WO2015006811A2

WO2015006811A2 - Diagnostic methods

Info

Publication number: WO2015006811A2
Application number: PCT/AU2014/000730
Authority: WO
Inventors: Marnie BLEWITT; Leonard Charles Harrison; Jovana MAKSIMOVIC; Gaetano NASELLI; Alicia OSHLACK; Yuxia Zhang
Original assignee: The Walter And Eliza Hall Institute Of Medical Research; Murdoch Childrens Research Institute
Priority date: 2013-07-19
Filing date: 2014-07-18
Publication date: 2015-01-22
Also published as: WO2015006811A3

Abstract

A method for identifying the level of T-regulatory cell activity in a test biological sample comprising immune cells, the method comprising screening a test sample for the methylation profiles of a pre-selected genetic locus/loci selected from the list consisting of: (i) at least one genetic locus or at least two genetic Loci defined m one or more of Table 1 to Table 5; (ii) at least one genetic locus or at least two genetic loci defined in one or more of Table 6 to Table 10; and (iii) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; and determining the degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample.

Description

DIAGNOSTIC METHODS

FIELD

[0001] The present specification describes an epigenetic approach to the identification and characterisat on of immune cells. Enabled herein are assays to determine the level of immune cell activity in a biological sample. The assays are suitable for integration into a wide range of diagnostic, prognostic, agent-screening and therapeutic protocols and reporting systems.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

[0003) Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0004] Various sub-groups of immune cells have traditionally been characterised via their cell surface staining patterns. More recently methods for stratifying immune cells include analysis of gene expression profiles typically by RT-PCR based protocols or mass spectrometry.

[0005] One immune cell type, of particular important is the regulatory T cell, referred to as Treg. These cells suppress immune responses and therefore their activity is critically important in T cell homeostasis and in establishing immune dysfunction such as found in various diseases including sepsis and cancer and in conditions involving immunosuppression and autoimmunity. Treg are recognised as playing a pivotal role preventing autoimmune disease (Wing K ei ah (2010) Nat Immunol; 1 1 :7- 13). Prototypic Natural Treg (nTreg), also referred to as thymus derived T reg cells are CD4^*'„ nTreg are derived from the thymus and programmed by the transcription factor Forkhead-box P3 (FOXP3) (Fontenot JD ei at. (2003) Nat. Immunol 4:330-336). nTreg are distinguished from peripherally derived Treg cells and from Treg induced in vhro. in human blood, FOXP3 ^" nTreg are separated into two groups according to their activation status: resting Treg (rTreg) and activated Treg ( Treg). rTreg are a relatively homogenous population defined by the surface expression of CD45RA and CD25. aTreg, derived from rTreg after antigen activation in vivo, are usually the CD25^ii!8¾ fraction of the memory T cell poo! (Miyara M et al (2009) immunity 30:899-91 1 ). [0006] However, in human blood, aTreg are not phenotypically distinct from activated conventional T cells (Tconv, CD45 O^' CD25 (MiyaraM et al. (2009) Immunity 30:899- 911) or from antigen activated naive CD4 T cells that also express FOXPS (Sakaguclii S et at (2010) Nai Rev Immunol 10:490-500).

[0007] Markers of human nTreg include the irjterleukin (IL)-7 receptor alpha chain (CD J 27), which is reduced on nTreg (Liu W et al. (2006) J Exp Med 203:1701-171 1 , Seddfki N et. ai (2006) J Exp Med 203: 1693-1700), and the Ikaros transcription factor family member Helios (Thornton AM et al. (2010) J Immunol 184:3433-3441 ). However, activation of Tconv also leads to down-re gulation of CD 127 (Sakaguclii S et al (2010 ) Nat Rev Immunol 10:490-500) and expression of Helios (Akimova T et al. (2001) PLoS One 6:e24226), The lack of consensus regarding the status of nTreg i human disease, (for example, see Zhang Y et al. (2012) Curr Opiti Endocrinol Diabetes Obes 19:271-278), underscores the need for more specific and stable markers of T-regulatory cells. Markers that identify nTreg are highly sought after.

[0008] In this regard. Baron et al. (Baron, U et al. (2007) Eur J Immunol 37:2378-2389) and Ploess et at, (Floess et al, (2007) PLoS .Biol 5;e38) describe a specific DNA hypomethylaiion pattern in the FQXP3-TSDR that distinguishes between Treg and non- regiilator T cells, independent of their activation status.

[0009] Demethylation allows chromosome relaxation and binding of EOXP3 itself as well as other transcription factors, including CREB/ATF, STAT5, ETs-1 , Cbf-p-Runxl to maintain the transcriptional activity of the FQAT3 locus (Zheng Y et al. (2007) Nature 445:936-940, Polarssky JK et at. (2010) J Mot Med (Berl) 88:1029-1040, Kim HP et al. (2007) J Exp Med 204: 1543-15 1 and Burchill MA et al. (2007) J Immunol 178:280-290).

[0010] The quality of nTreg is determined by its ability to .maintain its lineage, and expression of effector molecules required to suppress . There is need for further methods of screening immune ceils to facilitate diagnostic and therapeutic protocols, in particular, there is a need for further methods to determine the quality and quantity of T-regulatory cells.

SUMMARY

[0011] Throughout this specification and the claims whic follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0012 As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes single biomarker, as well as two or more biomarkers; reference to "an agent" includes a single agent or two or more agents; reference to "the disclosure" includes single and multiple aspects of the disclosure:, and so forth. Aspects taught and enable herein are encompassed by the term "invention". All suc aspects are enabled within the width of the present invention.

[0013] Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: I), <400>2 (SEQ ID NO:2), etc. Sequence identifiers are described in Table 12. A sequence listing is provided after the claims.

[0014] The present disclosure is of an epigenetic approach to the identification and characterisation of immune cells. In one aspect the epigenetic approach involves determination of the methyiation status of a preselected genetic locus or preselected loci.

[0015] In an embodiment, the pre-selected genetic locus/loc is/are selected from the list consisting of:

(i) a genetic locus/loci defined in one or more of Table 1 to Table 5 representing loci hypermethylated in T -regulatory cells;

(ii) a genetic locus/loci defined in one or more of Table 6 to Table 10 representing loci hypomethylated in T-regulatory cells; and

(iii) at least two genetic loci defined in one or more of Table I to Table 5, and one or more of Table 6 to Table 10 representing hypermethylated and hypomethylated genetic loci in T-regulatory cells.

[0016] In an embodiment, the pre-selected genetic locus/loci is/are selected from the list consisting of:

a genetic locus/loci defined in one or more of Table 1 to Table 5 representing loci hypermethylated in T-regulatory cells;

a genetic locus/loci defined in one or more of Table 6 to Table 10 representing loci hypomethylated in T-regulatory cells;

at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10 representing hypemiethylated and hypomethylated genetic loci in T-regulatory cells; and iv. a genetic locus/loci defined in Table 15 representing differentially methylated loci after activation of naive (CD4+, CD45RO-, CD25-).

[0017] In an embodiment, group (i) comprises at least two genetic loci defined in one or more of Table 1 and Table 2, In an embodiment, group (iii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7, These tables recite loci with generally greater deltabeta greater values than described in Tables 3 to 5 or Tables 8 to 10. and generally, loci with greater deltabeta values are employed. Within any of the tables recited herein, loci with or comprising CpG having greater deltabeta values are generally employed in the subject methods. In some embodiments., a locus or loci exhibiting a deltabeta value greater than 0.5 or at least 0.4 or at least 0.3 is selected. For Table 15, a locus of loci as described in Table 15 is/are selected fro listed loci/item numbers 1 to 183, or selected from item numbers 1 to 50, or 1 to 20 or 1 to 15. Again, in one embodiment, a locus or loci in Table 15 exhibiting a positive or negative deltabeta value greater than. 0.5 or at least 0.4 or at least 0.3 is selected.

[0018] In one embodiments, a locus or loci described in Table 1 5 is/are employed to assess selected effector cell activity, illustrative effector cells are activ ated CD4+ cells.

[0019] Accordingly, in one embodiment the present invention comprises a method for identifying the level of effector cell activity in a test biological sample comprising immune cells, the method, comprising screening the test sample for the methylation profile of one or more pre-selected genetic locus/loci selected from a locus loci defined in Table 15 and determining a degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the level of T-cell effector cell activity in the sample, in one embodiment, the locus defined in Table 15 is selected from the group of loci consisting KSR.L 1R2 K QFO , CSF2, NCF4, VMP3 , HSD17B8, IL13, ITGAX, RPTOR, AIM2, C (^"1.5 and CD59 loci as described in Table 15.

[0020] In some embodiments the test cells are enriched for CD4+ T cells. In some embodiments, the t-cells are activated. In one embodiment the reference methylation profile Is the methylation profile of naive CD4 T cells.

[0021 ] In one embodiment, CpG and DMP loci described in Table 15 are not employed.

[0022] In one embodiment, the single locus employed is not the F0XP3 locus.

[0023] In one embodiment, the FOXP3 locus is not employed.

[0024] In another embodiment, the specification enables a method for identifying the level of T-regulatory cell activity in a test biological sample comprising immune cells. In a embodiment, the method comprises screening the test sample for the methylation profile of a pre-selected genetic loci selected from the list consisting of:

(i) at least one genetic locus defined in one or more of Table 1 to Table 5;

(Ii) at least one genetic locus defined in one or more of Table 6 to Table 10; and

(iii) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; and determining a degi'ee of concordance betwee the test methyiation profiles and a reference methyiation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample.

[0025] In an embodiment, group (i) comprises at least one or two genetic locus/loci defined in one or more of Table 1 and Table 2. in an embodiment, group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

[0026] The subject specification teaches a method for identifying the acti ity of T- regulatory cells in a test biological sample comprising immune cells. In an embodiment, the method comprises screening a test sample comprising immune cells for the methyiation profiles of a pre-selected genetic locus/loci selected from the list consisting of:

(i) at least one genetic l ocus defined in one or more of Table 1 to Table 5;

(iii) at least two genetic loci defined in one or more of Table 1 to Table S, and one or more of Table 6 to Table 10; and determining the degree of concordance between the test methyiation profiles and a reference methyiation profile, wherein the degree of concordance identifies the level of T-regulaiory cell activity in the sample.

[Θ027Ι In an embodiment, group (i) comprises at least one or two genetic locus ioci defined in one or more of Table 1 and Table 2. In an embodiment group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7. As described in Example 1 , the TIGIT locus (see for example Table 6) was one of the most significantly differentially methylated regions, distinguishing naive from T-regulatory T cells, which was not affected by T-regulatory cell activation status. Accordingly, in an embodiment, the methyiation profile of the TIGIT locus is screened in accordance with the present invention. In some embodiments, 2, 3, 4, 5 or 6 CpG within the TIGIT locus are screened.

[0028| The ability of a subject to mount an appropriate immune response depends hi part upon the level T-regulatory cell activity in the subject. Test samples or test, biological samples include ny- tissue or biological sample that will comprise immune cells. Conveniently test samples comprising cells are derived from whole blood or a blood fraction comprising immune ceils, cells isolated on the basis of their expression products (phenotype or genotype) or other distinguishing features, such as size, binding characteristics etc. Suitable sources of biological samples include blood from any source such as artery or venous blood, cord blood. Biological samples include cells and tissues that have been maintained in viiro or ex vivo. Cells are typically derived from a subject but may have been maintained in ex vivo for some time, such as found with immortalised cell lines. Other bodily fluids and tissues comprise immune cells as are known in the art and are encompassed. Cells include immune stem cells, immune cells from the adaptive or innate immune systems. As described herein test sample may, for example, be processed to produce isolated or purified DN A, RNA or protein samples prior to or during the screening protocol.

[0029] The present specification is instructional for a method for facilitating an assessment of -whether or not a subject has developed or has the ability to develop an immune response or suppress a dysregulated or aberrant immune response based upon the level of T-regulatory cell activity.

[0030] The present specification teaches a method for determining whether or not a subject has or is at risk of developing an autoimmune condition.

[0031 ) The present specification teaches a method for determining whether or not a subject has or is at risk of developing an allergic condition/type .1 hyporesponsiveness. Such conditions are characterised by the production of IgE antibodies against essentially harmless antigens. Subjects with decreased T-regulatory cell activity are unable or have a limited ability to supress type 1 allergy by suppressing, for example, effector T cells and inhibiting activation of mast cells. Early detection of risk of type 1 allergy will enable early intervention strategies to increase the activity of T-regulatory cells. Allergic conditions include allergic and airwa inflammation, allergic rhinitis, allergic asthma, allergic dermatitis, allergic coiuunctitis, anaphylaxis, food or drug allergic reactions.

[Θ032] Autoimmune conditions involve aberrant immune responses, due in part to a deregulation of T-regulatory T cells. Autoimmune conditions include without limitation, systemic lupus erythematosus (S UE), multiple sclerosis (MS), arthritis including juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), coeliac disease ulcerative colitis (UC), type diabetes (T1D) and pre-diabetes. Other conditions, such as graft versus host disease, involve T-regulatory cells to suppress inappropriate allogeneic T cells and are also encompassed.

[0033] In an embodiment, the methods comprise screening a test sample comprising immune cells from a subject for the methylation profiles of a pre-selected genetic region selected from the list consisting of: (i) at least two genetic locus defined in one or more of Table 1 to Table 5;

(ii) at least one genetic locus defined i one or more of Table 6 to Table 10; and

(Hi) at least two genetic loci defined in one or more of Table 1 to Table 5 and one or more of Table 6 to Table ,10; and determining the a degree of concordance between the test methyiation profiles and a reference methyiation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample.

[0034] in an embodiment, group (i) comprises at least two genetic loci defined in one or more of Table 1 and Table 2. In an embodiment, group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

[0035] The present specification enables a method of treatment or prophylaxis of a subject, the method comprising screening a test biological sample comprising immune cells for the methyiation profiles of a pre-selected genetic region selected from the list consisting of;

(i) at least one or two genetic locus/loci defined in one or more of Table 3 to Table 5;

(iii) at least two genetic loci defined i one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; and determining the degree of concordance between the test methyiation profiles and a reference methyiation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample, and then providing therapeutic and/or behavioural modification to the subject.

[0036] In an embodiment, group (i) comprises at least one or two genetic locus/loci defined in one or more of Table 1 and Table 2. In an embodiment group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

[0037] The present invention enables a use of a panel or array of oligonucleotides specific to a pre-selected genetic region selected from the list consisting of:

(i) at least one or two genetic locus/loci defined in one or more of Table 1 to Table 5;

(iii) at least two genetic loci defined in one or more of Table 1. to Table 5, and one or more of Table 6 to Table 10;

in the manufacture of a kit or solid support for identifying the level of T-regulatory cell activity in a biological sample, tissue or subject, or the presence, risk, state, classification or progression of immune system deregulation such as found in an autoimmune condition in a subject.

[0038] The present invention enables a use of a panel or array of oligonucleotides specific to a pre-selected genetic region selected from the list consisting of:

(i) at least one or two genetic locus/loci defined in one or more of Table 1 to Table 5; (B) at least one genetic locus defined in one or more of Table 6 to Table 10; and

(iii) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10;

in the manufacture of a kit or solid support for identifying the level of T-regulatory cell activity in a biological sample, tissue or subject, or the presence, risk, state, classification or progression of immune system dysreguiation such as found in a type 1 allergic condition in a subject.

[0039] In some embodiments, type I hypersensitivity conditions are directed to allergens selected from the group consisting of weed pollens, grass pollens, tree pollens, mites, animals, fungi, insects, rubber, worms, human autoaller ens, and food allergens. 100401 In an embodiment, group (i) comprises at least one or two genetic loci defined i one or more of Table 1 and Table 2. in an embodiment, group (if) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

[00411 Further taught herein is a method of screening for an agent which modulates immune cell function, said method comprising screening an immune cell for a degree of concordance compared to a reference sample in the methylation profile of a pre-selected genetic region selected from the list consisting of;

(!) at least one or two genetic locus/loci defined in one or more of Table 5 t Table 5;

in the presence or absence of an agent to be tested wherein the agent is selected if it induces a change in the degree of concordance between the methylation profile of the immune cell and the reference sample methylation profile.

