WO2008028229A1 - Methods of identifying markers - Google Patents

Abstract

The present invention relates to methods of identifying markers, specifically those markers of antigen-specific regulatory CD4+ T cells and the use of such markers for identifying antigen-specific regulatory CD4+ T cells in bulk cell populations. The invention also relates to pharmaceutical compositions comprising the regulatory CD4+ T cells so identified, and use of the regulatory CD4+ T cells for treating and/or preventing diseases such as autoimmune disease.

Description

METHODS OF IDENTIFYING MARKERS

Field

The present invention relates to methods of identifying markers, specifically those markers of antigen-specific regulatory CD4⁺ T cells and the use of such markers for identifying antigen-specific regulatory CD4⁺ T cells.

Background

Immunological tolerance is a lack of harmful/undesirable responses towards certain antigens. Such tolerance in T cells is acquired first by central thymic mechanisms designed to eliminate self-reactive T cells by clonal deletion or to generate regulatory T cells, and second by peripheral mechanisms designed to eliminate or regulate undesirable T-cell responses towards self-antigens .

Regulatory T cells (hereinafter "T_reg cells") are subsets of T cells which are capable of suppressing or controlling immune responses. There is substantial evidence for an important role for these cells in maintaining tolerance to self-antigens, also referred to as autoantigens . T_reg cells have been shown to act as suppressor T cells that down-regulate other effector T cells and retard inflammatory processes in autoimmune diseases, allergic diseases and tissue transplantation responses .

Furthermore, it has been previously demonstrated that depletion of T_reg cells can promote tumour rejection in cancer and immunity to infectious agents in infectious disease .

Several distinct CD4⁺ T_reg subsets have been described, including naturally-occurring CD4⁺CD25⁺ T cells which develop during T cell maturation in the thymus, and

CD4⁺ T cells which are induced in the periphery and effect immuno-suppression via the anti-inflammatory cytokines IL- 10 or TGF- β .

Several studies have shown that CD4⁺CD25⁺ T cells in individuals with autoimmune disease have compromised function compared to CD4⁺CD25⁺ T cells isolated from healthy controls. This suggests that isolated T_reg cells with specificity for autoantigens may be of great therapeutic potential.

Expression of CD25 is not necessarily associated with T_reg cell function, since CD25 is also expressed by activated, non-regulatory effector T cells. Therefore in an organism in which many lymphocytes are being activated continually, simply identifying CD25 on a CD4⁺ cell does not provide much information about its regulatory capabilities . So far, the study of antigen-specific T_reg cells in humans has been indirect, and limited to identification of putative T_reg cells, e.g. based on cytokine expression. To date, it has not been possible to identify antigen- specific T_reg cells in bulk populations because markers which discriminate T_reg cells from non-T_reg cells following activation by antigen have not been identified.

There is therefore a need in the art to identify such markers. It is an aim of a preferred embodiment of the present invention to provide markers of antigen- specific regulatory CD4⁺ T cells, and to identify and isolate such cells from bulk cell populations.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Summary

In a first aspect the invention provides a method of identifying a marker of antigen-specific regulatory CD4⁺ T cells, said method comprising:

(a) generating antigen-specific CD4⁺ T cell clones from a cell sample; (b) screening said CD4⁺ T cell clones for suppressor function; and

(c) comparing expression of one or more markers in the CD4⁺ T cell clones which have suppressor function with expression of same markers in non- regulatory T cell clones, wherein a marker differentially expressed in the CD4⁺ T cell clones which have suppressor function when compared to the non-regulatory T cell clones is a marker of antigen-specific regulatory CD4⁺ T cells in the cell sample.

In a preferred embodiment, suppressor function is determined by measuring the ability of the CD4⁺ T cell clones to inhibit proliferation or cytokine production of autologous T cell lines/clones. In the method of the first aspect, expression of one or more markers may be greater or lower, or a combination of both, in antigen-specific regulatory CD4⁺ T cells than in non-regulatory T cells.

The expression of the one or more markers may be measured at the transcriptional level, or may be measured at the translational level such as by immunophenotyping . In a second aspect the invention provides a marker of antigen-activated regulatory CD4⁺ T cells, when identified by a method according to a first aspect of the invention.

In a preferred embodiment of the second aspect of the invention, the marker is CD52. In a further preferred embodiment, the marker is CD52 and the expression of CD52 is greater in antigen-specific regulatory CD4⁺ T cells than in non-regulatory T cells.

In a third aspect the invention provides the use of CD52 as a marker of antigen-specific regulatory CD4⁺ T cel l s .

In a fourth aspect the invention provides the use of a marker according to a second aspect of the invention, for identifying and/or isolating an antigen-specific regulatory CD4⁺ T cell in a cell sample. In a preferred embodiment, the marker is CD52.

In a fifth aspect the invention provides a method of identifying an antigen-specific regulatory CD4⁺ T cell, said method comprising: (a) detecting the level of expression of one or more markers according to a second aspect of the invention in a cell sample; and

(b) determining whether cells in the cell sample express said one or more markers in a manner which is characteristic for regulatory CD4⁺ T cells.

In a sixth aspect the invention provides a method of identifying an antigen-specific regulatory CD4⁺ T cell, said method comprising:

(a) generating antigen-specific CD4⁺ T cell clones from a cell sample;

(b) screening said CD4⁺ T cell clones for suppressor function; and

(c) comparing expression of one or more markers in the CD4⁺ T cell clones which have suppressor function with expression of same markers in non- regulatory T cell clones, wherein a CD4⁺ T cell clone which has suppressor function and which exhibits differential expression of said one or more markers when compared with expression of same markers in a non-regulatory T cell clone is identified as an antigen-specific regulatory CD4⁺ T cell in the cell sample.

In a method of the fifth or sixth aspect, an additional step can be performed wherein CD4⁺ T cells which express said one or more markers in a manner which is characteristic for regulatory CD4⁺ T cells are isolated.

In a preferred embodiment of the fifth or sixth aspect of the invention, expression of the CD52 marker is measured. Preferably, a level of expression of the CD52 marker which is greater indicates the presence of a regulatory CD4⁺ T cell.

In a seventh aspect the invention provides an isolated regulatory CD4⁺ T cell when identified by a method according to a fifth or sixth aspect of the invention.

In an eighth aspect the invention provides an isolated regulatory CD4⁺ T cell having greater expression of the marker CD52 than the expression of CD52 in a non- regulatory T cell.

In a preferred embodiment, the regulatory CD4⁺ T cell is a CD4⁺CD52^hi cell.

In a further preferred embodiment, the regulatory CD4⁺ T cell does not require direct cell-cell contact with non-regulatory T cells to suppress proliferation of the non-regulatory T cells. In the alternative, direct cell- cell contact may be required.

In a ninth aspect the invention provides the use of a regulatory CD4⁺ T cell according to a seventh or eighth aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of autoimmune disease, allograft rejection, graft-versus-host reactions, or allergic disease in a subject.

In a tenth aspect the invention provides a pharmaceutical composition comprising a regulatory CD4⁺ T cell according to a seventh or eighth aspect of the invention.

In an eleventh aspect the invention provides a method of treating and/or preventing autoimmune disease, allograft rejection, graft-versus-host reactions, or allergic disease in a subject, comprising administration of a regulatory CD4⁺ T cell according to a seventh or eighth aspect of the invention, or administration of a pharmaceutical composition according to a tenth aspect of the invention.

In a twelfth aspect the invention provides a method of treating and/or preventing autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease in a subject, comprising activating and/or inducing a regulatory CD4⁺ T cell in a subject, wherein said regulatory CD4⁺ T cell exhibits differential expression of one or more markers according to a second aspect of the invention when compared with expression of same markers in a non-regulatory T cell clone.

In a thirteenth aspect the invention provides the use of a marker according to a second aspect of the invention, for assessing the risk of developing an autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease, diagnosing an autoimmune disease, infectious disease, cancer, or allergic disease, measuring the progression of an autoimmune disease, infectious disease, cancer, or allergic disease, or monitoring the response to immunotherapy of an autoimmune disease, infectious disease, cancer, allergic disease or allograft rejection in a subject.

Brief Description of the Figures

Figure 1 shows a schematic of the suppressor assay described in Example 1. Figure 2 provides a graphical result of the screening of CD4⁺ T cell clones isolated from healthy donors for suppressor activity in response to the type 1 diabetes pancreatic autoantigens, glutamic acid decarboxylase molecular weight 65,000 isoform (GAD 65) or proinsulin, as described in Example 1.

Figure 3 provides graphical results of the testing of GAD 65-specific CD4⁺ T cell clones isolated from healthy donors and expanded in vitro for retention of suppressor function in the presence of GAD 65. Autologous GAD- specific T_reg (A) or non-T_reg (B) clones were co-cultured with non-T_reg clones at the indicated ratios in the absence or presence of GAD 65. Figure 4 shows graphically the ability of T_reg cells to proliferate in response to antigen in the absence or presence of IL-2 (A) . PKH26 dye-labelled T_reg (B, upper panels) or non-T_reg (B, lower panels) clones were co- cultured with autologous CFSE dye-labelled PBMCs in the absence (B, left panels) and presence (B, right panels) of GAD 65. The percentage of clones and PBMCs that divided in the absence of antigen (left panel) or in the presence of GAD 65 (right panel) are indicated (B) . Proliferation of the T_reg clones in response to GAD 65 (GAD) in the presence of PBMCs stimulated with tetanus toxoid (TT) is shown (C) .

Figure 5 represents graphically the results of testing a GAD 65-specific CD4⁺ T_reg clone for the ability to suppress proliferation of an autologous non-T_reg clone in the absence of direct cell-cell contact (A) . The cell division index (CDI) of PBMCs from a healthy HLA-DRl/4 donor that divided in response to human proinsulin when T_reg supernatant (SN) , pre- or post-ultracentrifugation was added to the culture (B) . Suppression following addition of neutralising IL-10- or TGF-βl-specific antibodies to co-cultures (C) .

Figure 6 shows graphically an example of the identification of markers differentially expressed on T_reg cells when compared to non-T_reg cells. Clones were analysed resting (A) and following stimulation during 24 hours with 5 μg/ml of plate-bound anti-CD3 (B) . CD52 expression was analysed by flow cytometry on T_reg and non-T_reg clones resting (C) and following stimulation during 24 hours with 5 μg/ml of plate-bound anti-CD3 (D) . Figure 7 shows that a high level of expression of

CD52 identifies antigen-activated (gated CD4⁺) T cells with regulatory function, (from an HLA-DRl, 4 healthy donor) (A) . Proinsulin-activated CD4⁺ cells were sorted into CD52^hl and CD52^lQ populations and tested for their ability to suppress proliferation of sorted autologous CD4⁺ tetanus toxoid (TT) -specific T cells (B) . The proportion of T_reg and non-T_reg clones specific for GAD 65 isolated from GAD- activated CD4⁺CD52^hi and CD4⁺CD52^l0 populations from two healthy donors is shown. Error bars represent mean+SEM (C) . The relationship between proliferation in response to GAD and GAD-dependent suppression by CD4⁺CD52^hl T-cell clones is shown (D) .

