing 10% heat-inactivated FBS (Foetal Bovine Serum), penicillin/streptomycin and 1-glutamine, in the presence of cytokines, proliferation activators and growth factors.

The cytokines in particular are interleukins, and more particularly IL- 2, preferably usable* at a concentration of approximately 20-100 U /ml, and IL- 15, preferably usable at a concentration of approximately 2500- 6500 U/ml, and mixtures thereof.

' The growth factor belongs to the TGF-β (Transforming Growth Factor β) family, and an be used at a concentration ranging from approximately 0.5 ng/ml to 2.5 ng/ml. It can be of human or animal origin, preferably human. Particularly preferred is TGF-β 1. For the purposes of the invention, also natural or synthetic variants of growth factors can be used, where what is meant by variants are the polypeptide chains which, by mutation, deletion, substitution and/or addition of one or more amino acids, conserve the ability to generate γδ Treg cells expressing Foxp3.

The synthetic activator is a specific compound for the proliferation of γδ, T lymphocytes, for example, it is a phosphoantigen selected from among those described in WO95/20673; particularly preferred is IPP (US5639653), used at a concentration ranging from 10 μg/ml to 50 μg/ml. It is present only in the initial stage of culture to trigger the proliferation.

According to the method of the invention, the lymphocytes are advantageously cultured initially in the presence of activator, cytokines and growth factors for at least three days, preferably for a period ranging from 3 to 10 days at 37°C. Every three days half of the spent culture medium is replaced by the same medium, but without the specific synthetic activator, for a period of time typically ranging from 3 to 30 days.

According to the method of the invention, after 4 days in culture, from lOOxlO⁶ total lymphocytes cultured at the concentration of 4xlO⁶ cells/ml are typically obtained:

10-15% Vγ9Vδ2 cells;

30-50% γδ Treg cells among the Vγ9Vδ2 cells.

The subpopulation of γδ Treg cells obtained by means of the method according to the invention can be used at the time of preparation, can be placed in cytapheresis bags, or can be stabilised for storage according to methods with which the expert in the field is familiar, possibly by cryopreservation. Generally, the cells are maintained in a medium containing a stabilising agent such as a polymer or a particular neutral protein. An example of a protein commonly used for this purpose is HSA (human serum albumin), preparations of which, also for parenteral use, are available on the market.

The cells obtained with the method according to the invention can be combined in a pharmaceutical composition additionally containing pharmaceutically acceptable carriers, vehicles, stabilising agents, preservatives and excipients which in themselves are known to the expert in the field. These compositions can be formulated in liquid form for parenteral use and can be prepared for the purposes of simultaneous, separate or temporally spaced use, possibly in combination with other pharmacological treatments. The methods of application/administration of the cells according to the invention can differ according to the type of use and depend on the particular therapeutic model implemented. If the physician, for instance, wishes to induce a specific tolerance for a solid organ subject to transplantation, a preventive treatment of the organ to be transplanted with inhibitory T regulatory cells obtained from PBMCs is advisable, preferably but not necessarily, from the recipient, so as to induce a state of tolerance confined only to the transplanted tissue site, without modifying in any way the systemic immunological competence. The treatment might consist in placing the organ to be treated, or suitably removed from its natural site, in contact with an effective amount of a culture of γδ Treg cells expressing Foxp3 for a period sufficient to prevent rejection of the organ once transplanted.

The cell populations according to the invention can be used in the preparation of pharmaceutical compositions for modulating the immune defences in patients suffering from autoimmune diseases in order to limit the damage caused by an excessive or abnormal response of the immune system. Any organ within the cardiocirculatory system, the digestive tract, the visual system, the endocrine system, the skin, joints, kidneys and lungs, the muscular system and the nervous system can be affected by an autoimmune disease (NIH Publication No. 02 4858, (2002) "Questions and answers about autoimmunity").

It is reasonable to propose that Treg infusional therapy can be administered in combination with traditional immunosuppressive pharmacological therapies. In that way one can envisage the use of a lower dose of immunosuppressive drugs, with a consequent reduction' in side effects, to the point where the immunosuppressive therapy can be totally eliminated as a result of successful achievement of a state of acquired immune tolerance. The advantages over therapy with α/β Treg cells are the greater number of cells obtainable by means of the expansion in vitro, greater efficacy depending upon the greater permanence of the Foxp3 marker on the γδ Treg cells and their ability to act regardless of the antigen specificity of the response to be inhibited.

The following examples are given to illustrate the invention and are not to be regarded as limitative of the scope of the invention.

Example 1. In- vitro generation of γδ Tree cells.