[0042] As described herein, and in accordance with another aspect, DNA methylatio in the promoter region of protein encoding genes is found to be inversely correlated -with gene expression. Thus the level of expression can be used as a surrogate marker of DNA methylation and of the level of T-regulatory cell activity in a sample. Furthermore, the level of expression is proposed for use in determining whether a subject has or is at risk of developing an autoimmune condition, or other condition associated with a dysregulated level or activity of T-regulatory cells. Illustrative examples of autoimmune conditions provided herein are coeliac disease, juvenile rheumatoid arthritis and diabetes/pre-diabetes. [0043] Conveniently, in one embodiment, T-regulatory cells are selected based upon the surface expression of TIGIT polypeptide. Thus they may be isolated, purified, selected, separated, sorted, or enriched using a TIGIT specific binding agent. Cells may subsequently or simultaneously be processed for DNA including DMA methylation, or another DNA epigenetie trait, N A or protein analysis as described herein,

[0044] In an illustrative embodiment, TIGIT is expressed in activated T-regulatory cells isolated from a subject with coeliac disease, whose FOXP3 levels are unstable and wherein the T-regulatory cells express an effector function (e.g., IFN-y expression),

[0045] In an illustrative embodiment, TIGIT is expressed in activated T-regulatory cells isolated from a subject with juvenile rheumatoid arthritis, whose FOXP3 levels are unstable and wherein the T-regulatory cells express an effector function (e.g. IFN-γ expression).

[0046] In an illustrative embodiment, TIGIT is expressed in activated T-regulatory cells isolated from a subject with pre -diabetes/diabetes disease, whose FGXP3 levels are unstable and wherein the T-regulatory cells express an effector function (e.g. 1L-4 expression).

[0047] In another illustrative embodiment, TIGIT is expressed in activated T- regulatory cells isolated from a subject with an allergic disease such as a food allergy, whose FOXP3 levels are unstable and wherein the T-regulatory cells express an effector function (e.g. IFN-γ expression).

[0048 In an embodiment, the results of assays to monitor expression product levels of TIGIT are used to determine the level of T-regulatory cell activity.

[0049] Accordingly, in an embodiment, the present description contemplates monitoring the expression of a pre-selected genetic region in a test sample comprising immune cells, which may be reflected in changing patterns of NA levels or protein production that are instructional concerning the level of T-regulatory cell activity. These methods generally comprise comparing the level of expression of a pre-selected genetic region hi the test sample to the level of expression of a corresponding genetic region in at least one reference sample, wherein a difference in the level of expression between the test sample and the at least one reference sample identifies the level of T-regulatory cell activity in the sample. In an embodiment, the pre-selected genetic region is a gene that expresses a protein selected from one or more of Tables 1 to 10. In an embodiment, the degree of similarity of expression between a test sample and reference sample is assessed. [0050] In an embodiment, the level of T-regulatory cell activity is determined by detecting in the sample a T-regulatory cell level-associated magnitude decrease in the level of expression of the gene/genetic region compared to tlie level of expression of the corresponding genetic region or the level in at least one reference sample,

[0051] In a embodiment, the level of T-regulator cell activity is determined by detecting in the sample a T-regulatory cell level-associated magnitude increase in the level of expression of the gene/genetic region compared to the level of expressio of the corresponding genetic region or the level in at least one reference sample,

10052] Accordingly, hi an embodiment, the present description contemplates monitoring the expression of a pre-selected. genetic region in a test sample comprising immune cells, whic may be reflected in changing patterns of RNA. levels or protein proditction that are instructional for the level of T-regulatory cell activity. These methods generally comprise comparing the level of expression of a pre-selected genetic region in the test sample to the level of expression of a corresponding genetic region in at least one reference sample, wherein a difference or similarity in the level of expression between the test sample and the at least one reference sample identifies the level of T-regulatory cell activity in the sample. In an embodiment, the pre-selected genetic region is a gene that expresses a protein selected from one or more of Table 7. In an embodiment, the preselected genetic region is TIGHT (T cell fg and ΓΠΜ domain polypeptide).

[0053 { Accordingly, in an embodiment, the present description contemplates monitoring the expression of at least two genes in a test sample comprising immune cells, which may be reflected in changing patterns of RNA levels or protei production that are instructional for the level of T-regulatory cell activity. These methods generally comprise comparing expression at least two genes in the test sample to expression of at least two corresponding genes in at least one reference sample, wherein a difference or similarity in the expression between the test sample and the at least one reference sample identifies the level of T-regulatory cell activity in the sample. In an embodiment, the at least two genes comprise T1GTT and a marker for an immune cell type (such as CD25, CD4, CD45, CD15S, CD.1 12, etc.) or immune cell effector function such IF -y or a molecule produced down-stream of TFN-y expression (e.g. IP 10),

[00541 In an illustrative example, the level or functional activity of an individual expression product is at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%, or no more than π about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0,1%, 0.01%, 0.001 % or 0.0001 % of the level or functional activity of an individual corresponding expression product, which may be referred to as "differential expression". 10055] The present invention contemplates a method of allowing a user to determine the status of a subject with respect to T-regulatory cells, the method including:

(a) receiving data in the form of the methyiation or RNA or proiein expression profiles of a pre-selected genetic region selected from one of the following groups:

(i) at least one or two genetic locus/loci defined hi one or more of Table 1 to Table 5;

(ii) at least one genetic locus defined in one or more of Tabl 6 to Table 10; and

wherein the degree of concordance between the methyiation or expression profiles of the test sample and a reference methyiation profile indicates the level of T- reguiatory cell activity in the sample,

from the user via a communications network;

(b) processing the subject data via multivariate analysis to provide a T-regulatory cell index value;

(c) determining the status of the subject in accordance with the results of the T- regulatory cell index value in comparison with predetermined values; and

(d) transferring a indication of the status of the subject to the user via. the eommunicati ons network.

[0056] The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of th present invention.

[0057| Each embodiment described here is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

[0058| Some Figures contain color representations or entities. Colour photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office,

[0059] Figure I provides graphical representations of CD4 T cell subsets: phenotype, function and DNA methyiation. (A) Naive and rTreg from male donors were sorted (top panel) and activated with soluble anti-CD3 and -28 antibodies (100 and 200 ng/ml, respectively) and autologous irradiated CD4 cell-depleted PBMCs for 4 days, supplemented with 20U/ml IL-2 for additional 2 days. Activated naive (Act-naive) and activated rTreg (Act-rTreg) cells were sorted as proliferating CTV^dtm CD25^'1' ceils (middle panel). FOXP3 expression was examined by intracellular staining (bottom panel). (B) Sorted CTV^d,m CD25^r ('suppressor') cells were sequentially diluted and incubated with Naive CD4^' T ceils (5x10⁴) at 1:1, 1 :2, 1 4, 1 :8 and 1 :16 ratios with anti-CD3 antibody (100 ng/ml), with or without IL-2 (20 U/nil) for 5 days. Dat are representative of results from 3 donors, all showing consistent results). (C) Multi-dimensional scaling (MDS) analysis of the normalized., filtered data. This plot is analogous to a principal components analysis plot. The axes of the MDS plot represent the major sources of variation in the data based on the top 1000 genes with the largest standard deviations between samples; dimension 1 represents the largest source of variation, dimension 2 represents the next largest orthogonal so urce of variation, followed by dimension 3, etc. The M DS plot on the left indicates that the largest source of variation between the samples were baseline methyiation differences between the donors. Data structure in higher dimensions revealed consistent methyiation differences between cell types and activation status. Each sample is labelled with the donor ID and coloured by cell type,

[0060] Figure 2 provides graphical representations illustrating the differential DNA methyiation between Naive and rTreg, (A) Functional annotations of the 450K array probes (left panel), 2,315 differentially methylated probes (DMP) between Naive and rTreg (middle panel) and 127 regions of differential methyiation (RDM) between Naive and rTreg (right panel), (B) Unsupervised clustering of the β values of both the significant DMPs and RDMs resulted in distinct grouping of Naive and rTreg samples.

[00611 Figure 3 provides graphical representations illustrating the activation- induced changes in DNA methyiation, (A) Act-naive and Act-rTreg displayed significant differences in 1,679 CpG probes (green cross), the majority of which overlap with the differences between these two T cell subsets before activation (red circles). The distinct DNA methyiation profiles- of Naive and rTreg were largely unaffected by activation. (B) Activation-induced methyiation changes in 466 CpGs in Naive (blue dots). (C) Activation-induced demethylation at the MIR21 locus. Significant regional methyiation differences (RDMs) are present between Naive (green circles) and rTreg (red crosses). ( D) Expression of miR-21 in Naive and Treg cells before and after activation (data are mean ± SEM of 3 donors and P values are derived from one-tailed Mann- Whitney tests).

[0062] Figure 4 provides graphical representation illustrating Treg-specific hypomethylation at the TIG/ 1 locus. (A) Depiction of the RDM in TIGIT locus. (B) Location of neighbouring DMPs in TIGIT (biFl-5) and FOXP3 genetic loci find clonal bisulfite sequencing results. Open circles, demethy!ated; closed circles, methylated. Ref.seq, reference sequence; Bis.seq, clonal bisulfite sequencing sites. (C) qPCR showing upregiiiation of TIGIT and FOXP3 transcripts in rTreg (data are mean ± SEM of 3 donors). (D) Combination of TIGIT and CD25 can be used to further identify rTreg and aTreg cells. Top panel, original gating strategy for identification of human liTreg (data are representative of 4 healthy individuals). (Data, are mean ± SEM and P values are derived from one-tailed Mann-Whitney tests.)

[0063] Figure 5 is a representatio of data illustrating Treg-speciflc hypomethylation of

TIGIT locus. (A) Expression of FQXP3 and TIGIT in Act-naive, rTreg and Act-rTreg cells. (8) Methylation status of TIGIT RDM (biF2) and FOXP3 TSDR. (C) Enrichment of FGXP3 in byponiethylatedTreg gene loci. AFM intron 1 is used as a control. Data are representative of 3 donors for A, represented as mean ± SE of 3 donors for B and C. Paired t-test was used to calculate the P value between iTreg and Act-rTreg cells.

[0064) Figure 6 is a graphical depiction of the inverse relationshi between gene expression and DNA methylation in genes with promoter-associated RDMs. This plot depicts the relationship between the average regional Δβ between naive and rTreg cells for each promoter-associated RDM and the log2 .ibid change in expression for the associated gene. A negative Δβ indicates hypomethylation in rTreg relative to naive cells and a positive Iog2 fold change indicates increased gene expression in rTreg, when compared to naive cells. The labelled points indicate genes that were significantly differentially expressed between the cell types (adjusted P value < 0.1). The gene expression data used in this analysis (GSE14232) were originally published by Schmidi et al, (2009) Genome Res, 1.9(7): 1 165-1 174. In this study, the raw data were analysed using the R (2.15.2) Btoconductor (2.11) package timma (3.14.4). The data were background corrected and normalized, and control probes and weakly expressed probes were removed. The expression level was then averaged for genes with multiple probes and a linear model was fitted. Thep-vaiues were adjusted to control the false discovery rate using the Benjamini- Hotchber method.

[0065] Figure 7 is a graphical representatio of data showing the forkhead-binding motif in promoter-associated RDMs. (A) The overrepresented motif in the promoter- associated RDMs and comparison with (B) the forkhead-binding motif. (C) qPCR- validated Treg-signature genes which contain hypomethylated promoter-associated RDMs and a putative forkhead-binding motif. Data are represented as mean ±■ SEM of 3 donors.

[0066] Figure 8 is a representation of TIG IT and FQXP3 loci methylation status in 5 donors. Bisulfite cloning sequencing results for TIG IT and FOXP3 loci in 5 male donors. Each closed circle represents a methylated CpG, open circle represent demethylated CpG. Circles in a row are CpGs from individual clones.

[0067] Figure 9 provides graphical representations of (A) how expression of TIGIT identifies activated nTreg cells from individuals with underlying autoimmune type 1 diabetes (pre-T!D). TIGIT and CD25 protein expression were analyzed by surface staining. FOXP3 and !FN-γ production were analyzed by intracellular staining after reactivation with PMA (100 tig/ml), lonomycin (1 μΜ½1) for 1 hr and monensi (2 μΜ) for 4 hrs; and (B) how identification on the basis of TIGIT expression enables loss of FOXP3 stability to be revealed, in individuals with pre-TlD.

[0068] Figure 10 shows a correlation of FOXP3 and 776-/7^" locus demethylation in cord blood CD4 ^;T cells. In total CD4^'T cells, methylation of FOXP3 and TIGIT loci are positively correlated. This demonstrates that demethylation of either locus can be used as surrogate markers of nTreg. Reduction of TIGIT md FOXP3 demethylation in cord blood CD4 T cells of children developed food allergy at 1 year of age. In CD4 T cells from cord blood of children who developed food allergy at 1 year of age, demethylation of 7 /07/ AND FOXP3 loci were significantly reduced. This suggests that a reduced nTreg frequency in cord blood may lead to the development of allergic disease and that raethylation of TIGIT and FOXP3 can be used as diagnostic biomarker. Methods: Total CD4 T cells were flow sorted from cord blood. Genomic DMA was isolated and bisulfite converted. Met ylation analysis of 776 7' and ΤΌΧΡ3 loci were as described in Example 1. 10069] Tables 1 to 10 list differentially hypermethylated and hypomethylated CpG sites derived by comparing the genome-wide methylation profiles of T-reguLatory cells and naive resting CD4+ cells.

[0070] Table 11 provides the ENTREZ identification and full names of genes represented by symbol in Tables 1 to 10.

[0071] Table 12 tabulates bisulfite sequencing primers and SEQ ID NDs for FQXP3 and TIGIT loci which are hypomethyl ted in T-regulatory cells.

[0072| Table 13 lists promoter-associated ROMs and putative FOXP3 binding sites identified herein.

[0073] Table 1.4 lists differentially methylated regions (RDM) that distinguish between naive and rTreg cells.

[0074] Table 1.5 lists DMPs between Naive and Act-Naive. DETAILED DESCRIPTION

[0075] It is described herein that hypomethylatkra (detsaet ylation) at the TIGIT and/or MIR21 loc characterize human Treg, TIGIT protein expression also facilitates the ex vivo differentiation of activated Treg cells from effector T cells. As shown herein, FOXP3 enrichment at the TIGIT locus in activated Treg cells, along with other Treg signature genes, provides evidence that FOXP3 directly binds to byporaethylated loci found in some Treg-signature genes and regulates their expression. In other loci, Treg cells display hypemieth lated CpG. It is proposed, without limitation, that the methyiation status of the Treg signature gene loci affects their expression by modulating FOXP3 accessibility and thereby influences Treg potency.