Figure 8 shows graphs demonstrating that high expression of CD52 defines antigen-activated CD4⁺ cells with low production of IFN-g and IL- 17. IFN-g production by FACS-sorted CD52^hi and CD52¹⁰ CD4⁺ cells that divided in response to tetanus toxoid (TT) (A) and GAD 65 (GAD) (B) . CD4⁺CD52^hi cells suppressed the IFN-g production of CD52¹⁰ cells when co-cultured at a 1:1 ratio in the presence of GAD 65 (B) . IL- 17 production by FACS-sorted CD52^hi and CD52^lD CD4⁺ cells that divided in response to GAD 65 (C) . Figure 9 shows the proportion of GAD-specific CD4⁺CD52^hl T cells in people with pancreatic islet autoantibodies at risk for type 1 diabetes (TlD) , people with established TlD, HLA-matched healthy controls and people with type 2 diabetes (T2D) . The proportion of CD52^hl cells in the CD4⁺ population responding to GAD 65 (GAD) (A) or tetanus toxoid (TT) (B) is shown. Cell division in response to GAD (C) or TT (D) relative to background (Cell Division Index) is shown.

Detailed Description

The inventors have recognised the need for methods which allow the identification of markers of antigen- specific regulatory CD4⁺ T cells in a cell sample.

As used herein, the term "marker" is taken to mean "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions." (NIH Biomarker Definitions Working Group, 1998) . An example of a suitable marker may include, but is not limited to, a gene and/or its encoded protein.

Another suitable marker may be a protein that has been post-translationally modified, the modified protein acting as a marker. The marker measurement may increase or be greater, or decrease or be lower, in response to particular biological processes; or, if a marker measurement typically changes in the absence of a particular biological process, a constant measurement may indicate occurrence of that process. Marker measurements can be represented in absolute or relative values (e.g. the relative concentration of two molecules in a biological sample) . As used herein, a reference to a "marker" includes a combination of two or more such markers .

An antigen-specific T cell is a cell that recognises a specific antigen through a clonal T-cell receptor that in practice is measured by proliferation, cytokine production or other molecular and cellular processes following engagement of the receptor by the antigen. Such cells include both 'protective' regulatory T cells ("T_reg") and 'potentially pathogenic' non-T_reg.

An autoantigen-specific T cell is a T cell that reacts to a self-antigen which is a normal constituent of the body .

T_reg are capable of suppressing or controlling types of immune responses that lead to undesirable clinical effects or disease. As used herein, the term "cell population" means a set of cells (e.g. a population of T_reg cells) having characteristics in common. The characteristics include without limitation the presence and level of one, two, three or more cell-associated molecules (e.g., cell- surface antigens) . One, two, three or more cell-associated molecules can thus define a T_reg cell population.

The activation of T_reg cells upon antigen presentation may lead to changes in expression of one or more markers in these cells when compared to expression of the same one or other markers in non-T_reg counterparts. This is known as differential expression and refers to the level or activity of a constituent (e.g. a gene and/or its encoded protein) in a first sample (or set of samples) as compared to the level or activity of the constituent in a second sample (or set of samples) , where the method used for detecting the constituent provides a different level or activity when applied to the two samples (or set of samples) . As an example, a protein which is measured at one concentration in a first sample, and at a different concentration in a second sample, is differentially expressed in the first sample as compared with the second sample. Essentially, the protein acts as a marker for distinguishing the two samples from each other.

A marker would be referred to as "greater" , or as having "greater", "higher", or "increased" expression, or as being "up-regulated" in a first sample, if the method for detecting the marker indicates that the level or activity of the marker is higher or greater in the first sample than in the second sample (or if the marker is detectable in the first sample but not in the second sample) . Conversely, the marker would be referred to as "lower", or as having "lower" or "decreased" expression, or as being "down-regulated" in a first sample, if the method for detecting the marker indicates that the level or activity of the marker is lower in the first sample than in the second sample (or if the marker is detectable in the second sample but not in the first sample) . In particular, a marker is referred to as "greater" or "lower" in a sample (or set of samples) obtained from a subject if the level or activity of the marker is higher or lower, respectively, compared to the level or activity of the marker in a sample (or set of samples) obtained from another subject or subjects or a reference value or range .

As used herein, the terms "fold increase" and "fold decrease" refer to the relative increase or decrease in the level or activity of a marker in one sample (or set of samples) compared to another sample (or set of samples) . A positive fold change indicates an increase in the level or activity of a marker while a negative fold change indicates a decrease in the level or activity of a marker. The increase or decrease may be measured by any method or technique known to those of skill in the art. As will be appreciated by one of skill in the art, the observed increase or decrease may vary depending on the particular method or technique that is used to make the measurement . For example, when the marker is a gene, the difference in the level of expression or activity of the gene between two samples (e.g. between a T_reg cell and a non-T_reg cell) can be measured at the transcriptional level, i.e. the conversion of the information encoded in the gene into messenger RNA (mRNA) , or at the translational level, i.e. the conversion of mRNA to protein, or a combination of both.

Changes in expression or activity of genes can be measured by any method known in the art . Such commonly used methods include differential display (Liang et al., 1992), representational difference analysis (Lisitsyn et al . , 1993), serial analysis of gene expression (Velculescu et al . , 1995), and more recently array based technologies (Schena et al., 1995) . For a review of these approaches see Carulli et al . , (1998).

By way of example only, the step of determining differential expression of a gene at the transcriptional level may be carried out by detecting the presence of mRNA by reverse transcription polymerase chain reaction (RT- PCR) , or using specific nucleic acid arrays utilising microchip technology. By way of example only, the step of determining differential expression of a gene at the translational level may be carried out by detection of the protein encoded by the mRNA, for example using ELISA, proteomic arrays, or surface or intracellular staining with specific probes such as antibodies as detected by flow cytometry. All of these methods are well known in the art. For the identification of differentially expressed proteins, preferably proteomic arrays are used. A preferred array is a protein microarray which will allow the identification of patterns of expression of proteins which are found on the surface of cells.

Essentially, genes or proteins which show differential expression in T_reg cells compared to non-T_reg cells represent markers for T_reg cells, and allow these cells to be distinguished and isolated from bulk cell populations.

As used herein, the terms "low" or "^lo" are used to refer to "down-regulation of" , or "a decrease in" , the expression or activity of a gene in T_reg cells when compared to non-T_reg cells, and the terms "high" or "^hl" are used to refer to "up-regulation of", or "an increase in", expression or activity of a gene in T_reg cells when compared to non-T_reg cells.

As used herein, terms such as "expression of a gene" and "expression of a marker" are to be used interchangeably.

The first aspect of the present invention provides a method of identifying markers of T_reg cells from a cell sample, wherein said method includes as an essential step determining the differential expression of one or more markers in the T_reg cells compared to non-T_reg cells.

For the purposes of the method of the first aspect of the present invention, the cell sample may be taken from a mammalian subject such as a human subject, and may originate from a number of sources, including for example, peripheral blood mononuclear cells (PMBC) , leukopheresis or apheresis blood product, bone marrow, cord blood, liver, thymus, tissue biopsy, tumour, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymph node tissue, spleen tissue, or any other lymphoid tissue. In a preferred embodiment, the cell sample originates from PBMC from a blood sample obtained from the peripheral blood of a subject. The method for the identification of T_reg markers first involves generating CD4⁺ T cell clones from the sample which are specific for an antigen.

As used herein, the term antigen refers to a molecule such as a protein, that is recognised by clonal receptors on T cells and B cells which then leads to activation/proliferation in a specific manner, i.e. elicits a specific immune response in a mammal. Suitable antigens to generate antigen-specific T-cell clones include, but are not limited to, infectious agents such as parasites, bacteria, viruses, and fungi, and tumour antigens. A suitable antigen may also be an autoantigen.

As used herein, the term "autoantigen" , also known as a self-antigen, refers to an antigen which is native to a mammal, but which elicits a specific immune response in the mammal .

Typical autoantigens which can be used in a first aspect of the present invention include, but are not limited to, those specific for type 1 diabetes such as proinsulin, glutamic acid decarboxylase (GAD 65) , insulinoma-like antigen-2 (IA-2) , islet-specific glucose- 6-phosphate catalytic subunit -related protein (IGRP) and heat shock protein 60 (hsp-60) ; those specific for pemphigus folacius and pemphigus vulgaris such as desmoglein-1 and desmoglein-3 , respectively; those specific for multiple sclerosis such as protein components in the myelin sheath including myelin basic protein (MBP) , myelin oligodendrocyte glycoprotein (MOG) , and proteolipid protein (PLP) ; those specific for celiac disease such as gliadin and glutenin protein families; those specific for rheumatoid arthritis such as type II collagen, filaggrin, vimentin, aggrecan Gl, and Gp39; those specific for myasthenia gravis such as the acetylcholine receptor; those specific for Hashimoto's thyroiditis such as thyroid peroxidase; those specific for scleroderma such as centromere proteins and topisomerase; and those specific for Graves' disease such as the thyrotropin receptor. Furthermore, an antigenic fragment of any autoantigen can be used in the present invention.

The generation of CD4⁺ T cell clones which are specific for an antigen can be achieved in a number of ways to enrich for T cells having the characteristic of expressing the CD4⁺ marker following presentation of the antigen. One way this may be achieved is by culturing cells from a relevant source with an antigen in an induction culture for a period of time sufficient to allow activation of CD4⁺ T cell clones which are specific for the antigen, then isolating the antigen-specific CD4⁺ T cell clones .

Such an induction culture will also require the presence of antigen presenting cells (APCs) in order for the antigen to be presented to the CD4⁺ T cells. In this regard, PBMCs and other heterogeneous lymphoid cell populations themselves containing different types of APCs

(dendritic cells, B lymphocytes and macrophages; alternatively, purified APC subsets such as dendritic cells, B lymphocytes, or macrophages), an APC cell line, or a crude APC mixture may be included in the induction culture .

The antigen may comprise the whole antigen, or a fragment or domain thereof. The antigen may be added to the induction culture in any suitable form, such as in the form of the whole antigen or a portion or fragment thereof .

Preferably the antigen is a purified or semi -purified protein or peptide. Accordingly, the protein/peptide antigen may be added to the culture medium in the form of an isolated polypeptide representing a whole protein antigen, or an isolated peptide (s) corresponding to a portion of a protein antigen.