The lymphocytes were isolated from buffy coats of healthy donors by means of the Ficoll-Hypaque technique (Pharmacia, Uppsala, Sweden) as described above, and were cultured at a concentration of 4xlO⁶ cells/ml in complete medium (RPMI 1640 with 10% heat-inactivated FBS, 2 mM 1-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin) in the presence of interleukin-2 (IL-2, 100 U/ml), interleukin-15 (IL-15, 4500 U/ml), Transforming Growth Factor-βl (TGF-βl, 1.7 ng/ml) and a specific stimulus for Vγ9Vδ2 T lymphocytes, such as isopentenyl pyrophosphate (IPP, 20 μg/ml). On day three, half the culture medium was replaced by fresh medium and the cytokines and growth factor were added again. On day four, the induction of γδ Treg cells was evaluated. The cells were washed with PBS buffer containing 1% BSA and 0.1% sodium azide and incubated for 15 minutes at 4°C with the following monoclonal antibodies to human proteins: anti-Vδ2 conjugated with FITC; anti-CD25 conjugated with PE-C5 and anti- CD 127 conjugated with APC. Intracellular staining with antibody to human protein FoxP3 conjugated with PE was later performed.

On day five, the separation of the total Vγ9Vδ2 T lymphocytes was carried out using a FACSVantage Flow Cytometer. Since the Foxp3 marker cannot be used for the cytofluorimetric selection of cells owing to its intracellular expression, the isolated Vγ9Vδ2 T lymphocytes, enriched or not in γδ Treg cells, were used directly for the proliferation tests. Figures IA and IB illustrate the results of the cytofluorimetric analysis on human leukocytes after 4 days in culture in the presence and in the absence of the stimulating agents according to the invention, suitable for generating γδ Treg cells expressing the Foxp3 transcription factor. In Figure IA the result is expressed as the amount of γδ T cells expressing Foxp3 ex vivo (i.e. in peripheral blood as collected), in the presence of standard culture conditions (IL-2/IPP) and according to the method of the invention (IL- 2 /IPP/ IL- 15/ TGF- βϊ) as a percentage of the total γδ T cell population. Figure IB shows the respective graphic representation of the results of the cytofluorimetric analysis in the different conditions described above.

Example 2: Phenotvpic analysis of γδ Treg cells.

The γδ Treg lymphocytes were analysed phenotypically with a flow cytometer using the following conjugated antibodies and fluorochromes: anti-Vδ2-FITC, anti-CD4-FITC, anti-Foxp3-PE, anti- CD3-PerCP, anti-CD25-PECy5, anti-CD 127-APC, anti-CD27-PC5, anti-CD45RA-APC, anti-CTLA-4-PE, anti-CD45RO-APC, CD69-APC. Figure 2 illustrates the phenotypic characterisation of the γδ Treg lymphocytes by means of cytofluorimetric analysis. It should be noted that the median peak fluorescence for the different markers (indicated in the figure) is greater in the γδ T cells according to the invention which also express the Foxp3 factor (top panel) than in the γδ T cells that do not express it (bottom panel).

Staining for cell surface antigens was done, washing the cells with PBS buffer containing 1% BSA and 0.1% sodium azide and incubating them for 15 minutes at 4°C with the above-mentioned antibodies.

The expression of Foxp3 was evaluated as follows. Peripheral blood mononuclear cells, stimulated for 4 days with IL-2, IL- 15 and TGF-β in the presence of IPP, were first labelled with a specific fluoresecent antibody for the surface molecule Vδ2, which identifies lymphocytes with TCR Vγ9Vδ2 and were subsequently labelled with a monoclonal antibody (BioLegend) with a different fluorescence than the previous one, specific for the cytoplasmic transcription factor Foxp3 according to the manufacturer's instructions. In short, the cells were fixed in Fix/Perm Buffer and incubated at room temperature in the dark for 15 minutes. The cells were then washed and permeabilised with Perm Buffer and incubated at room temperature in the dark for 10 minutes. Subsequently, in appropriate test tubes, the anti-Foxp3 monoclonal antibody conjugated with PE or the human anti-IgG control antibody, isotype k, conjugated with PE was added and the cells were incubated in the dark for 20 minutes.

The cells thus labelled were acquired with a FACScalibur Flow Cytometer and the percentage of cells with the Vγ9Vδ2 receptor expressing Foxp3 was analysed using CellQuest software (Bekyon Dickinson Immunocytometry Systems).

Example 3. Functional analysis of γδ Treg cells.

The antiproliferative capacity of the γδ Treg cells was measured by the method using Carboxyfluorescein Diacetate, Succinimidyl Ester (CFDA, SE), a fluorescent intracytoplasmic label whose intracellular concentration is halved at every cell division. This halving is measured cytofluorimetrically as peaks with a decreasing fluorescence intensity which represent the cells that have undergone one or more cell divisions.