[0076] In an illustrative embodiment, epigenetic profiles associated with pre-selected genomic loci within the genome of T-regulatory cells and naive CD have been established. The profiles include genetic loci that are substantially differentially hypermethylated in T-regulatory cells compared to non-T-regulatory T cells, as well as genetic loci that are differentially hypomethylated. Over two thousand CpG sites are identified herein as substantially differentially methylated in T-regulatory cells and naive CD4^' T cells. Cytosine methyiation is almost exclusively found in CpG dmucleotides. These CpGs are spread throughout the genome, but can be found clustered in so called CpG islands within the promoters of some genes. These data have permitted the identification of differentially methylated regions comprising one or more CpGs for use in the present assays. As a wide range of techniques are now available for high throughput methyiation profiling of the human genome, multiple loci as determined herein can be rapidly screened or re-screened to provide informative data relative to T-regulatory cells in a sample or subject. The pre-selected loci are designed to encompass one or more than one individual CpG within a specific region defined herein inter alia by Map Info numbers and start and stop sites, as well as flanking regions comprising one or more CpG. In an embodiment the observed methyiation profiles are substantially independent of the activation status of T-regulatory cells. The spectrum of stable methyiation markers facilitates the identification of cellular sub-types in a sample comprising a mixture of cells including immune cells. The instant methods also permit the stratification of T-regulatory cells based upon the methyia tion profile of a pre-sel ected locus or loci. [0077] As T-regulatory cells are critical in regulating immune responses, the present me hod has a broad range of diagnostic and prognostic applications in. the context of this cell type. Early or pre-symptomatic detection of a increased risk for immune system dysregulatioii as assessed by T-regulatory cell activity provides a useful tool to facilitate clinical management and improve the quality of decisions concerning treatment. Hence, the present description identities a correlation between an observed methylation profile of one or more pre-selected genetic loci derived from a cellular test sample and the level of T- regulatory cell activity in a sample or subject.

|0078] in an embodiment, the pre-selected genetic loci are selected from the list consisting of;

(i) a genetic locus/loci defined in one or more of Table 1 to Table 5 representing loci hyper ethyiated in T-regulatory cells;

(ii) a genetic locusloci defined in one or more of Table 6 to Table 10 representing loci hypomethylated in T-reguiatory cells; and

(iii) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10 representing hypennethyiated and hypomethylated genetic loci in T-reguiatory cells,

[0079] in an embodiment, a locus or loci that is/are hypennethyiated in T-regulatory cells are selected for analysis. Alternatively, a combination of loci is selected including hypomethylated and hypennethyiated loci to facilitate identification of T-regulator cell activity- In an illustrative example, hypermethyiation in a locus in. Table 3 or Table 2 or loci in Table 1 and Table 2 are used to establish a methylation profile that correlates with the level of T-regulatory cell activity.

(0080 j In an embodiment, particularly when multiple loci are employed, hypennethyiated. CpG are preferentially selected for the assay. The detection of methylation in a CpG island, for example, which is typically hypomethylated, makes the methylated CpG a particularly useful marker. In addition, methods of methylation detection are generally more sensitive to small amounts of methylation (e.g. approximately 1% rather than 0%) than to a small decrease in methylation (e.g. 100% to 99%). Hypomethylated regions are also more sensitive to increased methylation while methylated regions are fairly insensitive to small decreases in methylation, affording more biological significance to the detection of methylated CpG.

[0081 j In an embodiment, hypermethyiation and/or hypomethylation at a range of loci from any one or more of Tables 1 to 10 is used to generate a. methylation profile useful for stratifying T-regulatory cell activity into sub-groups.

[0082] Reference to "CpG" includes a CpNpG where N is a non-guanine nucleobase and p is a linking phosphate.

[0083] Reference to "genetic loci" or "genetic region" includes "pre-selected genetic loci" which are individual herein defined CpGs in the genome as well as a genomic region (locus) comprising a defined hyper- or hypomethylated CpG and flanking regions encompassing approximately one thousand nucieobases. "Flanking regions" encompass 50 to 100 or 10 to 600, or 20 to 300 bases on either side of a CpG dimicleotide.

|0084j Reference to "hypetmethylated" refers to a defined CpG that is methylated in T- regulatory cells and which is unmethylated in iron T-regulatory T cells. "Hypetmethylated. loci" also refers to a pre-selected genetic locus that is a genomic locus of approximately 1000 bases flanking a defined CpG, comprising further CpGs which are substantially more methylated compared to the corresponding region in non T-regulatory T ceils. "Defined" CpG are pre-selected genetic loci which are identified as known to those of skill in the art according inter alia to their genome map reference, a probe or primer or enzyme or other binding agent which selectively identifies the CpG or by an adjacent genetic region, or by their location in a domain of a gene or motif.

[0085] Reference to "hypomethylated" refers to a defined CpG which is unmethylated in T-regolatory cells and which is methylated in non T-regulatory T cells. "Hypomethylated loci" also refers to genetic loci of approximately 1000 bases flanking a defined CpG comprising CpG that are substantially less methylated compared to the corresponding region in a non T-regulatory T cells.

[0086] "Substantially more methylated" or "substantially less methylated" refers to a methy!ation difference in genetic regions comprising more than one CpG, which is preferably at least 30-50% or at least x2 to at least l.O (including 3x, 4x, 5x, 6x, 7x, 8x and 9x) more methylated or less methylated.

[0087] Non-limiting examples of non T-regulatory T cells include FOXP3^" T cells and naive CD4^÷ cells.

[0088| Tables 1 to 5 list hypermetliylated CpG residues that are methylated in T- regu!atory cells and which are unmethylated in naive CD4 T cells, Table 1 lists hypermetliylated CpG wherein the difference in β value (Αβ i.e., the difference between the proportion of methylated to unmethylated CpG deteeted between two cell types is at least or more than 0.6. Table 2 lists hypermethylated CpG wherein the difference between the proportion of methylated to unmethylated CpG detected between two cell types is between 0.5 and 0.6. Table 3 lists hypemiethylated CpG wherein the difference between the proportion of methylated to unmethylated CpG detected between two cell types is between 0,4 and 0.5. Table 4 lists hypennetliylated CpG wherein the difference between the proportion of methylated to unmethylated CpG detected between two cell types is between 0.3 and 0,4. Table 5 lists hypemiethylated CpG wherein the difference between the proportion of methylated to unmethyl ated CpG detected between two cell types is between 0.2 and 0.3. The greater the Δβ value the less likelihood of overlap between the two cell types. Accordingly, in an embodiment genetic loci are selected preferentially from Tables 1 and 2.

[0089] Tables 6 to 1 list Iiypomethylated CpG residues that are immethylated in T- regulatory cells and methylated in naive CD4 T cells. Table 6 lists hypomethylaied CpG wherein the difference in β value Δβ i.e., the difference between the proportion of methylated to unmethylated CpG detected between the two ceil types is at least 0.6 or more than 0.6. Table 7 lists Iiypomethylated CpG wherein the difference between proportio of methylated to unmethylated CpG detected between the two cell types is between 0.5 and 0.6. Table 8 lists bypomethylated CpG wherein the difference between proportion of methylated to unmethylated CpG detected between the two cell types is between 0.4 and 0.5. Table 9 lists bypomethylated CpG wherein the difference between proportion of methylated to unmethylated CpG detected between the two cell types is between 0.3 and 0.4. Table 10 lists hypomethylated CpG wherein th difference between proportion of methylated to unmethylated CpG detected between the two cell types is between 0.2 and 0.3. Genetic loci are selected preferentially from Tables 6 and 7.

[0090] The present invention is focussed upon the identification and stratification of T- regulatory cells. The skilled addressee will appreciate that the methods described herein are applicable to the identification and stratification of naive or active CD4^"' T cell whose profil es are also provided herein.

[0091] in an embodiment, the methods include screening for FOXP3 hypomethylation. In other embodiments, screens for hypomethylation or specifically hypomethylation. of FOXP3 loci is/are not included. As described herein, in an embodiment, FOXP3 hypomethylation is detected at MAP INFO 491 18313 or within the flanking region defined as START 491 17813 END 49118813 (see Table 6, No. 8). Hypomethylation of regions of the FQXP3 is described in the literature. See for example. Baron f l. Eur, J. Immunol. (2007) 37:2378-2389. However, detection of hyper methylated (methylated) CpGs, particularly for high density screening of multiple CpGs is preferred over hypomethylated CpG (non-meth lated) as they will tend to be of greater biological significance.

[0092J Reference to a "methylation profile" includes the methylation status at a single CpG locus, at a single CpG within a defined 1000 base pair locus, or multiple individual defined CpG or defined loci. As known in the art, several methods are available for analysing multiple samples at a large number of pre-s elected loci, based for example, upon microrrays, quantitative PCR methods, mass spectrometry and DNA sequencing protocols.

[0093 j Any methylation assay may be employed such as methylation sensitive PCR, methylation specific melting curve analysis (MS-MCA) or high resolution melting (MS- HRM) [Da &t al (2007) Supra; Wojdacz et al (2007) Nucleic Acids Res. 35(6):Q41]\ quantification of CpG methylation by MALDI-TOF MS (Tost et al (2003) Nucleic Acids Res J/(9j:e5G); methylation specific MLPA (Nygfen et al. (2005) Nucleic Acids Res. 33(J4}:el2$); methylated-DNA precipitation and methylation-sensitive restriction enzymes (COM ARE- S) (Yegnasubramanian et al (2006) Nucleic Acids Res, 34(3) : l9) or methylation sensitive oligonucleotide microarray (Gitan et al. (2002) Genome Res. 12(.1):158*-164), as well as via antibodies. Bisulfite patch PCR is expressly contemplated, as described in Varley et al. Genome Research (2010) 20:1279-1287. Other useful methods employ target enrichment as described by Ivanov et al in Nucleic Acids Research (2013) 1-9, published 15 January 2013 or target amplification (Mtacl) described by Nautiyal et al. PNAS 2010 107(28) 12587-12592, Other assays include NEXT generation (GEN) and DEEP sequencing or pyrosequencing.

10094 j Insofar as the methylation assay may involve amplification, an amplification methodology may be employed. Amplification methodologies contemplated herein include the polymerase chain reaction (PCR) such as disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195 including real-time PCR; the ligase chain reaction (LCR) such as disclosed in European Patent Application No. EP-A-320 308 and gap filling LCR (GLCR) or variations thereof such as disclosed in international Patent Publication No. WO 90/01069, European Patent Application EP-A-439 182, British Patent No. GB 2,225,1 12A and international Patent Publication No. WO 93/00447. Other amplification techniques include $ replicase such as described in the literature; Stand Displacement Amplification (SDA) such as described in European Patent Application Nos. EP-A-497 272 and EP-A- 500 224; Self-Sustained Sequence Replication (3SR) such as described in Fahy et al ( 1 91 ) PCR Methods Appl. /(/ ;25-33) and Nucleic Acid Sequence-Based Amplification (NASBA) such as described in the literature. [0095] Real-time quantitative PCR may conveniently be employed using fluorescently labelled probes. In an embodiment of this type of assay, a bisulfite treated target. DNA may be amplified with a single primer irrespective of its methyiation status and then probed with differentially labelled probes (for example, Taqman probes) that distinguish between methylated targets and unmethylated targets. To enhance specificity, each probe may cover more than one defined CpG. Bound probes are cleaved through the 5'nuciease activity of Taq DNA polymerase, releasing the fluorescent label for quantification. The amount of fluorescence (such as VIC or FAM) released during PCR will be directly proportional to the amount of PCR product generated from the methylated or unmethylated al lele. To calculate the ratio of methylated to unmethylated target sequence in the sample, the difference between the cycle-threshold values of both probes is determined. Cell numbers can be determined by means of a standard curve. (Tatura R el l. Plos ONE 7(1 1 ): 649962).

10096] A technology which can alternatively be employed for methyiation analysis utilizes base-specific cleavage followed by MALDi-TOF mass spectrometry on DNA after bisulfite treatment, where all the 5-methylCpG residues are converted to uracils or where all unmethylated CpG residues are not converted to thymine. Primers are designed based on particular regions around pre-selected CpG Primer sequence are designed to amplify' without bias both converted and unconverted sequences using the PCR amplification process under medium to high stringency conditions. The PGR products are in vitro transcribed and subjected to base specific cleavage and fragmentation analysis using ALDI-TOF MS. The size ratio of the cleaved products provides quantitative methyiation estimates for CpG sites within a target region. The shift in mass for non- methylated (NM) from methylated (M) fragments for a single CpG site is - 16 daltoiis due to the presence of an adenosine residue in the place of a guanosine. Software is then used to calculate methyiation for each fragment based on this difference in mass, where the output methyiation ratios are the intensities of methylated signal/[methylated+itnmethylated signal]. If the fragment size overlaps for different CpG, their methyiation output ratio is calculated based on the sum of intensities for methylated/ [methylated + unmethylated signal]. To distinguish how well the methyiation output ratio for multiple fragments of a similar size represented methyiation of separate CpG sites, for some amplicons both CpG and thymidine cleave reactions can be performed (that produced fragments o different size) prior to fragment analysis. Silent peaks (S) - fragments of unknown origin, should not be taken into consideration if their size does not overlap with the fragments of interest. Methylatio of CpG sites that have silent peaks (S) that overlap wi th the fragments of interest should be included in the analysis.

[0097! to ^m embodiment, a method is provided for determining the methylatio profile of one or more preselected genomic loci selected from Tables 1 to .10, the method comprising obtaining a sample of genomic DNA from a sample comprising immune or cells from a subject and subjecting the genomic DNA to primer-specific amplification and assaying for the extent of methylation in pre-selected genetic loci comprising CpG, including pre-selected CpG as defined herein, relative to a control including a change in the extent of methylation in a region, and associating a degree of concordance with the level of T-regulatory cell activity.

[0098] In another embodiment, the specification enables a method fo determining the proportion of T-regulatory cells in a test biological sample comprising immune cells . In an embodiment, the method comprises screening for the methylation profiles of a pre-selected genetic region selected from the list consisting of:

(i) at least one or two genetic locus/loci, defined in one or more of Table 1 to Table 5 ; or

(i i) at least one genetic locus defined in one or more of Table 6 to Table 1.0; or

wherein tire degree of concordance between the methylation profiles of the test sample and the reference methylation profile indicates the level of T-regulatory cell activity in the sample.

[0099] In an embodiment, group (i) comprises at least two genetic loci defined in one or more of Table 1 and Table 2. In an embodiment, group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

1001001 As described in the Examples, the defined loci in Table 1 are: I - MAPINFO 45406867(START 45406367-END45407367), and 2 - MAPINFO 1 13655841 (START 113655341 -END 1 13656341 ).

[001011 As described in the Examples, the defined loci in Table 2 are: 1 - MAPINFO 224564870 (Start 224564370-End 224565370),. 2 - MAPINFO 70387268 (Start 70386768- End 70387768), 3 - MAPINFO 108052093 (Start 108051593-End 108052593), 4 - MAPINFO 110731201 (Start 1 10730701 -End 1 10731701), 5 - MAPINFO 1823275 (Start 61822775-End 61823775), 6 - MAPINFO 1.0556294 (Start 10555794-End 10556794), 7 - MAPINFO 59064969 (Start 59064469-End 59065469), 8 - MAPINFO 60065489 (Start 60064989-End 60065989), 9 - MAPINFO 61835848 (Start 61835348-End 61836348), 10 - MAPINFO 29924694 (Start. 29924194-Ettd 299251 4), 11 - MAPINFO 78755379 (Start 78754879-End 78755879), 12 - MAPINFO 1 0978385 (Start 150977885-End 150978885), 13 - MAPINFO 69660603 (Start 69660103-End 69661 103), .14 - MAPINFO 101704898 (Start 101704398-End 101705398), aid 15 - MAPINFO 109387521 (Start 109387021 -End 109388021).

[001021 As described in the Examples, the defined loci in Table 6 are: I - MAPIN FO 1 14052269(START 1 14051769- END 1 14052769), 2 - MAPINFO 298485?9(START 29848079- END 29849079), 3 - MAPINFO 4911 8313( START 49117813- END 491 18813), 4 - MAPINFO 12230509(START 12230009- END 12231009), 5 - MAPINFO 33384179(START 33383679- END 33384679), 6 - MAPINFO 5G550589(START 50550089- END 50551089), and 7 - MAPINFO U 4012806(START 1 14012306- END 1 14013306).