If a fragment of an antigenic peptide or protein is used, the choice of the antigenic peptide from among the amino acids comprising the antigenic protein will depend on a number of factors which include the particular disease of interest and the interactions of specific amino acids derived from the antigenic protein with a T cell receptor. The antigenic peptides may be from about 9 amino acids to about 20 amino acids or more in length; however, this length may be smaller or greater as long as the peptide leads to induction of the CD4⁺ T cells.

The antigenic peptides may be prepared in a number of ways. The peptides may be synthesized, for example using an automated synthesizer (Hunkapiller et al . , 1984; Bodanszky, 1984) , or may be synthesized by proteolytic cleavage (e.g., by trypsin, chymotrypsin, papain, V8 protease, and the like) or specific chemical cleavage (e.g., by cyanogen bromide) . The peptides may also be synthesized by expression of nucleic acid sequences encoding a particular peptide.

In certain embodiments, the induction culture medium may contain CD4⁺ T cells and APCs that are inherently part of the same source, for example when whole tissue lysate or tissues containing APCs, such as PBMCs are used. The induction culture medium used in a preferred embodiment to induce antigen-specific CD4⁺ T cell clones preferably includes a suitable agent that will allow subsequent detection and isolation of the CD4⁺ cells. For example, the induction culture may include cells labelled with fluorescent dyes such as 5, 6-carboxylfluorescein diacetate succinimidyl ester (CFSE) , in which case cells in the cell sample are first labelled with CFSE before incubation with the antigen. Following antigen presentation, T cells which proliferate in response to the antigen will have a resultant reduction in CFSE intensity as measured using flow cytometry. This reduction in CFSE intensity is represented herein as "CFSE^dim" .

Other methods that allow detection and isolation of CD4⁺ T cells may also be used, for example the method of cytokine capture as disclosed in Akdis et al . , (2004) .

The time period of induction sufficient to generate CD4⁺ T cells which respond to a relevant antigen in accordance with this embodiment is typically from about 3 to about 14 days or longer. In a preferred embodiment, the time period of the induction is about 7 days. The specific amount of antigen used will vary according to a number of factors which will be appreciated by those of skill in the art, including, for example, the potency and other characteristics of the antigen used.

It is preferred that when the antigen is in the form of the whole antigenic protein, a concentration of from about 0.1 μg/ml to about 100 μg/ml , and more preferably about 5 μg/ml to about 50 μg/ml is used.

At the end of the incubation period, T cells which proliferate in response to the antigen can be identified by methods known to those skilled in the art, for example on the basis of a resultant reduction in CFSE intensity

(i.e. CFSE^dim) as measured using flow cytometry. Isolation of this subject cell population may be achieved by fluorescence activated cell sorting (FACS) . FACS can have varying degrees of sophistication, such as multiple colour channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. Furthermore, the cells may be selected against dead cells by employing dyes associated with dead cells (e.g. propidium iodide) . Any technique may be employed which is not unduly detrimental to the viability of the selected cells.

In addition to antibody reagents, peptide-MHC antigen, tetramers (as described in Nepom, 2003), and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like. Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc. The details of the preparation of antibodies and their suitability for use as specific binding members are well-known to those skilled in the art. Of particular interest is the use of antibodies as affinity reagents. Conveniently, these antibodies are conjugated with a label for use in separation. Labels include magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Fluorochromes that find use include phycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluorescein and Texas red. Frequently each antibody is labelled with a different fluorochrome, to permit independent sorting for each marker. In a preferred embodiment, an antibody to CD4 is used. The antibodies are added to a suspension of cells, and incubated for a period of time sufficient to bind the available cell surface antigens. The incubation will usually be at least about 15 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture, such that the efficiency of the separation is not limited by lack of antibody, i.e. using a saturating amount of antibody. The appropriate concentration can also be determined by titration. The medium in which the cells are separated will be any medium which maintains the viability of the cells. A preferred medium is phosphate buffered saline. Various media are commercially available and may be used according to the nature of the cells, including Dulbecco's Modified Eagle Medium (dMEM) , Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS) , RPMI, Iscove's medium, PBS etc., frequently supplemented with fetal calf serum, BSA, HSA, etc.

FACS may be used to separate the T cell population which is specific for the antigen based on the intensity of antibody staining, as well as other parameters such as cell size and light scatter. Although the absolute level of staining may differ with a particular fluorochrome and antibody preparation, the data can be normalized to a control. The staining intensity of cells is proportional to the amount of cell surface antigen bound by the antibodies . In a particular embodiment, the CD4⁺ T cells which are specific for the relevant antigen are selected and subsequently sorted based on the following characteristics: CFSE^dim, CD4⁺, and propidium iodide negative . Prior to screening these CD4⁺ T cells for suppressor or non-suppressor function according to the method of this aspect of the invention, the cells may be expanded by culturing in the presence of feeder cells, cytokines and/or mitogen. Feeder cells may be derived from the same cell sample which was used to generate the CD4⁺ T cell clones, or may be derived from alternative sources such as EBV-transformed JY cells or Jurkat cells. The feeder cell populations may be irradiated prior to culturing. The level of irradiation varies depending on the source of the cells as would be understood by the skilled person.

Typically, cells may be irradiated in the range of 1000 rad to 5000 rad. In a particular embodiment, feeder cells used for expansion comprise a combination of IXlO⁵ irradiated (2000 rad) allogenic peripheral blood mononuclear cells and 5X10⁴ irradiated (5000 rad) EBV- transformed JY cells.

Cytokines which may typically be used in the expansion culture include one or more of IL-2, IL-4, IL-7, and IL- 15. For example, IL-2 (20 U/ml) and IL-4 (5 ng/ml) may be used in combination.

Typical mitogens which act to activate the T cell population include phytohaemagluttinin (PHA) and anti-CD3 monoclonal antibodies. A number of anti-CD3 monoclonal antibodies are commercially available, such as OKT3 , G19- 4, Hit3a, and UCHTl (Pharmigen, San Diego, California) . To further activate a population of T cells, a co-stimulatory or accessory molecule on the surface of the T cells, such as CD28, can be stimulated with a ligand that binds to the accessory molecule. Accordingly, one of skill in the art will recognize that any agent capable of cross-linking CD28 molecules can be used to stimulate T cells, such as for example, an anti-CD28 antibody or a natural ligand for CD28. Exemplary anti-CD28 antibodies or fragments thereof include monoclonal antibody 93 (IgG2; Bristol Myers Squibb, Princeton, N.J.), monoclonal antibody KOLT-2 (IgGl), and CD28.2 (Pharmigen, San Diego, California). Exemplary natural ligands include the B7 family of proteins such as B7-1 (CD80) and B7-2 (CD86) (Freedman et al . , 1987) . In a preferred embodiment, the mitogen providing the activation signal is an anti-CD3 antibody. The CD4⁺ T cells are expanded in culture for a time period ranging from about 10 days to about 14 days with the addition of fresh cytokines to the culture medium about every 7 days .

According to the method of the first aspect of the present invention, once CD4⁺ T cells which respond to the relevant antigen have been generated, they are screened for suppressor function.

The term suppressor function in the context of an embodiment of the invention refers to the ability of one population of CD4⁺ T cells which respond to an antigen to block or significantly reduce the level of proliferation of another population of autologous T cells in a co- culture in response to the antigen, as compared to the proliferation in response to the antigen alone.

Suppressor function can be measured using a variety of in vitro assays as would be appreciated by the person skilled in the art. In one example of an assay, the autologous T cell may be a tetanus toxoid (TT) -specific T cell line. Proliferation of this cell line in response to TT is measured alone, in co-culture with a CD4⁺ T cell clone which responds to the relevant antigen, or in co- culture with a CD4⁺ T cell clone which responds to the relevant antigen together with the antigen itself. If the CD4⁺ T cell clone is regulatory (i.e. is a CD4⁺ T_reg cell), proliferation of the TT-specific T cell line will be suppressed only in the presence of the antigen. A non-T_reg clone will not suppress proliferation of the TT-specific T cell line but will proliferate in response to the antigen. In other examples of an assay, the autologous T cells may be fresh T cells, or T cell lines or clones to an antigen, including the antigen itself.

In a further example of an in vitro assay, the concentration of cytokine in a culture supernatant can be measured. For example, the presence of a CD4⁺ T cell clone which is regulatory in a cell culture may lead to the suppression of cytokine production by non-T_reg cells which are also present in the culture. Suppressor function can also be measured using a variety of in vivo assays in animal models, as would be appreciated by the person skilled in the art. One example of an in vivo assay is adoptive co-transfer, as described in WO 98/14203 and WO 01/30378. An autologous T cell as used herein is one that is derived from the same source from which the putative CD4⁺ T_reg cells that respond to the antigen have been isolated. Once CD4⁺ T cell clones with suppressor function are identified (i.e. CD4⁺ T_reg cells), the expression of one or more markers in these cells can be compared with expression of the same one or more markers in non-T_reg cells. Markers which are differentially expressed can be identified according to the methods described above. Preferably the differential expression is measured at the protein level by immunophenotyping, for example through the use of proteomic arrays. A protein microarray which will allow the identification of patterns of expression of proteins which are found on the surface of cells is particularly useful. Such arrays are known in the art (Belov et al . , 2003) and include commercially available arrays such as those available from MEDSAIC, Australia. Essentially, proteins which show differential expression in T_reg cells represent markers for these cells, which allow the cells to be distinguished and isolated from bulk cell populations.

Using the method according to a first aspect of the present invention, the inventors have identified a cell surface marker that exhibits differential expression in CD4⁺ T_reg cells. This marker is known as CD52, a target of therapeutic monoclonal antibody Campath-1H (Hale et al . , 1983) . The expression of CD52 was found to be consistently greater (high) in antigen-activated T_reg cells compared with antigen-activated non-T_reg cells.

As appreciated by the person skilled in the art, a well-characterised subset of T_reg has been identified by constitutively high expression of the marker CD25. These CD4⁺CD25⁺ cells have been demonstrated to suppress T cells in a contact -dependent manner. Interestingly, the CD25 marker was not expressed at a higher level in the regulatory CD4⁺ T cells of the present invention. Therefore, the markers identified in the present invention distinguish a novel subset of antigen-specific T_reg not previously described. This is further exemplified by the fact that the regulatory CD4⁺ T cells of the present invention did not require direct cell -cell contact with non-T_reg clones to suppress proliferation of these clones. This suggests that the regulatory CD4⁺ T cells of the present invention suppress by an as yet unknown soluble factor. More importantly, the regulatory CD4⁺ T cells of the present invention were able to suppress CD4⁺ T cells of other antigen specificities, indicating that while their activation is antigen-specific their suppressor effector function is non-antigen specific. The ability of the regulatory CD4⁺ T cells of the present invention to exert antigen-non-specific suppression (i.e. "bystander suppression") in response to specific antigen is of clinical importance. Bystander suppression resolves the dilemma of multiple autoantigens in human autoimmune diseases by obviating the need to know if the antigen used to induce tolerance is the major or primary pathogenic autoantigen. In contrast, tolerance based on anergy or deletion is limited to each individual antigen.