For this analysis, autologous peripheral lymphocytes were labelled with Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA SE; Pharmingen-BD), at room temperature in the dark for 5 minutes. The cells were then washed three times with PBS buffer supplemented with 5% FCS and resuspended in the complete culture medium. The proliferation of the labelled cells was subsequently induced by culturing them at a concentration of 2xlO⁶ cells/ml in the presence of IL-2 (100 U/ml), in plates precoated with anti-CD3 (5 μg/ml) and anti- CD28 (10 μg/ml) alone or in the presence of:

- γδ T cells isolated and enriched in γδ Treg (Foxp3⁺); or

- in the presence of isolated Vδ2 CD25 Foxp3- cells, or

- in the presence of γδ T cells. After 5 days in culture the proliferation of the autologous mononuclear cells was analysed cytofluorimetrically. Figure 3 shows the results of the proliferation inhibition experiment described above. The peaks on the right in the respective profiles represent the labelled αβ cells that have not proliferated. The intermediate peaks and those on the left in the respective profiles represent the labelled αβ cells that have proliferated and therefore show an increasingly less intense fluorescence. It will be noticed that only the γδ Treg cells obtained by the method according to the invention that express Foxp3 (γδ Treg) are capable of inhibiting the proliferation of the stimulated αβ T lymphocytes (profile No. 4). In fact, neither the addition to the stimulated lymphocytes of a total population of purified γδ T cells (profile 2), nor that of a selected population of T cells not expressing Foxp3 (Vδ2 CD25 cells; profile 3) is capable of inhibiting the proliferation of the stimulated lymphocytes.

Claims

1. Method for the in-vitro preparation of γδ T lymphocytes expressing the Foxp3 transcription factor with a regulatory function, comprising: (i) culturing a biological sample of peripheral blood mononuclear cells, possibly thawed, in the presence of a synthetic activator of lymphocytes, and in the presence of cytokines and growth factors in conditions such as to generate and expand said lymphocytes; and (ii) recovery the thus obtained cells.

2. Method according to claim 1, in which the peripheral blood mononuclear cell sample is obtained by cytapheresis.

3. Method according to claims 1-2, in which the cells are human.

4. Method according to claims 1-3, in which the cells are peripheral blood mononuclear cells.

5. Method according to claims 1-4, in which the cells are a subpopulation of γδ T cells, designated Vγ9Vδ2.

6. Method according to claims 1-5, in which the cells obtained are separated by flow cytometry or by immunomagnetic methods.

7. Method according to claims 1-6, in which the culturing is conducted in RPMI complete medium containing foetal bovine serum.

8. Method according to claims 1-7, in which the cyctokines are selected from the group consisting of IL-2, preferably usable at a concentration of approximately 20-100 U/ml, and IL- 15, preferably usable at a concentration of approximately 2500-6500 U/ml, and mixtures thereof.

9. Method according to claims 1-8, in which the growth factor belongs to the TGF-β family, particularly TGF-βl, and is usable at a concentration ranging from approximately 0.5 ng/ml to 2.5 ng/ml.

10. Method according to claims 1-9, in which the synthetic activator is a compound specific for the proliferation of γδ T lymphocytes of the phosphoantigen class, preferably IPP, usable at a concentration ranging from 10 μg/ml to 50 μg/ml.

11. Method according to claims 1-10, in which the lymphocytes are cultured initially in the presence of activator, cytokines and growth factors for at least three days, preferably for a period ranging from 3 to 10 days at 37°C; then every 3 days half the spent culture medium is replaced by the same culture medium, but without the specific synthetic activator.

12. Method according to claims 1-11, in which the total lymphocytes cultured are approximately lOOxlO⁶ at the concentration of 4xlO⁶ cells/ml, from which are obtained:

10-15% Vγ9Vδ2 cells;

30-50% γδ Treg cells among the Vγ9Vδ2 cells.

13. γδ Treg cells characterised by the fact that they express the Foxp3 nuclear transcription factor and present the following additional characteristics: being CD4 and CD8 negative; expressing the following surface markers: CD25, CTLA-4, CD127, CD45RO, CD69, and CD27.

14. Cell cultures obtained with the method according to claims 1-12, characterised by the fact that they contain at least 30% of γδ Treg cells out of the total population of γδ T lymphocytes after 4 days in culture.

15. Cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field.

16. Pharmaceutical compositions characterised by the fact that they comprise the cells and cell cultures according to claims 13 and 14.

17. Pharmaceutical compositions according to claim 16, further comprise a pharmaceutically acceptable vehicle such as a stabilising agent, e.g. human serum albumin.

18. Cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field for the treatment of diseases that benefit from negative modulation of the immune response.

19. Cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field for the treatment and prevention of transplant rejection.

20. Cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field for the treatment of autoimmune diseases.

21. Cells and cell cultures according to claim 20, in which the autoimmune diseases affect the cardiocirculatory system, the digestive tract, the visual system, the endocrine system, the skin, joints, kidneys and lungs, the muscular system and the nervous system.

22. Cells and cell cultures according to claim 20, in which the autoimmune diseases are selected from the group consisting of: autoimmune diabetes, multiple sclerosis, allergies, rheumatoid arthritis, asthma, rhinitis, and atopic dermatitis.

23. Use of cells and cell cultures according to claims 13 and 14 for pharmaceutical, cosmetic and research purposes.

24. Use of cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field for the treatment of diseases that benefit from negative modulation of the immune response.

25. Use of cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field for the treatment and prevention of transplant rejection.

26. Use of cells and cell cultures according to claims 13 and 14 to be used in the pharmaceutical field for the treatment of autoimmune diseases.

27. Use according to claim 26, in which the autoimmune diseases affect the cardiocirculatory system, the digestive tract, the visual system, the endocrine system, the skin, joints, kidneys and lungs, the muscular system and the nervous system.