[00103] As described in the Examples, the defined loci in Table 7 are: 1 - MAPINFO 34236648(START 34236148-END34237148), 2 - MAPINFO 8I 74148**(START S 173648-ENDS174648), 3 - M APINFO I 421273(STAR 16420773 -END 16421 773), 4 - MAPINFO 56432144(ST ART 56431644-END56432644), 5 - MAPINFO 121972412(START 121971912-ENDf 21972912), 6 - MAPINFO 238973024(START 238972524-END238973524), 7 - MAPINFO 61041.37(STAR.T 6103637-END6104637), 8 - MAPINFO 1 1401.2912(START 1 14012412-END 1 14013412), 9 - MAPINFO 12I 39206(START 12138706-END12139706), 10 - MAPINFO 201245077(START 201244577-END201245577), 1 1 - MAPINFO 38918253(STAR 38917753- END3891 8753), 12 - MAPINFO 8173451(START 8172951 -ENDS 173951 ). 13 - MAPINFO 37545423(START 37544923-END37545923), .14 - MAPINFO 1 138210(START 133137710-END1.331.38710), 1.5 - MAPINFO 38918135(START 3891.7635-E D3891 8635), 16 - MAPINFO 114027859(START 1 14027359- END1 14028359), 17 - MAPINFO 33593457(START 33592957-END33593957), 18 - APINFO 1 413624( START 164.13124-END16414124), 19 - MAPINFO 123173709(START 123173209-END 123174209), 20 - MAPINFO 37257404(START 37256904-END37257904), 2.1 - MAPINFO 149806635(START .149806135- EN 149807135), 22 - MAPINFO 19535154(START 19534654-ENDI9535654), 23 - MAPINFO 2690221 { { START 26901 71 1 -END2690271 1 ), 24 - MAPINFO 625089(START 624589-END625589), 25 - MAPINFO 149806502(START 149806002- END149807002), 26 - MAPINFO 204734181 (START 20473:3681 -END204734681 ), 27 - MAP INFO 2i457502(START 2.1457002-END21458002), 28 - MAPINFO 7634Q765(START 76340265-END76341265), 29 - MAPINFO 37394589(START 37394089-END37395089), 30 - MAPINFO 10512848S(START 105127988- END10S 128988), 31 - MAPINFO 12680897(START 1268G397-ENDI2681397), 32 - MAPINFO 33384337(START 33383837-END33384837), 33 - MAPINFO 1 1.4012659(START 114012.159-ENDl 14013.159), 34 - MAPINFO 44623459( START 44622959-END44623959), 35 - MAPINFO 39369720(STAR 39369220-END3937O220), 36 - MAPINFO 71200489(START 71 199989-END71200989), 37 - MAPINFO 521.50456(START 52149956-END52150956), 38 - MAPINFO 58S611 1 (START 588561 J -END58866 1), 39 - MAPINFO 20.9809(START 209309-END210309), 40 - MAPINFO 85827505(START 85827005-END85828005), 41 - MAPINFO 14858290(START 14S57790-END1485S79O),

42 - MAPINFO 1.23687270(STAR.T 123686770-ENDl 23687770), 43 - MAPINFO 245524769(START 245524269-END245525269), 44 - MAPINFO 55540725(START 55540225-ENDS5541225), 45 - MAPINFO 15.164788($TART 1 164288-END1.5165288), 46 - MAPINFO 70574295(START 70573795-END70574795), 47 - MAPINFO 115441S40(ST ART 1 15441 40-ENDi 15442340), 48 - MAPINFO 95358764(STAR.T 95358264-END95359264), 49 - MAPINFO 151 1.34876(START 1 1 134376- F.ND151 135376), 50 - MAPINFO 12188502(START 12188002-END12189002), 51 - MAPINFO 1S93.1082(START 15930582-END 15931582), 52 - MAPINFO 46I 89113(START 46188613-END46189613), and 53 - MAPINFO 801 1594(START 80 UO94-END8012094).

[00104] In an embodiment, methylation analysis includes an analysis of each occurrence of a defined CpG in a test sample. Accordingly, the proportion of methylated CpG, or unmethylate CpG, out. of the total number of methylated and unmethylated CpG provides the .number and proportion of the T cell, such as T-regulatory cell.

[00105] In one embodiment, the data described herein allows the skilled person to identify which CpG in the genome of a cell are methylated in T-regulatory cells and therefore allows the skilled person to the level of T-regulatory cell activity or that of non T-regulatory T cells in a sample, f om the methylation profile.

[00106] T-regulatory cells may be stratified using a range of functional and/or structural criteria. An illustrative functional criterion is the suppressor activity of T-regulatory cells displaying a pre-selected methylation profile. Suppressed activity may be assessed using methods such as cellular proliferation in autologous suppression assays. Other functional activities include metabolic or binding activity or secretary activity such as the secretion of cytokines or other immunomodulatory agents.

[00107] Reference to a "subject" includes a human subject, which may also be considered an individual, patient, host, recipient or target. Human subjects maybe asymptomatic or symptomatic relative to a condition associated with dysregulated T- regulatory cells. Subjects may nevertheless have been identified as at risk for such a condition.

[00ΪΘ8] Sample includes a sample of a single cell or multiple cells including a mixture of cells comprising immune cells. The sample may be processed or stored prior to use in the subject methods,

[00102] The ability of a subject to moun t an appropriate immune response depends in part upon the level of T-regulatory cell activity, in another aspect, the present specification provides methods for determining whether or not a human subject has developed or has the ability to suppress an autoimmune response based upon the level of T-regulatory cell activity. In another aspect, the present specification provides methods for determining whether or not a human subject has or is at risk of developing an autoimmune condition associated with low levels of T-regulatory cells.

[0103] in an embodiment, the methods comprise screening a test sample comprising immune cells from a subject for the rnethylatior! profiles of a pre-selected genetic region selected from the list consisting of:

(i) at least two genetic locus/loci defined in one or more of Table 1 to Table 5;

(i t) at least one geneti c locus defined in one or more of Table 6 to Table ί 0; and

(iii) at least two genetic loci defmed in one or more of Table 1 to Table 5 and one or more of Table 6 to Table 10; and determining the degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample.

[0104] in an embodiment, group (i) comprises at least one or two genetic locus loci defined in one or more of Table 1 and Table 2. In. an embodiment, group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

[0105] In an embodiment, the present specification provides a method of treatment or prophylaxis of a subject, the method comprising screening a test biological sample comprising immune cells for the methylation profiles of a pre-selected genetic region selected from the list consisting of:

(i) at least one or two genetic locus/loci defmed in one or more of Table 1 to Table 5; (ii) at least one genetic locus defined in one or more of Table 6 to Table 10; and

(iii) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; detemiinirtg the degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample, and then providing therapeutic and/or behavioural .modification of the subject.

[0106] in an embodiment, group (i ) comprises at least one or two genetic loci defined in one or more of Table 1 and Table 2. In an embodiment, group (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

[0107] In an embodiment, group (i.) comprises at least two genetic loci defined in one or more of Table 1 and Table 2. In an embodiment, group (ii) comprises at. least, two genetic loci defined in one or more of Tables 6 and Table 7.

[0108] The phrase "reference methylation profile" includes all or part of the methylation status data for pre-selected loci for T-regulatory cells or non-T-regulatory T cells. The phrase also includes methylation profile data for the pre-selected loci for T- regulatory cells or non-T-regulatory cells taken from the subject at an earlier time point. Reference methyation profiles may be derived from a control subject or group of control subjects.

[0109] The phrase "level of T-regulatory cell activity" includes cell numbers and a change or difference in cell numbers compared to a control or reference methylation profile. If the reference profile is from the subject at an earlier time point, the "level" could be expressed as a proportion, ratio, percentage degree of "fit" or divergence between test and reference profiles, etc. or exposed on a number on a scale of 1 to (say) 10 indication of a minimum activity (1) and a maximum activity (10) for a given population. The term includes the number, proportion and/or activity of T-regulatory cells,

[0110] In an embodiment, a control subject is a group of control subjects. The reference methylation profile in a control subject group may be a mean value or a preselected level, threshold or range of levels that define, characterise or distinguish a particular group. Thresholds may be selected that provide an acceptable ability to make conclusions concerning the number/level/strata of T-regulatory cells and predict diagnostic or prognostic risk, treatment success, etc. In illustrative examples, receiver operating characteristic (ROC) curves are calculated by plotting the value of one or more variables versus its relative frequency in two cell types (for example, functionally inimunosupressive T-regulatory cells and functionally non-immuno suppressive T-regulatory celts, or two populations (called arbitrarily "disease" and "normal" or "low risk" and "high risk" groups for example). For any particular methylation profile(s), CpG or CpG islands, a distribution of methylation levels (such as β values or p values) for subjects in the two populations wall likely overlap. Under such conditions, a test value does not absolutely distinguish between cell type or "disease" and "norma!" or "at risk" and "not at risk" with 100% accuracy, and the area of overlap indicates where the test cannot distinguish between groups. Accordingly, in an embodiment, a threshold or range is selected, above whic or below which, depending on the raethylation status the test is considered to be "positive" and below which the test is considered to be "negative" . Linear regression analysis is used to identify methylation profiles that are independent predictors of group assignment. Multivariate analysis is particularly suitable for developing a predictive model based on methylation profiles in populations. In anothe embodiment, the reference sample provides the level of TP-regulatory cell activity of a subject undergoing testing, at a earlier time point.

[0111] In an embodiment, the methylation profile or status of a single CpG in the genome is sufficient to determine the level or activity of T-regulatory cells or no T- regulatory T cells in a sample. The methylation status is determined and compared with a reference methylation profile of pre-selected regions as disclosed herein in order to draw conclusions concerning the level of T-regulatoiy cell activity, in other embodiments, the methods comprise determining or determining and comparing the methylation status of at least: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or more pre-selected regions including 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 1.04, 105, 1.06, 107, 108, 109, 110, 11 1 , 1 12, 1 13, 1 14, 115, 1 16, 1 17, 1 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 1.73, 174, 175, 176, 177, 1.78, 179, .180, 18.1 , 182, 183, 184, .185, 186, 1.87, 188, 189, 190, 191 , 192, .193, 194, 1.95, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 2 1, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275,. 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323, 324, 325, 326, 327, 328, 329 or 330 or more, pre-sef ected regions.

[0112] Primers and probes suitable for determinin the methylation status of CpGs in the genome are provided by applying the information provided in the instant specification using routine techniques.

[0113] in an illustrative embodiment, the reference sample methyfatio profile is derived from T-regulatory cells having the surfac markers CD45RA+, CD45 O-, CD25+. In aft embodiment, T-regulatory cells are thymus derived T-regtUatory cells (nTreg). In an embodiment, T-regulatory cells are induced iT-reguJatory cells (iTreg).

[0114] In another embodiment, the reference sample methylation profile is derived from resting naive CD4 T cells having the surface markers CD45 A+, CD45RO-, CD25- or activated forms thereof.

[0115] In an embodiment, the test sample methylation profile is derived using a methylation specific detection system such as using probes, primers or baits that indicate when a CpG is methylated and the reference sample primer or bait methylation profile is derived using an uimieth.ylati.on specific system such as using an oligonucleotide probe primer or bait that indicates when a CpG is unmethylated. In an embodiment, the test sample methylation profile is derived using an unmethylation specific detection system and the reference sample profile is derived using a methylatio specific system.

[0116] In an embodiment, the methods comprise amplifying TIGIT loci (see, fo example. Tables 10 and 12) and determining the methylation status of one or more preselected loci.

[0117] In an embodiment, the methods comprise isolating genomic DNA from a human biological sample comprising immune cells or preparing a test sample enriched for CD4+ T cells from a human biological sample comprising immune cells and then isolating genomic DNA therefrom. CD4 enrichment may conveniently be practised using positive or negative enrichment protocols known in the art.

[0118] In an embodiment, the methods comprise correlating the proportion of methylation at. each CpG nucleotide in each pre-seleeted genetic loci in the sample to the level and/or activity of T-regulatory cells in the sample. In an embodiment, the number of T-regulator cells i the same as the observed number of methylated CpG detected in the sample at any unique pre-selected CpG. The β value provides a direct indication of T- regxilatory cell number. Knowledge of the total number of cells in the sample, or using markers that together identify substantially ali cells in the sample, permits the number and proportion of T-regulatory cells and therefore .non T -regulatory T cells in the sample to be estimated.

[0119] In an embodiment, genome copy numbers may be calculated from calibration curves by linear regression. In some kit based embodiments, cell numbers in a biological sample may be independently estimated from pre-determined markers capable of providing a signal indicative of total cell numbers.

[0120] In another aspect, the specification enables a method for identifying the number, proportion and or activity of T-regulatory cells in a test biological sample comprising immune cells, in an embodiment, the method comprises

(a) screening a test sample for the methylation profiles of a pre-selected genetic locus/loci selected from one of the following groups:

(i) at least one genetic locus defined in one or more of Table .1 to Table 5;

(b) determining the similarities or differences between the test methylation profiles and a reference methylation profile, wherein the similarities or differences identifies the number, proportion and/or activity of T-regulatory cells in the sample.

[0121] In another aspect, the present invention provides a use of a panel or array of oligonucleotides specific to a pre-selected genetic region selected from one of the following:

(i) at least one or two genetic locus/loci defined in one or more of Table 1 to Table 5; fit) at least two genetic loci defined in one or more of Table 6 to Table 10; and

in the manufacture of a kit or solid support for identifying the number, proportion or activity of T-regulatory cells in a biological sample, tissue or subject, or the presence, risk, state, classification or progression of immune system dysregulation such as found in an autoimmune condition in a subject

[0.122] The subject assays, arrays or kits comprising or facilitating same may also be used to monitor the progression of treatment programs with reference to the number and/or activity/stratification of T-regulatory ceils,

[0123] Kits are contemplated in a range of different forms. They typically comprise reagents for determining the methylation status of pre-selected C G, such as for example, reagents for restriction digestion of DNA, buffers and wash solutions, sodium bisulfite, exonucleases, oligonucleotide probes or primers, universal oligonucleotide probe/primer pairs, sample specific bar codes or rags for sample identification, reagents for target enrichment and or capture, PCR amplification, visual detection systems for test samples and. control markers. Kits may also instructions for use. They may also comprise data collection components or communication devices or the means for transferring data to a communication device. Oligonucleotides probe and primers are designed to hybridise to or are substantially complementary to oligonucleotides that hybridise to the sequences adjacent to or flanking the pre-selected genetic loci including specific differentially methylated CpG identified tlierewithin.

[0124] The subject assays may also be used alone or in conjunction with assays to detect other epi enetic changes characteristic of T-regulatory cell levels or acti vity/strata. Suitable epigenetic changes include histone modification, and changes in acetytation, ubiquitylation, phosphorylation and sumoylation.

[0125] in an other aspect, the present invention provides a method of screening for an agent which modulates immune cell function, said method comprising screening an immune cell for a difference or similarity compared to a reference sample in the methylation profile of a pre-selected genetic region selected fro one of the following groups;

(i) at least two genetic loci defined in one or more of Table 1 to Table 5 ;

(ii) at least two genetic loci that are defined in one or more of Table 6 to Table 10; and (Hi) at least two genetic loci that are defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10;

[0126] The subject methods may also be used in a personalized or a population medicine approach in the management of pathology platforms and or of immunosuppression or autoimmune conditions such as, without limitation, systemic lupus erythematosus (SIT), multiple sclerosis (MS), arthritis including juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC), coeliac diseases, type 1 diabetes (TID) and pre- lD. Other conditions such as graft versus host disease in which T-regulatory cells suppress inappropriate allogeneic T cells are also encompassed.