The CD52 marker identified according to the method of the first aspect of the present invention provides a tool for the discrimination of T_reg cells in bulk cell populations. This marker may in turn be used to identify and isolate T_reg cells, and may also be used for assessing the risk of developing an autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease, diagnosing an autoimmune disease, infectious disease, cancer, or allergic disease, measuring the progression of an autoimmune disease, infectious disease, cancer, or allergic disease or allograft rejection, or monitoring the response to immunotherapy of an autoimmune disease, infectious disease, cancer, allergic disease or allograft rejection in a subject. Furthermore, T_reg cells identified with this marker can be used for the treatment and/or prevention of autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease in a subject.

Autoimmune disease as used herein includes but is not limited to insulin-dependent diabetes mellitus, insulin autoimmune syndrome, rheumatoid arthritis, psoriatic arthritis, chronic lyme arthritis, lupus, multiple sclerosis, inflammatory bowel disease including Crohn's disease, celiac disease, autoimmune thyroid disease, autoimmune myocarditis, autoimmune hepatitis, pemphigus, anti-tubular basement membrane disease (kidney) , familial dilated cardiomyopathy, Goodpasture's syndrome, Sjogren's syndrome, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Addison's disease, chronic beryllium syndrome, ankylosing spondylitis, juvenile dermatomyositis, polychondritis, scleroderma, regional enteritis, distal ileitis, granulomatous enteritis, regional ileitis, and terminal ileitis. Allergic disease as used herein includes but is not limited to a food allergy, airborne allergy, house dust mite allergy, cat allergy, or bee venom allergy.

Infectious disease as used herein includes but is not limited to Malaria and other tropical diseases, HIV, tuberculosis, measles, pertussis, tetanus, meningitis, and hepatitis .

With respect to cancer, the present invention enables the measurement/monitoring/manipulation of responses to tumour antigens or cancer causing agents, such as human papilloma virus in the case of cervical cancer, in a range of cancers. Such cancers may include but are not limited to cancers of the prostate, breast, lung, bladder, pancreas, ovaries, skin, blood, lymphoid tissue and cervix.

The identification and subsequent isolation of T_reg cells based on the CD52 marker of the invention will typically involve detecting expression of the gene which corresponds to the marker in a heterogeneous cell sample. The T_reg cells will express the gene in a characteristic pattern of expression as determined by the methods of the invention. For example, the T_reg cells can be identified based on greater expression of the CD52 marker. In a specific embodiment, expression of the CD52 marker is measured, wherein a level of expression of the CD52 marker which is greater than the expression of CD52 in other antigen-activated T cells indicates the presence of regulatory CD4⁺ T cells. Such cells are referred to herein as "CD4⁺CD52^hi" cells. The expression of markers may be determined by methods known to persons skilled in the art, and specific examples have been described above in relation to detection of CD4. For example, an anti-CD52 antibody may be used which will bind to the CD52 protein present on the surface of cells. Since the staining intensity of cells is proportional to the amount of CD52 bound by the antibody, those T cells for which the CD52 protein expression is greater (i.e. T_reg cells) will stain with greater intensity than T cells which express the protein as basal or "normal" physiological levels which are characteristic of non-T_reg cells. Such T_reg cells are therefore characterised as CD4⁺CD52^hl. Accordingly, T_reg cells can be easily identified and subsequently isolated according to the methods described above, such as through the use of flow cytometry.

Once the desired T_reg cells have been identified, isolated, and expanded, they may be used for a number of purposes including for the treatment and/or prevention of diseases including autoimmune diseases, infectious diseases, and cancer as previously defined, undesirable immune reactions such as allograft rejection and graft- versus-host reactions, as well as for the treatment and/or prevention of allergic diseases.

Preferably, treatment is preventative in nature, i.e. is begun before symptoms arise, wherein the subject treated is one at risk of developing such undesirable diseases or immune reactions.

As used herein, an "isolated" antigen-specific regulatory CD4⁺ T cell (or T_reg cell) is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the T_reg cell, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes .

The methods and CD52 marker described herein may be used to isolate antigen-specific regulatory CD4⁺ T cells.

These isolated T_reg cells may be formulated in a pharmaceutical composition suitable for administration to a subject.

Preferably the pharmaceutical composition contains an antigen-specific regulatory CD4⁺ T cell population which constitutes greater than 70%, such as 80%, 90%, and up to 100%, of the total cell population of the composition. Preferably, the pharmaceutical composition also comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA; adjuvants and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.

Such a pharmaceutical composition may be administered to a subject in a manner appropriate to the disease to be treated and/or prevented. The quantity and frequency of administration will be determined by such factors as the condition of the subject and the type and/or severity of the subject's disease. Appropriate dosages may also be determined by clinical trials. An "effective amount" of the composition can be determined by a physician with consideration of individual differences in age, weight, disease severity, condition of the subject, route of administration and any other factors relevant to treatment of the subject. Essentially, an "effective amount" of the composition is an amount which is sufficient to achieve a desired therapeutic effect. In general, a pharmaceutical composition comprising the antigen-specific regulatory CD4⁺ T cells may be administered at a dosage of about 10⁵ to 10⁸ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight, including all integer values within these ranges. The pharmaceutical composition may also be administered multiple times at these dosages. The optimal dosage and treatment regime for a particular subject can readily be determined by one skilled in the art of medicine by monitoring the subject for signs of disease and adjusting the treatment accordingly. Treatment and/or prevention of the diseases and conditions described herein may also be effected by in vivo activation or induction of endogenous antigen- specific regulatory CD4⁺ T cells of the present invention in a subject. For example such cells may be activated by administration to a subject of a relevant antigen in tolerogenic form, administration of a relevant antigen loaded on tolerogenic cells, or administration of a relevant antigen via a tolerogenic route.

The pharmaceutical composition can be administered by using infusion techniques that are commonly used in immunotherapy, and may be administered to a subject subcutaneously, intradermally, intramuscularly, or by intravenous injection.

In another aspect, the present invention provides methods for the treatment and/or prevention of autoimmune disease, allograft rejection, graft-versus-host reactions, or allergic disease. Such treatment methods comprise administering to the subject regulatory CD4⁺ T cells according to the present invention, or a pharmaceutical composition thereof.

The ability to identify regulatory CD4⁺ T cells using the markers of the present invention provides a mechanism for assessing the risk of developing an autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease, diagnosing an autoimmune disease, infectious disease, cancer, or allergic disease or allograft rejection, measuring the progression of an autoimmune disease, infectious disease, cancer, or allergic disease or allograft rejection, or monitoring the response to immunotherapy of an autoimmune disease, infectious disease, cancer, allergic disease or allograft rejection in a subject. For example, expression of any one or more of the markers of the present invention can be monitored in a sample of interest in order to establish the presence or absence of regulatory CD4⁺ T cells in the sample. The expression of the markers can be assayed according to methods which are known to the person skilled in the art, and which are described above. For example expression of the markers can be determined at the transcriptional (i.e. nucleic acid) or translational (i.e. protein) level. At the transcriptional level, any suitable method for detecting and comparing mRNA expression levels in a sample can be used. For example, mRNA expression levels of a marker in a sample can be determined by realtime reverse transcriptase PCR. Oligonucleotide primers specific for a particular marker as used to amplify the marker from mRNA isolated from a cell sample from the subject. The levels of amplification product are measured in real-time compared to the levels of amplification product in a control sample such as from a healthy subject .

Alternatively, marker expression in a test cell sample can be performed using serial analysis of gene expression (SAGE) methodology (Velculescu et al . , 1995) . SAGE involves the isolation of short unique sequence tags from a specific location within each marker transcript. The sequence tags are concatenated, cloned, and sequenced. The frequency of particular transcripts within the starting sample is reflected by the number of times the associated sequence tag is encountered with the sequence population.

Marker expression in a test cell sample can also be analysed using differential display (DD) methodology. In DD, fragments defined by specific sequence delimiters (e.g., restriction enzyme sites) are used as unique identifiers of markers, coupled with information about fragment length or fragment location within the expressed gene. The relative representation of an expressed marker within a sample can then be estimated based on the relative representation of the fragment associated with that marker within the pool of all possible fragments. Methods and compositions for carrying out DD are well known in the art, and are described for example in U.S. Pat. No. 5,776,683, and U.S. Pat. No. 5,807,680. Alternatively, marker expression in a test cell sample can be analysed using hybridization techniques, based on the specificity of nucleotide interactions. Oligonucleotides or cDNA can be used to selectively identify or capture DNA or RNA of specific sequence composition, and the amount of RNA or cDNA hybridised to a known capture sequence can be determined qualitatively or quantitatively, to provide information about the relative representation of a particular marker transcript within the pool of cellular messages in a sample. Hybridisation analysis can be designed to allow for concurrent screening of the relative expression of hundreds to thousands of markers by using, for example, array-based technologies having high density formats, including filters, microscope slides, microchips, or solution-based technologies that use spectroscopic analysis (e.g., mass spectrometry).

Suitable arrays can be produced according to a number of methods known in the art, for example as described in U.S. Pat. No.5, 445, 934, and WO 95/35505. Methods for collection of data from hybridisation of samples with arrays are also well known in the art. For example, transcripts from a particular cell sample can be generated using a detectable fluorescent label, and hybridisation of the transcripts in the samples to the array can be detected by scanning the array for the presence of the detectable label. Methods and devices for detecting fluorescently marked transcripts on devices are known in the art. Generally, such detection devices include a microscope and light source for directing light at a substrate. A photon counter detects fluorescence from the substrate, while an x-y translation stage varies the location of the substrate. A confocal detection device that can be used in the subject methods is described in U.S. Pat. No. 5,631,734. A scanning laser microscope is described in Shalon et al . , (1996) . Essentially, the ratio of the fluorescent signal from one cell sample is compared to the fluorescent signal from another cell sample, and the relative signal intensity determined.

At the translational level, any suitable method for detecting and comparing marker protein expression or activity in a sample can be used. For example, detection can utilise staining of cells or histological sections from a sample (e.g. from a biopsy sample) with labelled antibodies, performed in accordance with conventional methods. Cells can be permeabilised to stain cytoplasmic molecules. In general, antibodies which specifically bind to a marker of the present invention are added to a sample, and incubated for a period of time sufficient to allow binding to the epitope, usually at least about 10 minutes. The antibody can be detectably labelled for direct detection (e.g. using radioisotopes, enzymes, fluorescent markers, chemiluminescent markers, and the like) , or can be used in conjunction with a second stage antibody or reagent to detect binding (e.g., biotin with horseradish peroxidase-conjugated avidin, a secondary antibody conjugated to a fluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc.). The absence or presence of antibody binding can be determined by various methods, including flow cytometry of dissociated cells, microscopy, radiography, scintillation counting, etc. Any suitable alternative methods of qualitative or quantitative detection of levels or amounts of differentially expressed marker polypeptide can be used, for example proteomic or protein arrays as described above, ELISA, western blot, immunoprecipitation, radioimmunoassay, etc. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

It must also be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise .

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention encompasses any and all variations which become evident as a result of the teaching provided herein.

Example 1:

The inventors have recognised that it has not been possible to distinguish antigen-specific T_reg cells in bulk populations because surface markers that discriminate T_reg from non-T_reg, particularly after activation with antigen, are lacking. To address this problem, the inventors have taken an "inverted" approach to identify and phenotype regulatory CD4⁺ T cells specific for antigens. This entailed first generating CD4⁺ T cell clones specific for proinsulin or glutamic acid decarboxylase 65 (GAD 65) , pancreatic islet autoantigens in type 1 diabetes (TlD) . The inventors then screened the clones for suppressor function, the ability to suppress the proliferative response of "bystander" T cells or clones to the antigen or another antigen. Finally, the inventors analysed the suppressor/T_reg clones for phenotypic marker differences compared to non-T_reg clones. The inventors successfully isolated many T_reg and non-T_reg CD4+ T cell clones specific for proinsulin or GAD 65. Comparative analysis of clones has revealed differential expression of CD52, which can be used as a new marker to directly identify antigen-specific ■

Methods

Blood donors

Venous blood was obtained with informed consent and ethics committee approval from 11 subjects with pancreatic islet autoantibodies at risk for type 1 diabetes (TlD) , 12 subjects with established TlD, 9 healthy control subjects, and 6 subjects with type 2 diabetes (T2D) (Table 1) .

Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll/Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and washed twice in phosphate buffer saline (PBS) .

Reagents and antibodies

The cytokines IL-2 and IL-4 (Peprotech, Rocky Hill, NJ, USA) were used at final concentrations of 20 U/ml and 5 ng/ml , respectively. The following mitogens were used at the final concentrations indicated: phytohaemagluttinin (PHA) at 2.5 μg/ml and anti-CD3 (clone OKT-3) at 30 ng/ml.

Antigens

Tetanus toxoid (TT) was supplied by CSL (Parkville, Victoria, Australia) . Recombinant GAD 65 was purchased from Dr Peter van Endert, Hδpital Necker, Paris. The endotoxin concentration of the GAD 65 stock solution, measured by Limulus lysate assay (BioWhittaker,

Walkerville, MD, USA), was 1.2 EU/mg/ml . Recombinant human proinsulin was produced in-house as published (Cowley and Mackin, 1997) . Briefly, after refolding and reversed phase high performance liquid chromatography (RP-HPLC) purification, the protein resolved as a single species of expected molecular mass by matrix-assisted laser desorption/ionisation-time of flight mass (MALDI-TOF) spectrometry. The endotoxin concentration of proinsulin stock solution was 0.51 EU/mg/ml .

Isolation of antigen-specific CD4⁺ T-cell clones/lines PBMC were stained with 5 , 6-carboxylfluorescein (CFSE) as previously described (Mannering et al . , 2003). Stained cells (2 x 10⁵ in 150 μl) were cultured in 96-well round- bottom plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) either with medium alone, GAD 65 (5 μg/ml) , proinsulin (20 μg/ml) or TT (10 LFU/ml) . After 7 days of culture, cells for each antigen were pooled, washed in PBS and stained on ice with anti-human CD4-PE (BD Pharmingen, San Diego, CA, USA) . A single, viable (propidium-iodide negative) CFSE^dira CD4⁺ cell that had undergone division in response to antigen was sorted into each well of a 96-well plate as previously described (Mannering et al . , 2005). Each well contained 2xlO⁵ irradiated allogeneic PBMC, 2xlO⁴ irradiated JY EBV cells and 30 ng/ml anti-CD3 (OKT3) in medium supplemented with 20 U/ml IL-2 (NCIBRB Preclinical Repository, Fisher Biosciences, Rockville, MD) and 5 ng/ml IL-4 (Peprotech, Rocky Hill, NJ, USA) . Fungizone (Amphotericin B, Bristol-Myers Squibb, Princeton, NJ, USA) was added to cultures at a final concentration of 2 μg/ml. Cultures were supplemented on day 7 and 14 with fresh cytokines in 50 μl of medium, to the final concentrations indicated. Growing clones were identified by visual inspection, expanded into 48-well plates after ~2 weeks and then tested for antigen-specific regulatory function in a suppressor assay. TT-specific T-cell lines were generated by culturing IxIO⁷ PBMC with 10 LFU/ml TT. IL-2 (20 U/ml) was added on day 3 and the cells were used between day 7 and 10. Antigen-specific clones were expanded by re-stimulating with 2.5 μg/ml PHA (Sigma, St. Louis, MO, USA) in the presence of 20 U/ml IL-2 and 5 ng/ml IL-4. Suppressor T-cell assay- Clones were screened for antigen-specific suppressor function by measuring their ability to inhibit proliferation of autologous ("bystander") T-cell lines/clones. T_reg clones were identified based on their ability to suppress proliferation of bystander T cells in the presence of cognate antigen. In an example of the suppressor assay shown in Figure 1, the bystander cell is a tetanus toxoid (TT) -specific T cell line. Proliferation of the TT line in response to TT is measured alone (Figure IA) , in co-culture with a GAD 65-specific clone (Figure IB) , or in co-culture with a GAD 65-specific clone and GAD 65 (Figure 1C) . If the GAD 65-specific clone is regulatory, proliferation of the TT line is suppressed only in the presence of GAD 65 (Figure 1C) . A non-T_reg clone does not suppress proliferation of the TT line but proliferates in response to GAD 65 (Figure ID) .

For screening of antigen-specific suppressor function, 200 μl of each clone was taken from culture, washed once with PBS and once with medium. An equal volume of clones was added to 4 wells containing 5xlO⁴ irradiated autologous PBMC as antigen presenting cells (APCs) , IxIO⁴ autologous TT-specific T-cell line and 10 LFU/ml TT. The relevant antigen (GAD or proinsulin) , at the indicated concentration, was added to half of the wells.

Where indicated, neutralising antibodies to IL-10 (JES3-9D7, BD Pharmingen) and/or TGF-β (clone 141322, R&D systems) or the relevant isotype controls were added at a final concentration of 10 μg/ml . In some experiments, clones were cultured at a 1:1 ratio in 24-well plates separated by a transwell (0.4 μm, Costar) . Irradiated autologous APCs (7.5xlO⁵) were added to both chambers, 3.5xlO⁵ of non-T_reg was added to the bottom chamber, and 3.5xlO⁵ T_reg was added to the top chamber. GAD 65 was added at a final concentration of 5 μg/ml. ³H- thymidine incorporation assay

Cells were cultured in 96-well round bottom plates in replicates of six with or without antigen at the concentrations indicated. After 48 hours, ³H-thymidine was added at 0.5 μCi/well and the cells were harvested 16 hours later and proliferation was determined by ³H- thymidine incorporation. ³H-thymidine incorporation was measured by scintillation counting and expressed as counts per minute (cpm) . At this stage, antigen-specific T_reg clones were operationally defined if, in the presence of antigen, they inhibited proliferation of the TT line by >30%, a threshold that exceeded twice the coefficient of variation of the response of the line to TT.

Two-colour suppressor T-cell assay

T_reg or non-T_reg clones were labelled with the fluorescent dye, PKH26 (Sigma, St Louis, MO, USA) , at a final concentration of 2 μM and co-cultured at a 1:20 ratio with autologous PBMCs labelled with 0.5 μM CFSE. Following 5-6 days in culture, cells were analysed on a FACSAria flow cytometer and the proportion of PBMC or clone responding to antigen was determined.

Immunophenotyping For surface phenotyping, cells were collected and stained on ice with the appropriate concentrations of PE- labelled antibodies to GITR (eBioscience, San Diego, CA, USA) , HLA-DR and CD103 (BD Pharmingen, Franklin Lakes, NJ, USA) . Foxp3 expression was detected with a flow cytometry kit (Biolegend, San Diego, CA, USA) . In some experiments, PE-labelled antibody to CTLA-4 (BD Pharmingen) was added at the same time as antibody to Foxp3 to detect intracellular expression of CTLA-4. The expression of a panel of CD antigens was measured on the surface of T_reg and non-T_reg clones using a CD antigen array according to the manufacturer's instructions (MEDSAIC, Sydney, Australia) (Belov et al . , 2003). Clones (IxIO⁶) were taken directly from culture on day 10 and analysed resting or after stimulation for 24 hours with 5 μg/ml of plate-bound anti-CD3 (OKT3) . Cells captured by membrane-bound antibodies were quantified by densitometry.

Detection, isolation and functional analysis of antigen- specific CD4⁺CD52^hi and CD4⁺CD52^lα T cells

PBMC were stained with CFSE or PKH26 and cultured with and without antigen, as described above. One week later, cells were collected and incubated on ice with an unconjugated anti-CD52 antibody (Clone H24-930, BD Bisosciences) followed by APC-labelled anti-mouse IgG (Caltag) . After washing in FACS buffer (PBS, 0.1% pooled human serum), cells were stained with PE-Cy7-labelled anti-CD4 antibody (BD Biosciences) before a final wash with FACS buffer. In some experiments, anti-CD52 antibody directly conjugated with PE (clone CF1D12, Caltag) was used. GAD 65- or hPI-specific cells (CFSE^diltl) identified as CD52^hi and CD52^lD were FACS-sorted, washed in PBS and counted. CD4⁺ T cells that divided in response to TT were also sorted, washed and counted. CD52^hl and CD52¹⁰ cells were co-cultured with TT-responsive cells at a 1:1 ratio in 6 wells of a 96-well plate. All 6 wells contained 5xlO⁴ irradiated autologous PBMC as APCs and 10 LFU/ml TT antigen to stimulate proliferation of sorted autologous

TT-specific CD4⁺ T cells. The appropriate antigen, GAD or proinsulin, was added to 3 of the 6 wells to activate sorted "T_reg" or "non-T_reg" . As a control, irradiated PBMCs were also cultured with or without antigen. Antigen-specific CD4⁺ T cells were cloned from either the CD52^hl or CD52^lD fractions and expanded as described above. Clones were screened for antigen-dependent T_reg function in the suppressor assay: 10,000 cells from each clone were added at a 1:1 ratio with autologous TT line cells in the presence of TT alone or TT and antigen.