[0127] The present disclosure provides a computer program and hardware for monitoring methyiation profiles in a subject over time or in response to treatment or other affectors. Values are assigned to methyiation profile features or T-regulator ceil numbers/strata that are stored in a machine readable storage medium. A computer program product is one able to concert such values to code and store the code i a computer readable medium and optionally capable of assessing relationship between the stored data and incoming data and optionally a knowledge database to assess a potential relationship with a pathological condition such as immunosuppression or autoimmun related diseases, graft versus host type diseases,

[0128) The present specification therefore provides a web-based system where data on methyiation profiles are provided by a client server to a central processor which analyses and compares to a control and optionally considers other information such as patient age, sex. weight and other medical conditions and then provides a report, such as, for example, a risk factor for disease severity or progression or status or an index of probability of outcomes such as disease, immunosuppression, vaccination success in symptomatic or asymptomatic individuals.

[0129] The assay may, therefore, be in the form of a kit or computer-based system that comprises the reagents necessary to detect the methyiation status of pre-seleeted regions and the computer hardware and/or software including an algorithm to facilitate determination and transmission of reports to a clinician.

[Θ13Θ] The present invention contemplates a method of allowing a user to determine the status of a subject with respect to T-regulatory cells, the method including:

(a) receiving data in the form of the methyiation profile of a pre-selected genetic region selected from one group from the following:

(ii) at least two genetic loci that are defined in one or more of Table 6 to Table 10; nd

(in) at least two genetic loci that are defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; wherem the similarities or differences between the raefhylation profiles of the test sample and a reference methylation profile indicates the number, proportion and/or activity of T- regulatory cells in the sample,

from the user via a communications network;

(b) processing the subject data via multivariate analysis to provide a T-reguiatory cell index value;

(c) determining the status of the subject in accordance with the results of the T- regulatory ceil index value in comparison with predetennined values; and

(d) transferring an indication of the status of the subject to the user via tire communicati Ons netw ork.

[0131] Conveniently, the method generally further includes:

(a) having the user determine the data using a remote end station; and

(b) transferring the data from the end station to the base station via the communications network.

[0132] As used herein, the term "binds specifically," "specifically mrmuno-interactive" and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen -binding molecules bind to a particular antigen and do not bind in a significant amount to other proteins or antigens present in the sample. Specific binding to an antigen under such conditions may require an antigen-binding molecule that is selected for its specificity for a particular antigen. For example, antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample. A variety of immunoassay formats may be used to select antigen-binding molecules specifically immuno-interactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno-interactive with a protein. See Harlow and Lane (1 88) Antibodies, A Laborator Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreacti v ity .

[01.33] By "corresponds to" or "corresponding to" is meant a polyn cleotide (a) having a nucleotide sequence that is substantiall identical or complementary to all or a portion of a reference polynucleotide sequence or (b) encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein. This phrase also includes within its scope a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.

[0134] The terms "expression" or "gene expression" refer to either production of RNA message or translation, of K.NA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention.

[0135] The term "gene" as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions. The gene is also intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In. this regard, the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The D^'NA sequences may be cDNA or genomic DNA or a fragment thereof.

[0136] The term "oligonucleotide" as used herein refers to a polymer composed of multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof including nucleotides with modified or substituted sugar groups and the like) linked via phosphodi ester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring, it will be understood that, the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids tPNAs), phosphorothioate, phosphorodithioate, phophoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoramiadate. phosphoroamidate, methyl phosphonates, 2-0- methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. Oligonucleotides are a polynucleotide subset with. 200 bases or fewer in length. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 12, 33. 14, 15, 16. 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g. , for use in the construction of a variant nucleic acid sequence. Oligonucleotides of present description can be either sense or antisense. Oligonucleotides are selected based upon the naturally occurring genetic sequence, but may be modified to enhance binding, selectivity and for use in various assay. Various modifications of naturally occurring sequences tor use as probes or primers etc are known generally in the art and are made and tested using routine methods known in the art.

[0137| By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of file primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the primer may be at least about 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, 17, 18, 1 , 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end of the primer to allow extension of a nucleic acid chain, though the 5' end of the primer may extend in length beyond the 3' end of the template sequence. In certain embodiments, primers can be large polynucleotides, such as from about 35 nucleotides to several kilobases or more. Primers can be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridize wit a target polynucleotide. Desirably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non- complementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non-complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarit with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.

[0138] "Probe" refers to a molecule that binds to a specific sequence or sub-sequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope.

[0139] Variants of the pre-selected genetic region expressio products are contemplated.. Nucleic acid variants can be naturally-occurring, such as allelic variants (same locus), homologues (different locus), and orthologues (different organism) or can be non naturally-occurring. Naturally occurring variants such as tliese can be identitied with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as known in the art, Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions., deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product). For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a pre-selected genetic/gene product, such as TIG IT. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a pre-selected genetic region product polypeptide of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 70%, 75%, 80%, 85%, desirably about 90%, 91%, 92%, 93%, 94% to 95% or more, and more suitabl about 96%. 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

[0140] Variants of protein expression products such as TIG1T are enabled. "Variant" polypeptides include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-temiinal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed b the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biological ly active variants of a native polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a proteinaceous expression product from a pre-selected gene may differ from that protein generally by as much 1000, 500, 400, 300, 200, 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue,

[0141] Consistent with the present description, a difference in ie level of RNA expression from a pre-selected genetic region/gene, such as TIGI is indicative of the level of T-regiilatory cell activity. Nucleic acid used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et at, 1989. supra; and Ausubel et at, 1994, supra). The nucleic acid is typically fractionated {e.g., poly A RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template -dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the pre-selected genetic region present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR), described in detail in U.S. Pat Nos. 4,683,195, 4,683,202 and 4,800,1 59, Ausubel et at (supra), and in Innis et at, ("PGR Protocols", Academic Press, Inc., San Diego Calif.. 1990). Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxyn cleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Tag polymerase. If a cognate pre-selected genetic region is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated. A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et at, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.

[0142] In certain advantageous embodiments, the template-dependent amplification involves the quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et at, Biotechnology .10: 413-417). By determining tire concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA .mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mi and is independent of the original concentration of target DNA, In specific embodiments, multiplexed, tandem PCR (MT-PCR) is employed, which uses a two-step process for gene expression profiling from small quantities of RNA or DNA, as described for example in US Pat. Appl, Pub. No. 20070190540. In the first step. RNA is converted into cDNA and amplified using multiplexed gene specific primers. In the second step each individual gene is quantitated by real time PCR.

[0143] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPO No. 320 308. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposit complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR ibr binding probe pairs to a target sequence.

[0144] Q RepKcase, described in PCT Application No. PCT US87/O088O, may also be used. In this method, a replicati ve sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the repKcative sequence that can then be detected.

[0145| An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide S'tt-thio-trtphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention. Walker el «/., (1992, Proc. Natl. Acad Set. U.S.A 89: 392-396).

[0146] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand dis lacement and synthesis, i.e., nick translation, A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases ca be added as biotinylaied derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). in CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific UNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction i treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is- annealed to another cycling probe and the reaction is repeated,

[0147) Still another amplification method described in GB Application No. 2 202 328, and in PCT Application No, PCT/US89/G1025, may be used. In the former application, "modified" primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers ma be modified by labeling with a capture moiety (e.g., biotm) and/or detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.

[0148) Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh t ah, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1 173; Gmgeras el ah, PCT Application WO 88/10315). in NASBA, the nucleic acid can be prepared for amplification by standard phenol/chloroform extraction, heat denaturatio of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidiiHum chloride extraction of RNA. These amplification techniques involve annealing a primer that has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single sti'anded DNA is made fully double stranded by addition of second target specific primer, followed by polymerisation. The double- stranded DN A molecules are then multipley transcribed by an RN A polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into single stranded DNA, which is then converted to double-stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

[0149] Vincent and Kong disclose a method termed he!icase-dependent isothermal DNA amplification (HDA) (Vincent and Kong, E BO Reports, 5(8):795-800, 2004). This method uses DNA helicase to separate DNA strands and hence does not require thermal cycling. The entire reaction can be carried out at one temperature and this method should have broad application to point-of-care DNA diagnostics.

[0150] Davey ef al., EPO No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssR A"), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRMA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA.RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "K!enow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA. ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

10151 j Miller ei al. in PCT Application WO 89/06700 disclose a nucleic acid sequence am lification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, Le., new templates are not produced .from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, M A., In: "PCR Protocols: A Guide to Methods and Applications", Academic Press, Ν.Ϋ., 1990; Ohara e/ o/., 1989, Proc. Natl Acad. ScL U.S.A., 86: 5673-567).

[0152] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleoti.de", thereby amplifying the di-oligonitcleotide, may also be used for amplifying target nucleic acid sequences. Wu ef l. , (1989, Genomics 4: 560).

[0153] Depending on the .format, the pre-selected gene product of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with, a second, known nucleic acid following amplification. Next, the identified product is detected. In certai applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of tire product via chemilumiiiescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, J Macromol. Set. Pure, Appi. Chem., A31(1): 1355-1376).

[Q154J In some embodiments, amplification products or "afflplicons" are visualized in order to confirm amplification of the pre-selecte genetic region product. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or iluorometrically-labelied nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In some embodiments, visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified pre-selected genetic region. The probe is suitably conjugated to a chromophore but may be radiolabeled. Alternatively, the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member of the binding pair carries a detectable raoiety or reporter raolecule. The techniques involved are well known to those of skill in the art and can foe found in many standard texts on molecular protocols (e.g., see Sambrook et a/,, 1989, supra and Ausubel et al 1994, supra). For example, chromophore or radiolabel probes or primers identify the target during or following amplification.

[0155] in certain embodiments, target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of UNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDN A blotting is analogous, in many aspects, to blotting or RN A species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysts. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturmg conditions. Unbound probe is then removed, and detection is accomplished as described above.

[0156] Followin detection/quantification, one may compare the results seen in a given subject with, a control reaction or a statistically significant reference group or population of control subjects as defined herein. In this way. it is possible to correlate the amount of a pre-seiected gene marker nucleic acid detected with a change in the level of T-regulatory cell activity.

[0157] Also contemplated are genotypittg methods and allelic discrimination methods and technologies such as those described by Kristensen et al. (Biotechniques 30(2): 318- 322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan®, liquid chromatography, and mass spectrometry.

[0158] Also contemplated are biochip-based technologies such as those described by Hacia ei al. (1996, Nature Genetics 1.4: 441 -447) and Shoemaker et al. (1996, Nature Genetics 14; 450-456). Briefly, these techniques involve quantitative methods for analysing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high-density arrays and screen these molecules on the basis of hybridization. See also Pease et al (1994, Proc. Natl. Acad. Sri. LISA. 91 : 5022-5026); Fodor et al. (19 1, Science 251 : 767-773). Briefly, nucleic acid probes to pre-seiected gene polynucleotides are made and attached to biochtps to be used in screening and diagnostic methods, as outlined herein. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed pre-seiected gene nucleic acids, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect; there may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two. three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (te._t have some sequence in common), or separate,

[0159] As will be appreciated by those of ordinary skill in the art, nucleic acids can be attached to or immobilized on solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non- covaient. By "non-covalent binding" and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included, i non- covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the tw moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules, Immobilization may also involve a combination of covalent and non-covalent interactions.

[0160] In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.

[0161] The biochip comprises a suitable solid or semi-solid substrate or solid support. By "substrate" or "solid support" is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by practitioners in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalised glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluoresce. [0162] Generally the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well ^'For example, the probes ma be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.

[0163] In certain embodiments, oligonucleotides probes are synthesized on the substrate, as is known in the art. For example, photoactivation techniques utilizing photopolymerisation compounds and techniques can be used, in an illustrative example, the nucleic acids are synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/251 16; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymefri GeneChip™ technology.

[0164] In an illustrative biochip analysis, oligonucleotide probes on the biochip are preselected gene such as TIGIT polynucleotides under conditions favoring specific hybridization. Sample extracts of DNA or RNA, either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment wit SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyroe. Suitable DNA, which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, ei al, 1 94, supra, and Sambrook, et al, et a!. , 1989, supra.

[0165| Suitable RNA, which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1 94, supra and Sambrook. et at. 1989, supra).

[0166] cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.

[0167] Usually the target pre-selected gene, such as TIGIT, polynucleotides are detectably labeled so that their hybridization to individual probes can be determined. The target polynucleotides are typically detectably labeled with a reporter molecule illustrative examples of which include cliromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, biolumioescent molecules, lanthanide ions («.#., En'⁴), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrati ve labels of this type includ large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In some embodiments in which an enzyme is used as a direct visual label, biotinyiated bases are incorporated into a target polynucleotide. Hybridization is detected by incubation with streptav i din-reporter molecules.

[0168] Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (F1TC), tetrainetliylihodaniine isothiocyanate (TR.ITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower e( al. (International Publicatio WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer ei a!), 5,326,692 (Brink!ey el al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commerciall available fluorescent labels include, for example, fluorescein phosphorarnidites such as Fluoreprime™ (Pharmacia), Fluoredite™ (Miliipore) and FAM (Applied Biosystems faternationa! ).

[0169] Radioactive reporter molecules include, for example, ^~'P, which can be detected by an X-ra or phosphoirnager techniques.

{0170] The hybrid-forming step can be performed under suitable conditions for hybridizin oligonucleotide probes to test nucleic acid including DNA or RNA. in this regard, reference may be made, for example, to NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH (Homes and Higgins, eds.) (IRL press, Washington D.C., 1985). In general, whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridization. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.

[0171] in certain advantageous embodiments, high discrimination hybridization conditions are used. For example, reference may be made to Wallace et al. (1979, Nucl. Acids Res. 6: 3543) who describe conditions that differentiate the hybridization of 1 1 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch. Reference also may be made to Wood et !. (1 85, Proc. Natl, Acid. Sci. USA 82; 1585) who describe conditions for hybridization of 1 1 to 20 base long oligonucleotides using 3M tetramethyl ammonium chloride wherein the melting point of the hybrid depends only on the length of the oligonucleotide probe, regardless of its GC content. In addition, Drmanac et al (supra) describe hybridization conditions that allow stringent hybridization of 6-10 nucleotide long oligomers, and similar conditions may be obtained most readily by using nucleotide analogues such as locked nucleic acids (Christensen et at.,, 2001 , Biochem j 354: 481 -4).

[0172] Generally, a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization-optimizing agent, such as an isostabilizing agent, a denaturing agent and/^'or a renaturation accelerant. Examples of isostabilizing agents include, but are not restricted to, betaines and lower tetraalkyi ammonium salts. Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents include, but are not restricted to, fomiamide, formaldehyde, dimethylsulfoxide, tetraethyl acetate, urea, guanidfum isothiocyanate, glycerol and chaotropic salts. Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyitrimethyla monium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanoL Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM.and 2 nM.

[0173] A hybridization mixture containing the pre-selected gene, such as T1G1T polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridization betwee the target, sequences in the target polynucleotides and any complementary probes. Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C. For probes longer than 14 nucleotides, 20° C to 50° C is desirable. For shorter probes, lower temperatures are preferred. A sample of target, polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes For example, the hybridization may be carried out at about 45° C +/-10⁰ C in formamide for 1-2 days.

[0174] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimizing agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridized to a target polynucleotide.

[0175] The hybridization reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be insirumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer: or detection of a dye particle or a colored colloidal metallic or non metallic particle using a ref!ectometer; in the case of using a radioactive label or chemiluminescent molecule employing radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focused beam or laser light, in such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probc:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymati catty generated color spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means is suitably interfaced ^'with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific fo a pre-selected gene, such as TIGIT products are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung el al (U.S. Patent No. 5,143, 854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplar}-' chip readers are described for example by Fodor et al (U.S. Patent No,, 5,925,525). Alternatively, whe the array is made using a mixture of individually addressable kinds of labeled mierobeads, the reaction may be detected using flow cytometry.