Proliferation of the TT line without antigen, with TT or with TT plus antigen was also measured. ELISpot assay

IFN-g and IL-17 production by CD4⁺CD52^hi and CD52^lD cells was measured using ELISpot kits from MABtech (IFN-g) and eBioscience (IL-17) . Antigen-specific CD4⁺CD52^hl and CD4⁺CD52^l0 cells were isolated as described above; CFSE- labelled PBMCs were cultured with tetanus toxoid (10 Lfu/ml) or GAD65(5 μg/ml) for 7 days, washed and stained with PECY7-anti-CD4 and PE-anti-CD52 antibodies. The cells were then sorted into CD4⁺CD52^hi and CD4⁺CD52^l0 populations and 5,000 cells of each, or no added cells, were incubated in duplicate for 20 hours with irradiated (2000 rad) autologous PBMCs (30,000 cells/well) as APCs, with or without antigen, in the presence or absence of IL-2 (10 U/ml) , in wells of ELISpot plates pre-bound with antibody to IFN-g or IL-17. Cells were cultured at 37°C, 5% CO₂ for 20 hours. Coating of wells and detection of cytokine was carried out according to the manufacturer's instructions.

Results Isolation and identification of GAD 65-specific T_reg and non-T_reg clones

CD4⁺ T-cell clones were isolated from bulk PBMCs that had divided in response to GAD 65 or proinsulin by FACS sorting 1 cell/well and were subsequently expanded over 2 to 3 weeks. Each clone was co-cultured with an autologous T-cell line specific for TT (TT line) in the presence of TT antigen and either in the absence (nil) or presence of GAD 65 (GAD) or proinsulin. Clones that suppressed proliferation of the TT line by at least 30% in response to antigen, such as clones 3.3 and 3.7 from donor #3 ; clones 4.3 and 4.4 from donor #4; clones 5.1 and 5.10 from donor # 5 (Figure 2), were operationally defined as T_reg. Clones that did not suppress the TT line and proliferated in response to antigen, such as clone 3.11 from donor #3; clone 4.1 from donor #4; clone 5.8 from donor # 5 (Figure 2), were operationally defined as non-T_reg. GAD 65 -specific T_reg and non-T_reg clones can be expanded in vitro and retain suppressor function. Clones from 2 healthy donors (donors #3 and #4) classified as GAD 65-specific T_reg and non-T_reg at screening were further expanded and subjected to phenotypic and functional analysis. Autologous GAD 65-specific T_reg and non-T_reg clones were co-cultured at different T_reg:non-T_reg ratios in the absence or presence of GAD 65. GAD-specific T_reg clones from donor #3 (clone 3.3) or donor #4 (clone 4.4) suppressed the proliferation of autologous GAD-specific non-T_reg clones (clones 3.73 and 4.19, respectively) when co-cultured at 0.1:1, 1:1 and 2:1 ratios (Figure 3A, left panel), with suppression reaching over 80%. No suppression was observed when two non-T_reg clones (clones 4.15 and 4.19) were co-cultured under identical conditions (Figure 3B, left panel) . When the proliferative responses of individual T_reg and non-T_reg clones to GAD was measured, T_reg clones did not proliferate whereas both non-T_reg clones (clones 3.73 and 4.19) had strong proliferative responses (Figure 3A, right panel). Both clones 4.15 and 4.19 proliferated in response to GAD (Figure 3B, right panel) . T_reg clones that elicit their suppressor function in response to GAD 65 could also suppress CD4⁺ T cell clones of other antigen specificities. This demonstrates that although the T_reg clones are specific for GAD 65, their suppressor effector function is non-antigen specific.

An inability of T_reg to proliferate to antigen has been described. The inventors have found, similar to previous reports, that this T-cell anergy could be reversed with low concentrations of IL-2. However, non- T_reg clones proliferated in response to GAD65 under both conditions (Figure 4A) . Furthermore, by co-culturing PKH- labelled clones with autologous CFSE-labelled PBMC they demonstrate that T_reg clones divide in response to antigen while maintaining suppressor function. The percentages of clone and PMBC that divided in response to no antigen (left panel) or GAD 65 (right panel) are indicated in Figure 4B. In the presence of TT, an antigen that elicits a stronger PBMC response than GAD, the T_reg clone exhibited even more robust proliferation to GAD (Figure 4C) . As expected, the non-T_reg clone proliferated strongly in response to GAD (Figure 4B, lower right panel) . Although the anergic state of the T_reg clone was reversed, its suppressor function was not, as reflected by the lower proportion of PBMCs that underwent division in response to GAD (Figure 4B, upper right panel) compared to the proportion in the presence of the non-T_reg clone (Figure 4B, bottom right panel) .

A well -characterised subset of T_reg identified by constitutively high expression of CD25 (CD4⁺CD25⁺ Tr) has previously been demonstrated to suppress T cells in a contact-dependent manner in vitro. To address whether the inventor's T_reg clones functioned similarly, they tested their ability to suppress proliferation of autologous non- T_reg clones in the absence of direct cell-cell contact. Suppression still occurred when clones were prevented from coming into direct contact by a semipermeable membrane, indicating that T_reg induce suppression by a soluble factor (Figure 5A) . Supernatant harvested from T_reg clones stimulated with GAD 65 for 48 hours was centrifuged for 30 minutes at 100,000xg. Confirming the presence of a soluble suppressor factor, the cell division index (CDI) of PBMCs from a healthy HLA-DR1/4 donor dividing in response to hPI was suppressed when T_reg supernatant (SN) , pre- or post- ultracentrifugation was added to the culture at a final SN: medium ratio of 1:1. No suppression was observed when non-Treg SN was added (Figure 5B) .

Prime candidates for the suppressor factor were IL-10 and TGF-β, because T cells secreting these molecules are known to be regulatory. Addition of neutralising antibodies to IL-10 or TGF-βl did not however alter suppressor activity (Figure 5C). Furthermore, supernatant harvested from T_reg clones stimulated with GAD 65 for 24 hours and screened for cytokines and chemokines did not contain immunoassayable IL-IO or TGF-β (data not shown) . Therefore, suppressor function of the inventor's regulatory CD4⁺ T cells is mediated via a soluble factor (s) which is not IL-IO or TGF-β.

Identification of CDS2 as a marker of antigen-specific T_reg

Two pairs of GAD 65-specific T_reg and non-T_reg clones from donors #3 and #4 were compared phenotypically with the aim of identifying differentially expressed cell surface molecules. Clones were immunophenotyped using a CD antigen array (MEDSAIC Pty Ltd.) that allows the expression of >80 CD antigens to be determined simultaneously (Belov et al . , 2003) . Clones were analysed resting (Figure 6A) and following stimulation for 24 hours with 5 μg/ml of plate-bound anti-CD3 (OKT3) (Figure 6B) . T_reg and non-T_reg clones were similar at rest but a difference in one CD antigen, CD52 , was observed following stimulation; CD52 expression was consistently higher on CD4⁺ T_reg clones (Figure 6B) . This phenotype was confirmed by flow cytometry at resting (Figure 6C) and following activation with 5μg/ml of plate-bound anti-CD3 (Figure 6D) . Interestingly, CD25, a marker of the naturally- occurring Treg subset, was not higher on T_reg than non-T_reg. The ultimate test of a T_reg marker is its ability to identify and isolate specific cells with regulatory function from a heterogenous bulk cell population.

CD52 delineates antigen-specific CD4⁺ T_reg and non-T_reg

PBMC were isolated from the blood of a healthy HLA- DRBl*010l/0401 donor, labelled with CFSE and cultured with no antigen (nil), human proinsulin (hPI) or TT, as previously described (Mannering et al . , 2003). One week later, cells were collected and CD4⁺ and CD52⁺ cells analysed by flow cytometry. A proportion (8-10%) of the CD4⁺ cells that divided in response to hPI maintained higher expression of CD52 compared to the remainder of the dividing population (Figure 7A) . hPI -responsive cells were sorted into CD52^hi and CD52¹⁰ populations by FACS and analysed for their ability to suppress proliferation of sorted CD4⁺ TT-specific T cells, in response to hPI . The CD52^hl population suppressed proliferation of the TT- specific T cells in an hPI -dependant manner, whereas suppression was not observed in the presence of CD52¹⁰ cells (Figure 7B) . Similar results were found for GAD- activated T cells. This indicates that CD52 can be used to select CD4⁺ cells with suppressor function from an antigen- responsive CD4⁺ T-cell population, irrespective of which antigen led to their initial induction. This finding was further supported by analysis of CD4⁺ T-cell clones isolated from CD52^hi and CD52¹⁰ PBMCs that responded to GAD. GAD-specific T_reg clones were predominantly found within the CD52^hi population (Figure 7C) . Thus, of 151 CD52^hi clones tested from two donors, 21 (14%) suppressed proliferation of an autologous TT-specific T-cell line, in a GAD-dependent manner, by greater than 30%. In contrast, only 3/101 (3%) CD52¹⁰ clones were T_reg. As predicted from earlier results, the proliferative response of these clones to GAD was inversely correlated with their suppressor function (Figure 7D) .

Furthermore, high CD52 expression denoted antigen- activated CD4⁺ cells with low production of IFN-g AND IL- 17. To demonstrate this, the inventors incubated CFSE- labelled PBMCs with tetanus toxoid (10 Lfu/ml) or GAD65 (5 μg/ml) for 7 days, washed the cells and stained them with PECY7-anti-CD4 and PE-anti-CD52 antibodies. The cells were then sorted into CD4⁺CD52^hi and CD4⁺CD52^l0 populations and 6,000 cells of each, or no added cells, were incubated in duplicate for 20 hours with irradiated (2000 rad) autologous PBMCs (30,000 cells/well) as APCs, with or without antigen, in the presence or absence of IL-2 (10 U/ml) , in wells of ELISpot plates pre-bound with antibody to IFN-g or IL-17. Less than 3 IFN-g-producing cells/well were observed with irradiated PBMCs and tetanus alone. CD52 ^hl cells expressed significantly fewer IFN-g spots than CD52^lσ cells (Figure 8) . Data (mean+SD) are representative of results in two normal individuals (Figure 8A, B) . Similar results were seen when FACS-sorted CD52^hl and CD52^lD CD4⁺ cells that divided in response to GAD 65 were tested by ELISpot for IL-17 production. Whereas some IL-17 producing cells were detected in the CD52¹⁰ fraction, particularly in the presence of IL-2, this was not the case for CD52^hl cells (Figure 8C) .

CD4⁺CD52^hl cells suppressed the IFN-g production by CD4⁺CD52^l0 cells when co-cultured at a 1:1 ratio (Figure 8C) .

The frequency of GAD-specific CD4⁺ CD52^hl T cells is decreased in type 1 diabetes Based on the finding that CD52 was a marker of autoantigen-activated T_reg, the inventors investigated whether the proportion of CD4⁺CD52^hl T cells responding to GAD was different in people at risk for, or with, TlD compared to HLA-matched healthy controls or people with type 2 diabetes (T2D) . To standardise comparisons between individuals, CD52 expression on undivided CD4⁺ cells was used as an internal reference. GAD-activated (divided) cells with CD52 expression ≥ the upper 5% of CD52⁺ cells in the undivided CD4⁺ population were taken to be CD52^hl. In three consecutive weekly experiments on the same healthy donor the coefficient of variation of the CD52^hl/1° ratio in response to GAD was 26%. In comparing the subject groups, individuals at risk for, and with, TlD had a significantly lower proportion of CD52^hl cells in the CD4⁺ population responding to GAD, compared to healthy controls or individuals with T2D (Figure 9A) . This difference was not observed with the control antigen TT (Figure 9B) . Importantly, the number of cells dividing in response to GAD or TT relative to background (cell division index) was similar between the groups (Figure 9C, D) . Discussion

In this study the inventors define islet antigen- specific T cells with regulatory properties that can be identified by expression of a particular marker, CD52 , within a population of CD4⁺ T cells dividing in response to antigen. CD52 for example could be used to isolate T_reg from a healthy individual, a person at risk for TlD and an individual with clinical diabetes. Using a novel approach to identify CD4⁺ T cells with regulatory function, islet antigen-responsive CD4⁺ T cells were cloned and classified as T_reg or non-T_reg based on suppressor function. T_reg clones were demonstrated to have potent suppressor function (Figure 3) and, once activated in an antigen-specific manner, could suppress cells of other specificities, e.g. TT-specific T-cell lines. This demonstrates that although these cells are activated in an antigen-specific manner, their suppressor function is non-antigen specific.

T_reg clones were anergic, in that they failed to divide in response to antigen, but anergy could be reversed by exogenous IL-2. This implies that, given appropriate conditions, T_reg can divide in response to antigen and explains why they could be first isolated from a population of cells dividing in response to antigen. In support of this, co-culture of the PKH-labelled T_reg clones with autologous CFSE-labelled PBMC clearly demonstrated that T_reg undergo division in response to GAD 65 yet maintain suppressor function. These observations are in line with previous studies in mice which demonstrated that CD4⁺CD25⁺ T_reg are anergic in vitro but proliferate in vivo. T_reg clones did not require direct cell-cell contact with non-T_reg clone/cells to suppress proliferation of the latter. This suggests that these T_reg clones are not related to the CD4⁺CD25⁺ T_reg subset but are more similar to T_regs induced in the periphery. However, in contrast to the TrI or Th3 types of induced T_reg, neutralisation of IL-10 or TGF-βl, respectively, did not reverse suppression and neither IL-10 nor TGF-β was detected in T_reg supernatants, indicating that these T_reg clones suppress by an as yet unknown soluble factor.

Comparative phenotyping with a CD antigen array identified a nominal T_reg marker, CD52 , differentially expressed following stimulation of clones through the T- cell receptor (TCR) , suggesting that CD52 expression could be used to distinguish T_reg and non-T_reg within a population of cells dividing in response to antigen. CD52 is a very small glycopeptide molecule, the peptide core of which is composed of only 12 amino acids. The immunological role of CD52 has yet to be determined. Watanabe et al . , 2006 indicate that a monoclonal antibody to CD52 provides a co- stimulatory signal to human CD4⁺ T cells leading to induction of T_reg cells. However they do not provide evidence that CD52 is a marker of T_reg cells.

Analysis of CD52 expression on antigen-responsive CD4⁺ T cells demonstrated that a small proportion of cells responding to antigen maintained expression of CD52 (CD52^hl) , whereas the majority of dividing cells lost CD52 expression (CD52^lQ) .

CD52^hl cells could suppress bystander TT-specific CD4⁺ T cells in an antigen-dependant manner, unlike the CD52^lσ population. Isolation of clones from CD52^hl and CD52¹⁰ populations demonstrated that T_reg were predominantly in the CD52^hl population. Furthermore, CD52^hl cells produced less of the pro-inflammatory cytokines, IFN-g and IL-17, than CD52^lQ cells. Interestingly, CD52^hl cells underwent fewer rounds of division compared to CD52¹⁰ cells overall, determined by mean fluorescence intensity of PKH (48 for CD52^hi versus 29 for CD52^lD) . However, CD52^lQ cells that had undergone the same number of divisions as CD52^hl cells did not demonstrate suppressor properties (data not shown) . Kubota et al . , (1990) noted that CD52 is normally down-regulated as cells undergo division. In the two- colour assay, T_reg clones divided in response to antigen but not as rapidly as PBMC or non-T_reg- Retention of CD52 expression in response to antigen stimulation may therefore denote relative anergy, a feature of cloned T_reg. This is the first report of markers that enable human antigen-specific T_reg to be identified in a bulk population of cells. The ability to directly identify autoantigen- specific T_reg should facilitate monitoring of immune function during the natural history of autoimmune disease and in response to intervention/prevention treatment, as well as the isolation of antigen-specific T_reg for autologous cell -based therapy.

Example 2 :

It is hypothesized that subjects with autoimmune disease, or who are at -risk of developing autoimmune disease, have a defect in the number and/or function of antigen-specific CD4⁺CD52^hl T_reg when compared to healthy controls .

The present invention has enabled the identification of such T_reg cells in subjects and therefore enables one to determine for example whether the number and/or function of such T_reg cells is decreased in people with, or at risk of, type 1 diabetes, or other immuno-inflammatory diseases such as coeliac disease and rheumatoid arthritis, compared with healthy controls. For example, the number and function of CD4⁺CD52^hl T_reg cells specific for GAD 65 or proinsulin in people with or at risk for type 1 diabetes, or specific for gliadin and deamidated gliadin in people with or at risk for coeliac disease, or specific for synthetic collagen type II or fillagrin peptides in people with or at risk for rheumatoid arthritis, can be compared with the number and function of these cells in matched healthy control subjects .

Example 3 ;

It has previously been demonstrated that injection of NOD-SCID mice with splenic T cells from a diabetic NOD mouse results in development of diabetes, which can be prevented by the co- injection of T cells with regulatory properties (see WO 98/14203, WO 98/14203) . It is hypothesised that injection of the antigen-specific CD4⁺CD52^hl T_reg cells of the present invention may suppress/delay disease development .

The present invention therefore enables one to test for example the ability of CD4⁺CD52^h:L T_reg cells to prevent and reverse disease. The present invention also enables one to evaluate whether the ex-vivo expansion of antigen- specific CD4⁺CD52^hl T_reg cells is a potential cell-based therapy for autoimmune diseases.

Example 4 ; The present invention has identified markers that can be used to identify and/or isolate antigen-specific regulatory CD4⁺ T cells. For example, these cells can now be characterised as CD4⁺CD52^hl T_reg. The inventors have found that these cells do not require direct cell -cell contact with a target T cell in order to exert their suppressor function as shown in Figure 5A.

The mechanism by which the T_reg cells of the present invention exert their suppression and how they influence the phenotype, function and activation of antigen- presenting cells can be determined.

For example, supernatants can be harvested from T_reg and non-T_reg clones stimulated with antigen and can subsequently be subjected to a number of physico-chemical treatments in order to determine the nature of the suppressor factor (i.e. whether it is a protein, lipid or sugar) . Furthermore, non-protein factors known to suppress T-cell function, namely nitric oxide (NO) , prostaglandins and the tryptophan metabolites, kynurenine and 3- hydroxyanthranilic acid, generated by indoleamine-2 , 3- dioxygenase (IDO) , can also be tested for by blocking their generation in the T_reg suppressor assay with inhibitors of NO synthase (nitro-L-arginine methylester, L-NAME), cyclooxygenases (indomethacin) and IDO ( 1 -methyl - D-tryptophan) , respectively.

If the suppressor factor is peptidic in nature, the cytokine, chemokine and growth factor profiles of supernatants from T_reg and non-T_reg clones can be determined using commercially-available protein arrays. Furthermore, the nature of the soluble factor can be elucidated by its purification and analysis using techniques such as chromatography and amino acid sequencing.

Example 5 :

The inventors have used a CD antigen array to identify markers which are differentially expressed in antigen-specific regulatory CD4⁺ T cells (T_reg cells) when compared to non-regulatory T cells (non-T_reg cells) . Additional methods of identifying markers which are differentially expressed at the translational or protein level can be explored. For example proteomic analysis using the technique of two-dimensional difference gel electrophoresis (2-DIGE) can be used as outlined in Van der Bergh and Arckens et al . , (2004) . Using this technique, total cell lysate or the cell membrane fraction of T_reg and non-T_reg cells can be analysed by 2-DIGE to identify proteins differentially expressed in T_reg cells when compared to non-T_reg cells. This method is advantageous in that it also allows the identification of proteins which have different post -translational modifications in T_reg and non-T_reg cell types.

Example 6 :

It has been previously demonstrated that depletion of regulatory T cells can promote tumour rejection and immunity to infectious agents. It is hypothesised that depletion of antigen-specific CD4⁺CD52^hl T_reg cells of the present invention may enhance tumour rejection or clearance of infectious agents. Furthermore, monitoring of CD4⁺CD52^hl cells specific for tumour antigens or infectious agents could aid prognosis of these conditions.

The present invention therefore enables one to test for example the effect of CD4⁺CD52^hl depletion on tumour size, tumour immunity and tumour rejection as well as protection against and/or clearance of infectious agents.

Table 1

Subjects investigated for antigen-responsive CD4⁺CD52^hl T cells .

Donor Sex Age HLA GAD Ab ^A

Pre-TID

1 M 13 ND +

2 M 8 ND +

3 M 1 1 ND +

4 F 1 1 ND +

5 F 7 ND +

6 F 19 ND +

7 M 8 ND +

8 M 9 ND +

9 M 7 ND +

10 M 8 ND +

11 M 13 ND +

TlD

12 M 19 DR 3,3 +

13 F 56 DR 4,4 +

14 M 49 DR 4,13 +

15 F 25 DR 3,4 +

16 M 30 DR 4,11 +

17 M 30 DR 3,3 +

18 M 28 DR 3,4 -

19 F 64 DR 3,4 +

20 M 38 DR 4,13 +

21 M 9 ND +

22 M 5 DR 3,4 -

23 F 20 DR 3,13 -

Healthy

24 M 43 DR 3,4 ND

25 F 48 DR 3, 4 ND

26 F 51 DR 4,7 ND 27 M 52 DR 3,7 ND

28 M 30 DR 4,6 ND

29 M 45 DR 3,13 ND

30 M 26 DR 3,1 1 ND

31 M 39 DR 1 ,4 ND

32 M 32 DR 3,4 ND

T2D

33 M 54 DR 3,3 -

34 M 58 DR 4,8 -

35 M 53 DR 1,6 -

36 F 52 ND -

37 F 59 ND -

38 M 41 DR 2,10 -

A. + = positive; - = negative; ND = not done

References

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Claims

Claims

1. A method of identifying a marker of antigen-specific regulatory CD4⁺ T cells, said method comprising:

(a) generating antigen-specific CD4⁺ T cell clones from a cell sample;

(b) screening said CD4⁺ T cell clones for suppressor function; and

(c) comparing expression of one or more markers in the CD4⁺ T cell clones which have suppressor function with expression of same markers in non- regulatory T cell clones, wherein a marker differentially expressed in the CD4⁺ T cell clones which have suppressor function when compared to the non-regulatory T cell clones is a marker of antigen-specific regulatory CD4⁺ T cells in the cell sample .