[0176] Consistent with the present description, a difference in concentration of a preselected gene expression protein product, such as TIGIT polypeptide, between a test subject or sample and a control subject or reference sample is indicative of the presence or level of T-regulatory cell activity in the sample or subject. TIGIT or other pre-selected polypeptides levels in biological samples can be assayed using any suitable method known hi the art. Convenient methods include monitoring th level of surface expression of surface expressed polypeptides such as TIGIT polypeptides. For example, if appropriate, the protein can be quantified based upon its catalytic activity or based upo the number of molecules of the protein contained in a sample. Antibody-based techniques ma be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a protein of interest in a tissue sample. For example, specific recognition is provided by a primary antibody (polyclonal or .monoclonal) and a secondary detection system is used to detect presence (or binding) of the primary antibody. Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) that produces a quantifiable, e.g., colored, product, hi another suitable method, the primary antibody itself can be detectably labeled. As a result, immunolustological labeling of a tissue section is provided. In some embodiments, a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis. Such an extract (e.g., a detergent extract) can be subjected to western-blot or dot/slot assay of the level of the protein of interest, using routine immunoblotting methods (lalkanen el al., 1985, J. Cell. Biol. 101 : 976-985; Mkaiien e/ ., 1987, J. Cell. Biol. 105: 3087-3096).

[0177| Other useful antibody-based methods include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RJA). For example, a protein-specific monoclonal antibody, can be used both as an im unoadsorbertt and as an enzyme-labeled probe to detect and quantify a pre-seleeted gene expression polypeptide product. The amount of such protein present in a sample can be calculated by reference to the amount present in a standard or reference preparation using a linear regression computer algorithm (see Lacobilli et «/,, 1988, Breast Cancer Research and Treatment 11: 1.9-30). In other embodiments, two different monoclonal antibodies to the protein of interest can be employed, one as th immunoadsorbent and the other as an enzyme-labeled probe.

[0178] Additionally, recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins. For example, low-density protein arrays on filter membranes, such as th universal protein array system (Ge, 20O0 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immune-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-dru treatment.

[0179] Protein capture arrays typically comprise a plurality o protein-capture agents each of which defines a spatially distinct feature of the array. The protein-capture agent can be an molecule or complex of molecules that have the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array. The protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor. Alternatively, the protei -captu e agent may instead be a partially or wholly synthetic or recombinant protein that specifically binds a protein. Alternatively, the protein-capture agent, may be a protein that has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target. The selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art. Alternatively, the protein-capture agent, obtained via in vitro selection may be a DNA or RNA aptamer that specifically binds a protein target (see, e.g., Potyrailo et al, 1998 Anal. Chem. 70:3419-3425; Cohen et al, 1998, Proc. Natl. Acad. Sei. USA 95:14272-14277; Fukuda, ei i, 1997 Nucleic Acids Syrap. Ser. 37:237-238; available from SomaLogic). For example, aptamsrs are selected from libraries of oligonucleotides by the Sele™ process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers}, Aptamers have the advantages of ease of production b automated oligonucleotide synthesis and the stability and robustness of DNA; universal f uorescent protein stains can be used to detect binding, Alternatively, the in vitro selected protein -capture agent may be a polypeptide (e.g. , an antigen) (see, e.g., Roberts and Szostak, 1997 Proc. Natl Acad. Sci. USA 94: 12297- 12302).

[0180] An alternative to an array of capture molecules is one made through 'molecular imprinting^* technology, in which peptides {e.g., from tire C-terrainal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymer isabie matrix; the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g. , available from ProteinPrint™ and Aspira Biosystems).

[0181] Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteorne. Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibod arrays have been reported (see, e.g., Lopez el al, 2003 J. Chromatogr. B 787: 19-27; Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat, App. Pub, 2003/0003599; PCT publication WO 03/062444; PCX publication WO 03/077851; PCT publication WO 02/59601 ; PCT publication WO 02/39120; PCT publication WO 01/79849; PCT publication WO 99/39210). The antigen -binding molecules of such arrays may recognise at least a subset of proteins expressed by a cell or population of cells, illustrative examples of which include growth, factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras-like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucme-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-relate factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C vims (HCV) proteases and HIV proteases.

[0182] Antigen-binding molecules for antibody arrays are made either by conventional immunization (e.g. , polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosonie display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, Affitech and Biosite). Alternatively; 'combibodtes' comprising non-covalent associations of VH and VL domains, can be produced i a matrix format created from combinations of diabody- produdng bacteria! clones (e.g., available from Domantis). Exemplary antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyciona! antibodies, Fv, Fab, Fab' and F(ab*)2 immunoglobulin fragments, synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.

[0183] Individual spatially distinct protem-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.

[0184] While microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifitgation devices based on developments in microfluidics (e.g., available from Gyros) and specialized chip designs, such as engineered microchannels in a plate (e.g. , The Living Chip™ , available from Biotrove) and tiny 3D posts on a silicon surface e.g., available from Zyomyx).

[0185] Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g.., QDots™, available from Quantum Dots), and barcoding for beads (UltraPlex™, available from Smartbeads) and multimetal microrods (Nanobareodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). Where particles are used, individual protem-capture agents are typically attached to an individual particle to provide the spatial definition ox separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a rnicTotiter plate or in separate test tubes.

[0186] n operation, a protein sample, which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub, 2002/0055186), is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifiealiy bound components of the sample from the array. Next, the presence or amount of protein or peptide boirad to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the arra may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, tile amount of the second protein in the sample is already known or known to be invariant.

[0187] For analyzing differential expression o proteins between two cells or cell populations, a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding. In an analogous manner, a protein sample of a second cell or population of cells to a second array, is delivered to a second array which is identical to the first array. Both arrays are then washed to remove unbound or non- specifiealiy bound components of the sample from the arrays. In a final step, the amounts of protein remaining bound to the features of the first array are compared to the amounts of protein remaining bound to the corresponding features of the second array. To determine the differential protein expression pattern of the two cells or populations of ceils, the amount of protein bound to individual features of the first array is subtracted from the amount of protein bound to the corresponding features of the second array.

(0188! In an illustrative example, fluorescence labeling can be used for detecting protein bound to the array. The same instrumentation as used for reading DMA microarrays is applicable to protein-capture arrays. For differentia! display, capture arrays (e.g. antibody arrays) can be probed with fluorescently labeled proteins from two different cell states, i which cell lysates are labeled with different fluorophores (e.g. , Cy-3 and Cy-5) and mixed, such that the color acts as a readout, for changes in target abundance. Fluorescent readout sensitivity can be amplified .10-100 fold by tyramide signal amplification (TSA) (e.g., available from PerkinElraer Lifesciences). Planar waveguide technology e.g., available from Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot), Fluorescence resonance energy transfer has been adapted to detect binding of unlabel!ed ligands, which may be useful on arrays (e.g., available from Affibody). Several alternative readouts have been developed, including adaptations of surface plasmon resonance (e.g._f available from HTS Biosystems and intrinsic Bioprobes), rolling circle D A amplification (e.g., available from Molecular Staging), mass spectrometry (e.g.., av ailable from Sense Proteomic, Ciphergen, Intrinsic and Bioprobes), resonance light scattering (e.g., available from Genicoti Sciences) and atomic force microscopy (e.g., available from BioForce Laboratories). A niicrdfluidics system for automated sample incubation with arrays on glass slides and washing has been co-developed by NextGen and Perkin Elmer Life Sciences.

[0189| In certain embodiments, the techniques used for detection of TIGIT or other preselected expression products will include internal or externa! standards to permit quantitative or semi-quantitative determination of those products, to thereby enable a valid comparison of the level or functional activity of these expression products in biological sample with the corresponding expression products in a reference sample or samples. Such standards can be determined by the skilled practitioner using standard protocols. In specific examples, absolute values for the level or functional activity of individual expression products are determined.

[01901 In specific embodiments, the diagnostic method is implemented using a syste as disclosed, for example, in International Publication No. WO 02/090579 and in copending PCT Application No. PCT/AU03/015I 7 filed November 14, 2003, comprising at least one end station coupled to a base station. The base station is typically coupled to one or more databases comprising predetermined data from a number of individuals representing the level or functional activity of TIGIT or of other pre-selected gene expression products, when the predetermined data was collected, hi operation, the base station is adapted to receive from the end station, typically via a communications network, subject data representing a measured or normalized level or functional activity of at least one expression product in a biological sample obtained from a test subject and to compare the subject data to the predetermined data stored in the datahase(s). Comparing the subject and predetermined data allows the base station to determine die status of the subject in accordance with the results of the comparison. Thus, the base station attempts to identify individuals having similar parameter values to the test subject and once the status has been determined on t e basis of that identification, the base station provides an indication of the diagnosis to the end station.

[0191] The present invention is further described by the following non-limiting Examples. Materials and Methods used i these Examples are provided below.

Human blood T eel! isolation, activation and pheno.t ping

[0192] Blood buffy coats from healthy male donors were donated by the Australian Red Cross Blood Service. Individuals with 'preclinicaT type I diabetes (pre-T ID) (Harrison LC (2001) P&iiatr Diabetes 2:71-82) had a first-degree relative with Tl.D and circulating autoantibodies to > 2 pancreatic islet antigens (insulin, GAD65, IA2). CD4' T ceils were enriched with anti-human CD4 microbeads on LS columns (Miltenvi Biotec), labelled with a«ti-CD4-Pacific Blue, -CD45 A-APC, -CD45R.O-PE (BD) and -CD25-PE-Cy7 (eBiosciences) antibodies and flow-sorted into Naive (CD45RA ^' CD45 O^" CD25^") and rTreg (CD45RA^> CD45RO^"CD25^'+). Cells were cultured in. IPS medium (Iscove's modified Dulbecco's medium [Gibco] supplemented with 5% heat-inactivated pooled human serum, 2mM glutamiiie. O.QSmM 2-mercaptoethanol, I0QU/ml penicillin, 100pg ml streptomycin and ΙΟΟμΜ non-essential amino acids). For activation, freshly sorted Naive and rTreg were labelled with CellTracer Violet (CTV) dye (invitrogen) and co-cultured with irradiated (3000 rad) autologous CD4 ^! T cell-depleted PBMCs at a 1 :4 ratio for 3 days in the presence of soluble an£i-CD3 (100 ng ml) (clone OKT3) (Walter and Eliza Hall Institute Monoclonal Lab) and anti-CD28 (200ng ml) (clone CD28.2) (BD) antibodies. On day 4, fresh IPS medium containing recombinant human IL-2 (final concentration 20 LVml) was added and cells were cultured for a further 2 days. On day , cells that had proliferated (CTV^dSm) and were CD25 were sorted for preparation of genomic DNA. For phenot pic analysis, cells were first stained with anti-C D4-PE-Cy7 and anti-CD25-FiXC antibodies (BD), followed b intracellular staining with anti- H^'XH^>-?-Alexa647 antibody (Biolegend).

Suppressor assay

[0193] Activated naive ( Act-Naive) and activated rTreg (Act-rTreg) were generated as described above except they were labelled with carboxyfluorescein succinimidyl ester (CFSE) dye (invitrogen)- CFSE^dns CD25¹ cells were sorted as "suppressor cells". Autologous naive CD4 ^* T cells were isolated with a human narve CD4 ^' T cell isolation kit (Miltenvi Biotec) and labelled with CTV as "responder cells". 'Suppressor' cells were added to 'Responder' cells (5 l 0⁴) at 1 : 1, 1 :2, 1:4, 1 :8 and 1:1.6 (suppressor: responder) ratios, together with autologous CD4 T cell-depleted irradiated PBMCs (lxlO⁵) as antigen presenting cells. Cells were stimulated with soluble anti-CD3 antibody (1.00 ng/ml) for 5 days and analyzed by FACS for CTV dilution.

Chromatin immunopre ipitation and quantitative RT-PCR

[0194] Sorted naive and rTreg cells were activated with plate-bound anti-CD3 (3 g/rnl), soluble anti-CD2S (1 g nd) antibodies and IL-2 (200 U/ml) to obtain Act-Naive and Act-rTreg cells, induced Treg cells (iTreg) were differentiated from naive cells with the same stimulation with the addition of TGF-βΙ (5 ng/ l). Ten to twenty million cells were used for FOXP3 CMP. Briefly, cells were restimulated. with anti-CD3/CD28 antibodies for 2 hours before fixed with 1% Formaldehyde solution, sonicated with 0.25% SDS lysis buffer (50 ra Tris-HCL PH8.0, !OroM EDTA, 0.25% SDS). Lysates were diluted to contain 0.1 % SDS before antibody iaununoprecipitation (IP). Polyclonal goat- anti-human FOXP3 (10 g ChIP), Biotinylated-donkey-anti-goat gG and Streptavidin Ferrofluid beads (R & D Systems) were used to enrich DNA fragments. FOXP3-pass- thro gh lysates were IPed with normal goat-lgG as controls. Relative abundance of regions of interest in precipitated DNA was measured by qPCR using GoT^'aq SYBR (Prarnega).ChlP-q PCR primers are listed in Table 12. Relative FGXP3-binding abundance was first normalized to input DNA and fold change was calculated to an intergenic region known void FOXP3 binding.

Genomic DNA isolation, bisulfite con version and sequencing

[0195] Genomic DNA was isolated with the DNeasy blood & tissue kit (Qiagen) following the protocol for cultured animal cells and precipitated with ethanol. Purified DNA (500-750 ng) was subjected to sodium bisulfite conversion with an EZ DNA Methylation Kit (Zymo Research) using the recommended protocol for the Illumina Infinium methylation assay: 16 cycles at 95°C for 30 sec and 50°C for 60roin, followed by hold at 4°C. For sequencing, bisulfite converted DNA was used to amplify TIG1T and FOXP3 fragments at an annealing temperature of 52°C (or 65°C for the TSDR), for 35 cycles with a final extension at 72°C for 5 min, Amplified products were ligated into the pGEM-T vector (Promega) and sequenced with the Ml 3 -forward primer. Bisulfite sequencing primers for FOXP3 and TKrlT loci are listed in Table 12 and the sequence listing. RT-PCR and quantitative PCR

[0196] Total NA was extracted with. TRI Reagent solution (Invitrogen). AMV reverse transcriptase and random hexamers (Qiagen) were used for e^'DNA synthesis following product instructions. Quantitative PCR was performed on the AB1 790OHT system in 384 well plates (Applied Biosystems) with a total volume of 1.5μ1 reaction mix using GoTaq qPCR master mix (Promega) in duplicate. Hypoxanthine phosphoribosyitrans erase- 1 (HPRTI) was amplified as a housekeeping gene. Primers are provided in Table 12. miRNAs were detected by quantitative RT-PCR using Taqman miRNA assays (Ambion) according to the manufacturer's protocol. U6 snRNA was used as a reference gene.

Taqman PCR for detection of differential methylation

[0197] Bisulfite converted genomic DNA from Act-naive, Act-rTreg and iTreg cells were subjected to qPCR using HEX and FAM-labelled probes thai recognize methylated and demeihylated CpG sites, respectively. Primers and probes were listed in Table 12. Methylation level was calculated with the formula: % methylation = 100/ [1 +2^{c¾metl yl}*'^ed" iwmcihy ted!j

Taqman PCR for detection of mhroRNA

[0198! Total RNA was extracted using TRI Reagent following the manufacturer's protocol with one modification: RNA was precipitated with efhanol at -80°C (instead of isopropanol on ice) in order to obtain all RNAs including miRNAs. miRNAs were detected by quantitative RT-PCR using Taqman miRNA assays (Ambion) according to the manufacturer^'s protocol. 0¾snRNA was used as a reference gene, lllumina 450k arrays

( 019 1 4μΙ of bisuifite-converted DNA was used for hybridization on Infinium HumatiMethylation450 Bead Chips, following the lllomina Infinium HD methylation protocol, processed by the Australian Genome Research Facility (AGR.F). The latter consisted of a whole genome amplification step followed by enzymatic end-point fragmentation, precipitation and re-suspension. The re-suspended samples were hybridized at 48°C for 16 hr. Washing to remove non-hybridized and non-specifically hybridized DNA, was followed by a single nucleotide extension using the hybridized bisulfite-treated DNA as a template. The nucleotides incorporated were labelled with biotm (ddCTP and ddGTP) and 2₅ 4-dimtrophetioL (D P) (ddATP and ddTTP), After the single-base extension, repeated rounds of staining were performed with a combination of antibodies that differentiated DNP and biotfn by fixing them with different fluorophores, Finally, the BeadChip was washed and protected prior to scanning, niuniin iScan was used to scan the BeadChips. The raw intensity data (IDAT) files were imported into the R statistical environment (see the Comprehensive R Archive Network), where subsequent processing and qualit control of the 450k data were performed using the minft Biocondnctor package (see the Bioconduetor open source software for biomformatics). The data were initially assessed for outlying samples and significant, technical variation. All samples passed quality control. Data were, then normalized for technical variation between type 1 and ΪΙ Infraium probes (see Bibikova M ei ai. (201 1) Genomics 98:288-295, Dedeurwaerder S et al (2011) P/pigenomies 3:771 -784) using S WAN (see Maksimovic J et ai. (2012) Genome Biol 13:R44). Poorly performing probes, defined as having a detection P-value > 0.01 in one or more samples were removed from further analysis. Probes included on the array to interrogate SNPs were also excluded at this stage. This reduced the size of the final data set to 481,473 probes.

Differential metliylation analysis

[02001 The proportion of methylation at eac CpG is represented by the β-va^'lue, defined as the proportion of the methylated signal to the total signal [β = methylated / (unmethy!ated + methylated)] and is calculated from normalized intensity values. However, all of the statistical analyses were performed on M-values [M - Iog2 (methyiated/uiimethylated)] as recommended in Du P et at (2010) .Biomformatics 1 1 :587. For probe-wise differential metliylation analysis, a linear model was fitted taking into account the paired samples within an individual, using the limma package (see Smyth GK (2005) Biomformatics and Computational Biology Solutions using R and Bioconduetor 397-4.20). P-values were adjusted to control for the false discovery rate using the Benjaniini-Hochberg method of Benjamini Y et at. (1995) ./ R Statist So 57:289-300. Significantl di ferenti lly methylated probes (DM Ps) were defined as those having an adjusted P-value < 0.05 and an absolute difference in mean β values |Δβ|≥ 0.2.

Identification of regions of differential methylation

[0201 ] Regions of differential methylatio (RDMs) were identified using the "dmrFmd" algorithm available in the Biocondiictor package charm (see Aryee MJ et ah (2011) Biostaiisiics 12:197-210). Default parameters were used, except for the linear modelling parameter, for which the limma option was selected. False discovery rates (FDR) were calculated using the "qval" function, also available from the charm package, with m iiter ^" 1000 permutations. The list of RDMs was further refined by the exclusion of regions with an absolute average percentage methylation difference across probes within the RD of <15%.

Functional annotation

[0202] Functional annotation was performed using the functional annotation clustering tool and gene ontology (GO) terms available from the online Database for Annotation, Visualization and integrated Discovery (DAVID) v6.7 Huang da W ef al. (2009) Nat Protoc 4:44-57. As functional annotation operates at the level of "genes", in cases where multiple DMPs were annotated to a single gene that gene was represented only once. Unannotated DMPs were excluded from the analysis. In the case of RDMs, only those that could be annotated to genes were used for functional annotation analysis. The DAVID tools were run using default parameters.

Focused gene sets and gene set testing

[0203{ To test whether the RDMs were putative FOXPS targets potentially relevant to Treg function, gene sets were assembled from the literature. These encompassed targets of FOXPS in mice (see Zheng Y el al. (2007) Nature 445:936-940, Marson A ei al (2007) Nature 445:931 -935) and humans (see Sadlon TJ et at. (2010) J Immunol 185: 1071 -1081 ) from FOXPS ChlP experiments, genes regulated in Treg by ectopic expression of FOXPS in mice (see Hill JA, et al (2007) Immunity 27:786-800) and from expression microarrays in humans (see Pioertner S et al. (2006) Genome Biol 7:R54) as well as cytokines and transcription factors (see Wei G, et al. (2009) Immunity 30 : 1 5- 167) (data not included).

[0204] Gene set enrichment analysis was performed on all. significant RDMs annotated to genes. One-tailed Fisher's exact tests were performed on contingency tables for overrepresentation of differentially methylated genes in a set. All RDMs were tested against all gene sets. RDMs were also separated into two groups: hypermetbylated in rTreg compared to Naive and hypomethylated in rTre-g compared to Naive, which were then independently tested against the selected gene sets. Motif identification

[0205] The sequence underlying each RDM associated with a gene promoter region was extracted from the human genome (hgl ). An RDM was deemed to be promoter- associated if it contained at least one CpG probe annotated to any of the following categories: TSS200, TSS 1500, 5'UTR or Γ^! exon. The sequence boundaries of the selected RDM.s were extended if significant DMPs were within 150bp of the original RDM. The RDM sequences were then submitted to the MEME motif discovers' tool see Bailey TL ei al (1994) Proc fat Con Iniell Syst Mol Biol ;2:28~36. MEME was run using default parameters. The identified motifs were submitted to die TOMTOM motif comparison tool see Gupta S et al (2007) Genome Biol 8:R24. TOMTOM was run using default parameters in conjunction with the J AS PAR FAM motifs database Sandelin A et al. (2004) J Mol Biol 338:207-215. containing models describing shared binding properties of structural classes of transcription factors.

EXAMPLE 1

C 4 T ceil subsets: phenotype, function and methyiation

[0206] Human rTreg are a relatively homogenous population which, after activation m vitro, resemble activated nTreg (aTreg) in peripheral blood Miyara M el al. (2009) immunity 30:899-91 1. Naive and rTreg were sorted from buffy coats of 3 healthy male donors (M28, 29, 30) (Figure 1A, top panel) and activated them for 6 days with anti-CDS and anti-CD28 antibodies, supplemented with 1L-2 at day 4. Both populations proliferated and were CD25' (Figure ί A . middle panel). The majority (> 85%) of activated rTreg (Act-rTreg) and some (~ 30%) of the activated naive (Act-naive) cells expressed FOXP3 (Figure 1 A, bottom panel), in an autologous suppression assay, Act-rTreg cells were more potent than Act-na^'ive cells as shown by the inhibition of proliferation, except in the presence of lL-2 when suppression by Act-rTreg or Act-naive cells was similar (Figure I B).

[0207] DNA was prepared from rTreg, Naive, Act-rTreg and Act-naive for DNA methyiation analysis on ^'lllumina Infinium HumanMethylation450 (450k) aiTays. Following quality control and preprocessing (see Methods), mufti-dimensional scaling (MDS) analysis of the normalized, filtered data indicated that most of the variation in methyiation between samples was inter-individual. However, higher dimensional analysis also revealed consistent differences in methyiation depending on cell type and activation status (Figure IC left panel). The statistical analysis therefore included a factor to account for inter-individual differences when testing for methylation differences between cell types. Thus, when the individual biological variation is taken into account statistically, the methylation differences in cell type and activation are clearly apparent (Figure 1 C right panel).

Differential DN A methylation between Naive ami rTreg

[0208J Between Naive and rTreg, 2,315 CpGs were significantly differentially methylated (P-value < 0.05, ]Δβ| > 0.2) (see Tables 1 to 20). These represented less than 0.5% (2,315/481,473) of the filtered set of CpG probes. More than 70% (1,681/2,315) of the differentially methylated probes (DMPs) wer associated, with 1 , 137 genes, whilst the remaining 634 were not annotated. With respect to genomic context, 33% (747) of the DMPs were located in promoter regions, 40% (920) in gene bodies and 4% (86) in 3 'untranslated regi ons (UTBLs) of known genes; 24% (541 ) were not assigned to a genom ic feature. Compared to the proportions of annotations of all probes on the 450k array, the list of DMPs showed a significant enrichment for probes in the gene body (P-value < 2.2x l0^"16, Fisher's exact test [FET]) and significant depletion of probes in promoter regions (P-value < 2,2xl0^{"i f>}, FET), whilst the proportions of 3'UTR and unannotated probes were not significantly different from a randomly selected set (Figure 2A). Using DAVID functional annotation clustering analysis (Huang da W et al. (2009) Nal Protoc 4:44-57, Huang da W era!. (2009) Nucleic Acids Res 37: 1 -13), the most significant cluster of genes associated with DMPs was for the terms T cell activation and T cell differentiation, followed by Regulation of cell death (data not included).

[0209] Neighbouring CpGs are often correlated in methylation status (Eckhardt F ei al. (2006) Nat Genet 38: 1378-1385, Irizarry RA, et al. Nat Genet 009 41 : 178-186) and genomic regions rather than individual CpGs can be more functionally relevant (Hansen KD et al (201 1) Nat Genet 43:768-775, Jaeniseh R et al. (2003) Nat Genet 33 Suppl:245- 254, irizarry ei al (2009) Nat Genet 41: 178-186, Lister R et al. (2009) Nature 462:315- 322). An analysis was therefore performed to identify regions of differential methylation (RDM), defined as clusters of physically proximal CpGs (within 300bp) that are significantly different in their methylation between cell types (Jaffe AE et al (2012) hit J Epidemiol 41 :200-209). One hundred and twenty seven RDMs were identified that distmguished Naive and rTreg (included as Table 14). To explore the relationship between the observed, methylation changes and gene expression, the methylation data for promoter- associated RDMs was correlated with publicly available gene expression data from Schmidl et at**. A negative correlation was observed between the methylation and gene expression data for these loci, with a Pearson's correlation coefficient of -0.5 and p- value==0.0026 (Figure 6), suggesting that changes in methylatio at the promoter-associated ROMs are related to gene expression changes. The 12Mb methylation data from Schmidl et a 1. ² has a minimal (<0.0l%) overlap with our 450k array CpGs, however, five ROMs intersected with their regions of interest, 3 of which exhibited differential methylation consistent in both studies (data not shown).

[02ΪΘ] The RDMs encompassed a total of 608 individual CpG probes and were associated with 97 genes and 23 unannotated genomic regions. Of the 60S individual probes found in RDMs, 65% (385) were located in promoter regions, 23% (134) in gene bodies and 2% (11) in 3'UTRs; 1 1 % (65) were unannotated (Figure 2A right, panel). In contrast to the individual DMPs, the probes located in RDMs showed a significant enrichment in promoter annotations (P-vatue < 2.2x10^"u>), when compared to the full set of 450k array annotations, and a corresponding decrease in the proportions of gene body (P- value < 1.823χ10^",,ΐ) and unannotated (P-value < 1 .348x10^"14) probes, whilst the proportion of 3'UT probes did not differ significantly. As with the probe-wise analysis, DAVID functional annotation of RDM genes revealed significant enrichment for the terms T cell activation and T cell differentiation. However, in contrast to the functional annotation of DMPs, the next most significant terms were Repressor activity and the Plasm membrane (data not included).

[0211] Unsupervised clustering of the β values of both the significant DMPs and RDM probes resulted .in distinct grouping of the Naive and rTreg samples (Figure 2B). In both eases, relative hypennethylation and hypomethylation were distributed evenl between Naive and rTreg. in contrast to the previously published observation of relative hypomethylation of nTreg determined by other methods (see Ohkura N et at. (2012) Immunity 37:78 -799) .

Activation-induced changes in DMA methylation profiles

[0212] The ability of the immune system to combat diverse, potentially pathogenic insults yet maintain tolerance to self is reflected by the lineage and functional plasticity of naive T cells, countered by Treg. Following activation, conventional T cells may express markers in common with Treg such as CD25, FOXPS, CD i.27 and HELIOS. The fidelity of distinct DNA methylation profiles was therefore examined after antigen activation of Naive cells and rTreg. Act-Naive and Act-rTreg displayed significant differences in 3 ,689 CpG probes (data not shown), the majority of which overlapped with the differences found between these two T cell subsets before activation (Figure 3A). Thus, the DNA methyiation profiles of Naive and rTreg were largely unaffected by activation,

[0213] The activation-induced DNA methyiation differences in naive CD4 T cells and rTreg cells were examined. In aive T cells, inethy!ation at 466 CpGs was significantly different after activation (see Table 15). 36.7% (171 ) of these CpGs were altered such that they had similar methyiation patterns to rTreg, and 60.7% (283) were unique to activation (Figure 3B). The genes associated with the DMPs unique to activation were enriched for tenns related to transcription, DNA binding and positive regulation of various cellular processes in the most significant cluster, followed by clusters containing terms associated with DNA binding, nucleotide binding, phosphorylatio and kinase (data not included). Eighteen RDMs were also identified, 13 of which were associated with known genes (^"J SR1 , 1R21 , GFE1 , CSF2, NCF4, V P1 , eSD17B8, IL13, ITGA^'X, RPTO , AIM2, CCL5, CD59) and 5 were imannotated (data not shown). The genes associated with the activation RDMs were enriched for terms related to cytokine-cytokine receptor interaction and regulation of T ceil differentiation, indicating changes in DNA methyiation are associated with naive CDC T cell activation and plasticity, Sitrprisingly, activation did not induce any significant DNA methyiation changes in rTreg.

[0214] Methyiation changes in activation associated RDMs may regulate gene expression. At the mieroRNA MIR21 locus, activation induced significant detnethylation in naive CD4^" T cells (Figure 3C). The expression of mlR-21 in naive and rTregs and after antigen activation was examined. Activation induced significant upregulation o mi -21 in naive CDC T cells. In vitro activation did not induce significant changes in rTreg, however aTreg isolated directly ex vivo exhibited significantly increased miR~2l expression (Figure 3D). These may reflect the differences in the activation process of rTreg, it also implies that other mechanisms besides DNA methyiation contribute to the regulation i iR-21 expression in Treg cells.

Hypomethylation in Treg signature genes and identification of Forkhead-hinding mnttfs in promoter-associated RDM

[0215] FOXP3 binds to the deroethylated TSDR at the FOXP3 locus in Treg to maintain its transcriptional activity, underlying the fimctional cooperation between FOXP3 and epigenetic modifications in shaping Treg function. To test whether other regions of altered DMA methyiation are putative FOXP3 targets potentially relevant to Treg function gene set testing was performed on genes associated with RDMs that were significantly more (hyper Treg)or less (hyper Treg) methylated in Treg. Eleven gene sets were identified from the literature, as described in Methods. A gene set test analyzes a set of genes as a functional unit A one-tailed Fisher's exact test, was performed to determine whether genes from the compiled sets were enriched in the pool of genes associated with RDMs. The common Treg signature genes, Treg signature genes directly u -regulated by FOXP3 and FQKP3 targets were significantly enriched in both the full set of RDM-associated genes and the subset of RDMs that were hypomethylated in Treg compared to Naive (data not shown). This suggests that FQXP3 binding to hypomethylated regions shapes Treg signature gene expression.

[0216] It was then determined whether potential transcription factor binding sites were present in the sequences of promoter-associated RDMs (83/127). An overrepresented motif was identified in promoter-associated RDM sequences using MEME (see Figure 7). This motif was then compared to known binding motif models for various families of transcription factors using the TOMTOM tool and was identified as a candidate Forkhead- binding motif that could potentially bind FOXP3 (Figure 7B). This motif is similar to one previously described (Sadlon TJ ei al, (2010) J Immunol 185:1071-1081). The genomic positions of all 127 RDMs was compared with published human FOXP3 ChlP-Chip data (see Sadlon TJ, ei al. (2010) / Immunol; 185(2): 1071-108 ! ) and found a direct overlap of FOXP3 enrichment with 15 of the RDMs. Given the ChlP-Chip FQXP3 peaks covered only 8331 ( <2%) of the CpGs on the 450k array the overlap of genomic bases covered by both the RDMs and FOXP3 ChlP-chip peaks is highly significant (P-value < 2.2x10^"16 ₅ Pearson's Chi-squared Test), suggesting a relationship between methylation and FOXP3 binding in nTreg. The positions and relative methylation status of the R DM s, along with evidence for the presence o FOXP3 binding in is summarised in (Tabl 13). The expression of several other demethylated Treg genes in Treg was selected and validated by qR -PCR, controlled by FOXP3 (Figure 7C). Bioinformatic analysis thus far suggests that demethylatton, especially in promoter regions of Treg genes, contributes to increased gene expression. FOXP3 potentially binds to the demethylated regions via the Forkhead- binding motif, thereby further shaping Treg function.