2. A method according to claim 1, wherein suppressor function is determined by measuring the ability of the CD4⁺ T cell clones to inhibit proliferation or cytokine production of autologous T cell lines/clones.

3. A method according to claim 1 or claim 2, wherein the antigen is one or more autoantigens selected from the group including proinsulin, glutamic acid decarboxylase (GAD 65), IA-2, islet-specific glucose-6-phosphate catalytic subunit-related protein (IGRP) , heat shock protein 60 (hsp-60) , desmoglein-1 , desmoglein-3 , myelin basic protein (MBP) , myelin oligodendrocyte glycoprotein (MOG) , proteolipid protein (PLP) , gliadin and glutenin protein families, yype II collagen, filaggrin, vimentin, aggrecan Gl, Gp39, acetylcholine receptor, thyroid peroxidase, thyroglobulin, topisomerase, and thyrotropin receptor, or any other disease-associated antigen.

4. A method according to any one of claims 1 to 3, wherein the cell sample is selected from the group consisting of peripheral blood mononuclear cells (PMBC) , leukopheresis or apheresis blood product, bone marrow, cord blood, liver, thymus, tissue biopsy, tumour, lymph node tissue, gut associated lymphoid tissue, mucosa associated lymph node tissue, spleen tissue, or any other lymphoid tissue.

5. A method according to any one of claims 1 to 4 , wherein expression of the one or more markers is greater in antigen-specific regulatory CD4⁺ T cells than in non- regulatory T cells.

6. A method according to any one of claims 1 to 4 , wherein expression of the one or more markers is lower in antigen-specific regulatory CD4⁺ T cells than in non- regulatory T cells.

7. A method according to any one of claims 1 to 6, wherein expression of the one or more markers is measured at the transcriptional level.

8. A method according to any one of claims 1 to 6, wherein expression of the one or more markers is measured at the translational level.

9. A method according to claim 8, wherein expression of the one or more markers is measured by immunophenotyping.

10. A marker of antigen-specific regulatory CD4⁺ T cells when identified by a method according to any one of claims

1 to 9.

11. A marker according to claim 10, which is CD52.

12. A marker according to claim 11, wherein expression of CD52 is greater in antigen-specific regulatory CD4⁺ T cells than in non-regulatory T cells.

13. Use of CD52 as a marker of an antigen-specific regulatory CD4⁺ T cell.

14. Use of a marker according to any one of claims 10 to 12, for identifying and/or isolating an antigen-specific regulatory CD4⁺ T cell in a cell sample.

15. A use according to claim 14, wherein the marker is CD52.

16. A use according to any one of claims 13 to 15, wherein the antigen is one or more autoantigens selected from the group including proinsulin, glutamic acid decarboxylase (GAD 65), IA-2, islet-specific glucose-6- phosphate catalytic subunit-related protein (IGRP), heat shock protein 60 (hsp-60) , desmoglein-1 , desmoglein-3 , myelin basic protein (MBP) , myelin oligodendrocyte glycoprotein (MOG) , proteolipid protein (PLP) , gliadin and glutenin protein families, Type II collagen, filaggrin, vimentin, aggrecan Gl, Gp39, acetylcholine receptor, thyroid peroxidase, thyroglobulin, topisotnerase, and thyrotropin receptor, or any other disease-associated antigen.

17. A method of identifying an antigen-specific regulatory CD4⁺ T cell, said method comprising:

(a) detecting the level of expression of one or more markers according to any one of claims 10 to 12 in a cell sample; and (b) determining whether cells in the cell sample express said one or more markers in a manner which is characteristic for regulatory CD4⁺ T cells .

18. A method of identifying an antigen-specific regulatory CD4⁺ T cell, said method comprising:

(a) generating antigen-specific CD4⁺ T cell clones from a cell sample;

(b) screening said CD4⁺ T cell clones for suppressor function; and

(c) comparing expression of one or more markers in the CD4⁺ T cell clones which have suppressor function with expression of same markers in non- regulatory T cell clones, wherein a CD4⁺ T cell clone which has suppressor function and which exhibits differential expression of said one or more markers when compared with expression of same markers in a non-regulatory T cell clone is identified as an antigen-specific regulatory CD4⁺ T cell in the cell sample.

19. A method according to claim 17 or claim 18, comprising the additional step of isolating CD4⁺ T cells which express said one or more markers in a manner which is characteristic for regulatory CD4⁺ T cells.

20. A method according to any one of claims 17 to 19, wherein expression of the CD52 marker is measured.

21. A method according to claim 20, wherein a level of expression of the CD52 marker which is greater indicates the presence of a regulatory CD4⁺ T cell.

22. An isolated regulatory CD4⁺ T cell when identified by a method according to any one of claims 17 to 21.

23. An isolated regulatory CD4⁺ T cell having greater expression of the marker CD52 than the expression of CD52 in a non-regulatory T cell.

24. An isolated regulatory CD4⁺ T cell according to claim

23. which is a CD4⁺CD52^hi cell.

25. A regulatory CD4⁺ T cell according to any one of claims 22 to 24, wherein said cell does not require direct cell -cell contact with non-regulatory T cells to suppress proliferation of the non-regulatory T cells.

26. Use of a regulatory CD4⁺ T cell according to any one of claims 22 to 25, in the manufacture of a medicament for the treatment and/or prevention of autoimmune disease, allograft rejection, graft-versus-host reactions, or allergic disease in a subject.

27. A pharmaceutical composition comprising a regulatory CD4⁺ T cell according to any one of claims 22 to 25.

28. A method of treating and/or preventing autoimmune disease, allograft rejection, graft-versus-host reactions, or allergic disease in a subject, comprising administration of a regulatory CD4⁺ T cell according to any one of claims 22 to 25, or pharmaceutical composition according to claim 27, to said subject.

29. A method of treating and/or preventing autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease in a subject, comprising activating and/or inducing a regulatory CD4⁺ T cell in a subject, wherein said regulatory CD4⁺ T cell exhibits differential expression of one or more markers according to any one of claims 10 to 12, when compared with expression of same markers in a non-regulatory T cell clone.

30. Use of a marker according to any one of claims 10 to 12, for assessing the risk of developing an autoimmune disease, allograft rejection, graft-versus-host reactions, infectious disease, cancer, or allergic disease, diagnosing an autoimmune disease, infectious disease, cancer, or allergic disease, measuring the progression of an autoimmune disease, infectious disease, cancer, or allergic disease, or monitoring the response to immunotherapy of an autoimmune disease, infectious disease, cancer, allergic disease, or allograft rejection in a subject.

31. A use according to claim 26 or claim 30, or a method according to claim 28 or claim 29, wherein the infectious disease is selected from the group consisting of Malaria and other tropical diseases, HIV, tuberculosis, measles, pertussis, tetanus, meningitis, and hepatitis.

32. A use according to claim 26 or claim 30, or a method according to claim 28 or claim 29, wherein the cancer is selected from the group consisting of cancers of the prostate, breast, lung, bladder, pancreas, ovaries, skin, blood, lymphoid tissue and cervix.

33. A use according to claim 26 or claim 30, or a method according to claim 28 or claim 29, wherein the autoimmune disease is selected from the group including insulin- dependent diabetes mellitus, insulin autoimmune syndrome, rheumatoid arthritis, psoriatic arthritis, chronic lyme arthritis, lupus, multiple sclerosis, inflammatory bowel disease including Crohn's disease, celiac disease, autoimmune thyroid disease, autoimmune myocarditis, autoimmune hepatitis, pemphigus, anti -tubular basement membrane disease (kidney) , familial dilated cardiomyopathy, Goodpasture's syndrome, Sjogren's syndrome, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Addison's disease, chronic beryllium syndrome, ankylosing spondylitis, juvenile dermatomyositis, polychondritis, scleroderma, regional enteritis, distal ileitis, granulomatous enteritis, regional ileitis, and terminal ileitis.

34. A use according to claim 26 or claim 30, or a method according to claim 28 or claim 29, wherein the allergy is selected from the group including food allergy, airborne allergy, house dust mite allergy, cat allergy, or bee venom allergy.

35. A method of identifying a marker of regulatory CD4⁺ T cells specific for the autoantigens GAD 65 or proinsulin, or specific for the antigen tetanus toxoid, said method comprising :

(a) generating CD4⁺ T cell clones specific for GAD 65, proinsulin, or tetanus toxoid from a cell sample;

(b) screening said CD4⁺ T cell clones for suppressor function; and

(c) comparing expression of one or more markers in the CD4⁺ T cell clones which have suppressor function with expression of same markers in non- regulatory T cell clones, wherein a marker differentially expressed in the CD4⁺ T cell clones which have suppressor function when compared to the non-regulatory T cell clones is a marker of regulatory CD4⁺ T cells specific for GAD 65, proinsulin, or tetanus toxoid in the cell sample.

36. A marker of regulatory CD4⁺ T cells specific for the autoantigens GAD 65 or proinsulin, or specific for the antigen tetanus toxoid, when identified by a method according to claim 35.

37. Use of a marker according to claim 36, for identifying and/or isolating regulatory CD4⁺ T cells specific for the autoantigens GAD 65 or proinsulin, or specific for the antigen tetanus toxoid, in a cell sample.

38. A method of identifying a regulatory CD4⁺ T cell specific for the autoantigens GAD 65 or proinsulin, or specific for the antigen tetanus toxoid, said method comprising :

(a) detecting the level of expression of one or more markers according to claim 36 in a cell sample; and

(b) determining whether cells in the cell sample express said one or more markers in a manner which is characteristic for regulatory CD4⁺ T cells.

39. An isolated regulatory CD4⁺ T cell when identified by a method according to claim 38.

40. An isolated regulatory CD4⁺ T cell according to claim 39 having greater expression of the marker CD52, than the expression of CD52 in a non-regulatory T cell.