[0217] Tr gspecifk hypomethylalion of Tcell ig and ΓΠΜ domain (TIGIT)

TIGIT is a recently discovered receptor that suppresses activation of T cells, dendritic cells and N cells (Yu X et al (2009) Nat Immunol 10:48-57, Joller N ei al. i) J Immunol 186: 1338-1342, Lozano E et al (2012) J Immunol 188:3869-3875). Demethylation of the TIGIT locus was one of the most significantly differentially methylated regions that distinguished Naiv and rTreg, not affected by activation (Figure 4A), Furthermore, a putati ve Forlchead-binding motif was found in the RDM (hiF2) of the TIGIT promoter. Differential hypomethylation of TIGIT was further confirmed by clonal bisulfite sequencing of CpG dinucleotides neighbouring the differentially methylated probes on 5 donors. As a control, clonal bisulfite sequencing of the FOXP3 promoter and TSD was performed on the same samples. CpG dtnucleotid.es of TIGIT were hypomethylated in rTreg across several regions, most apparent in biF!, .2 and 5 where putative promoter and enhancer regions are located (Figure 4B left panel). The (hypo)methylation status of the TSDR st ihe OXPS

locus indicated the purity of isolated Naive and rTreg (Figure 4B, right panel). Interestingly by methyl ation of TIGIT was nevertheless heterogeneous within rTreg cells (Figure 8). TIGIT was described as being expressed in human Treg and activated Tconv cells (Yu X et al (2009) Nat Immunol 10:48-57). It was confirmed by qRT-PCR that TIGIT transcription was upregulated in Treg, and to lesser degree in Act-naive (Fi gure 4C). At protein level, a combination of TIGIT and CD25 provided a better means to identify ex vivo human rTreg and aTreg cells (Figure 4D).

[0218] To test whether FOXP3 binds to the demethylated TIGIT promoter and contributes to TIGIT expression in huma Treg ceils, naive and rTreg cells were activated respectively to obtain Act-Naive, iTreg and Act-rTreg cells. Intracellular FOXP3 staining indicated expression of FOXP3 in. all activated cells, lowest in Act-Naive, similar high level at iTreg and Act-rTreg cells. TIGIT exhibited the highest amount of expression in Act-rTreg cells and modest upregulation in Act-Naive and iTreg cells (Figure 5A). Methylation-sensitive Taqman-qPCR further confirmed raethylatton of the TIGIT locus (biF2) in Act-Naive and iTreg cells and demethylation in Act-rTreg cells (Figure 5B). By ChlP-qPCR, enrichment of FOXP3 at the TIGIT locus in Act-rTreg cells was observed, which is demethylated, as well as other hypomethylated loci (FOXP3 (TSDR), MIR2I, CTL4-4, I Ra (CD25j) known to bind to FOXP3. As a control, at intron 1 of AFM, no enrichment of FOXP3 binding was observed (Figure 5C).

EXAMPLE 2

[0219] Naive resting natural Treg (rTreg) were flow-sorted from blood of indi viduals at high-risk to develop autoimmune type 1 diabetes (preT 1 D). They were activated with plate-bound a&ti.-C.D3 (3

soluble anti-CD28 antibodies (1 p.g/ml) and recombinant human IL-2 (200 U/ml) in 96-well flat-bottom plate. Cells were transferred to 24-well plate at day 3, supplemented with IL-2 (200 U/ml) for an additional 3 days,

[0220j The data show (see Figure 9) that TIGIT protein expression can be used as a surrogate marker of TIGHT locus demethylatton and true nTreg identity. FOXP3 and IFN-γ production in TIGIT+ nTre cells reveals nTreg stability and suppressor/effector function. This combination can be used inier alia to predicate TID progression, reveal treatment efficacy and as a platform for drug screening. Similar results were also observed in individuals with Coeliac Disease and Juvenile Idiopathic Arthritis and the instant methods are relevant to other inflammatory conditions.

^'EXAMPLE 3

[0221] As enabled herein, the methylation status of TIGIT in cord blood is used as a diagnostic marker for Tregulatory cell activity and a diagnostic for, or for risk of, developing an allergic disease such as food allergy. Total CD4^'!T cells were flow sorted from a test sample of cord blood. Genomic DNA. was isolated and bisulfite converted. Methylation analysis of TIGIT and FOXP3 loci were as described in Example 1. Figure 10 shows a correlation of FOXP3 and TIGIT deinethyfation in cord blood CD4 T cells. In total CD4 T cells, methylation of FOXP3 and TIGIT loci are positively correlated. This demonstrates that demethvlation of either locus can be used as a surrogate marker of nTreg. in CD4 T cells from cord blood of children who developed food allergy at 1 year of age, demethvlation of TIGIT AND FOXP3 loci were significantly reduced. This indicates that a reduced nTreg frequency in cord blood is related to a risk of development of allergic disease and that methylation of TIGIT by itself or togtber with FOXP3 can be used as a diagnostic biomarker for diseases or conditions associated with T-regulatory cell activity. This method can be used inter alia to predicate food allergy development and progression, reveal treatment efficacy and as a platform for drug screening.

[0222] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0223] Many modifications will apparent to those skilled in the art without departing from the scope of the present invention. |{⁾22 ί This appiication claims priority from Australian Provisional Application No. 2013902753 entitled "Diagnostic Methods" filed on 1 July 2013. The entire contents of the application are hereby incorporated by reference.

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TABLE 11

i ProbdD SYMBOL I ENTRE2ID i OES C

j i siai ttransferase 1 i 311 cg008074?5 FLOT1 ] 10211 i ffotiin 1

i 312 cg02835823 NA ί NA i NA

! 313 cg07107916 PPP2R5A I 5525 j protein phosphatase 2, regulatory subunit B', alpha

! 314 cg10296205 SYTL3 94120 i synaptotagmin-iike 3

[ 315 cg23196549 CCDC109B I 55013 j coiled-eoii domain containing Ί09Β

Hie^" ^"cg014W86 ^"e es ^~~ Τ 5Ϊ523 i CXXC finger protein 5

i 317 cg07933926 SC N1A 6337 I sociium channel non-voitage-gated 1 aipha subunit

1 318 cg13440795 MSRA j 4482 1 methionine sulfoxide reductase A

! 319 cg121?1761 NA j NA i NA

i 320 cg21483092 RG812 6002 j regulator of G-protein signaling 12

! 321 cg21015805 NA j NA i NA

j 322 cg23572944 SLC15A3 51296 i solute carrier family 5, member 3

! 323 cgG1343692 NA j NA i NA

j 324 eg 12832726 LRP5 4041 I fow density lipoprotein receptor-related protein 5 i 325 cg07391141 NA j NA i NA

j F-box and WO repeat domain containing 7. E3

326 og05D52463 FBXVV7 j 552S4

i ubiquitin protein iigsse

j 327 eg 12789173 A fCAI j 120425 j adhesion molecule, interacts with CXADR antigen 1 ί 328 cg27338353 NA NA ! NA

i 329 cg011 ^'56249 GRN1 j 23295 j mahogunin ring finger 1, E3 ubtquitin protein !igase

! 330 cg00857S21 TGFBR3 j 7049 I transforming growth factor, beta receptor III

I , cation 331 cg13300580 SLC9A1 6548 j solute carrier family 9, subfamily A (NHE1

i proton antiporter 1), member 1

I 332 cg13725825 NA j NA i NA

j 333 cg02417427 SERINC5 j 256S87 j serine incorporator 5

i 334 og16755833 TBCD I 6904 j tubulin folding cefaclor D

i 335 og080160§2 RT72 j 140807 i keratin 72

j 336 CQ02609337 ST3GAL1 I 6482 j ST3 beta-galactoside aSpha-2,3-siaiyitransfera$e 1 j 337 cg209S0926 NA j NA j NA

j 338 cg175l2187 TRABD2A j 129293 j TraS domain containing 2A

i 339 cg23931Q49 LAMAS j 3909 i iarninin, alpha 3

j leucine rich repeat and fibronectin type Hi domain

! 340 cg25289028 LRFN3 J 79414

j containing 3

j 341 cg27190014 NA NA i NA

j 342 cg07917644 CHSY1 j 22856 i chondroiSin sulfate synthase 1

i 343 cg27iS6013 HPGDS I 27306 j hematopoietic prostaglandin D synthase i 344 ^' cg2S9806S2 SLC15A3 j^" 51296 i solute carrier family 15, member 3

j 345 cg01814191 DUSPS j 1848 j dual specificity phosphatase 6

! 346 cg20496896 LRRC2 79442 i leucine rich repeat containing 2

j 347 cg21936454 AD1L1 j 8379 i MAD1 mitotic arrest deficient-like 1 (yeast)

! 348 cg20937934 LAMAS j 3909 i laminin, aipha 3

j 349 cg27202779 CARD11 I 84433 j caspase recruitment domain family, member 1

I 350 cg03653601 b-famify G (CFTR/ P),

ABCC1 j 4363 I ATP-binding cassette, su

i member 1

[35Γ^" ^"¾ 7ΪΪ54Ϊ9 NA j^" NA ^"JNA

g i¾971 TG?] i^"7050 I TGFB-induced factor homeobox Ϊ

i 353 cgQ168S177 NA j NA i NA

j 354 cgQ6994324 AK4 j 205 i adenylate kinase 4

ProbdD SYMBOL 1 ENTRE2ID i OES C

! 621 eg03724368 NA NA j MA

j 622 cgG078S138 RERE j 473 i arginine-glutarrsfc add dipeptide (RE) repeats i 623 cg16310192 ETV6 j 2120 i efe variant 6

eg2 0O4368 TLQTT Ί02ΤΪ ^~ Γ Yoilri

[ 825 cg02772266 NA j NA i NA

! 626 eg 16635948 SH3RF3 j 344558 i SH3 domain containing ring finger 3

! 627 cg02945192 CNN2 1 1285 j ca!portln 2

i 628 _ ¾23629187 TSS1 j 9788 j_ metastasis suppressor 1

^~cgft4650653 TSPT ^~ j^~4046 i ymphoc te-speciic protein i

i 630 cgQ 46401 NA I NA i NA

! 631 cg170K807 NOP2 j 4839 j NOP2 nucleolar protein omotog (yeast)

I 632 cg2Q178976 NA j NA i NA

i brain and reproductive organ-expressed (TNFRSFIA

633 cg254226?8 BRE I 9577

i modulator}

i 634 cg2747S883 NA j NA i NA

! 635 cg19903805 TC2N I 123036 I tandem C2 domains, nuclear

I i deformed epidermal autoregulatory factor 1 636 cg21156386 DEAF1 j 10522

i (Drosophila)

! 637 cg24401737 CASP10 j 843 i caspase 10. apoptesis-related cysteine peptidase j 638 cg04701181 NA j NA ! NA

I 639 cg08209294 NA j NA i NA

I 640 og04920761 NA j NA I NA

I i UDP-N-acetyi-alpha-D-gaiactosamirie:polypeptid6 N- 641 cg06240690 GALNT5 j 11227

i acetylgalacfesa inyltransferase 5 (GalNAc-T5) j 642 cg01642550 NA j NA ! NA

j 643 cg08645907 ATXN7L1 j 222255 i ataxin 7-lifte 1

j microtubule associated serine/threonine kinase

644 cg05Q88356 AST4 375449

j family member 4

j C-type lectin domain family 2, member D

645 cg032&9764 LOC374443 374443

I pseudogene

i 646 og03467087 SYNJ2 j 8871 i synaptojanin 2

j 647 cg134Q8655 COL15A1 i 1306 i collagen, type XV. aipha 1

j j kiilsr csli immmoglobuiirt-like receptor, three 648 cg03602500 IR3DX1 j 90011

i domains, X1

j 649 eg 14562523 NA I NA i NA

I 650 cg02381279 NA ί NA i NA

j 651 cg15 36975 CAPZB j 832 j capping protein (actin filament) rnusc!e Z-!ine, beta i 652 cg2l 747310 NA NA i NA

j 653 cg17895608 NA I ^m i NA

! 654 cg05337681 UPC j 3990 i lipase, hepatic

j i solute carrier family 12 (potassium/chloride 655 og22772691 SLC12A7 10723

i transporters), member 7

j 656 cg23281123 SPRED2 j 200734 I sproi!ty-related, EVH1 domain containing 2

I 657 cg004591l9 SNX29 j 92017 i sorting nexin 29

i 658 eg 19059056 DG Z j 8525 j diacylgiyceral kinase, zete

[ 659 eg 16704703 NA j NA ! NA

i 660 og09614389 N1NL j 22981 i rtineirHike

j 661 cg00804354 SH3BP5L j 80851 j SH3-binding domain protein 5-like

j j I complement component (3d/Epstein Barr virus) 662 Cg05508558 CR2 1380

j receptor 2

! 663 cg2465)940 ZNF608 j 57507 j zinc finger protein 808

i Probe!D SYMBOL I ENTRE2ID j OESC I

! 1290 eg16341836 STAM8PL1 57559 i STAM binding: profein-ifte 1

j 1291 cg25767433 NA j NA j NA j

I 1292 cg00004667 ZBT817 j 7709 I zinc finger and BTB domain containing 17 j ft 29$ ^"cg15608714 ^~ARMC2 T^"S4Q71 aimadiTlo repeat containing 2_ ^™ j

[l294~ cg0140CI040 RORA j 6095 j RAR-reiated orphan receptor A

! 1295 cg274571S1 PHTF2 j 57157 i putative homeodornain transcription factor 2 j

! 1296 cg24 14325 I ZF4 I 64375 I JKAROS family zinc finger 4 (Eos) j i 1297 cg16S22174 AEBP2 j 12 536 i AE binding protein 2 j

ΓΪ29 ^"¾2¾02δ46 ^"PRD S^" ^~~ I ^"9588 i peroxifedoxin 1 i 1299 cg213S7735 RPS6 A2 I 6196 i ribosomal protein S6 kinase, 90kDa, polypeptide¹ 2

! 1300 cg03128029 NOP58 j 51602 j NOP58 ribonucleoprotein homo log (yeast) j j 1301 cg1Q550882 NA j NA j NA

j 1302 cg18Q52528 TNi j 23043 j TRAF2 and NC interacting kinase j j i guanine nucleotide binding protein (G protein), 1303 cg24723883 GNG7 j 2788 j

j gamma 7 j

! 1 04 cg03464224 IAA0922 I 23240 1 iAA0S22 j j 1305 cg27209072 NA j NA ! NA

i 1306 cg15774510 NA j NA NA

1 1307 cg2S427498 NA j NA I NA

! 1308 cg23175023 NA I NA 1 ^A

! 1309 cg0519 839 NA j NA ! ^m 1 j 1310 og11213983 CEP112 201134 1 centro$omal protein 112RDa j i 1311 cg0801Q865 SPATA13 j 221178 I spermatogenesis associated 13

j 1312 cg2492122i LONRF1 91694 I ION peptidase N-terminai domain and ring finger 1 j i 1313 cg22014112 TNFAIP3 j 7128 I tumor necrosis factor, alpha- induced protein 3 j i 1314 cg18146927 NA I NA i NA j i 1315 cg14003416 NA NA i NA

j 1316 og09717585 ZCCHC14 j 23174 i zinc finger, GCHC domain containing 14 ]

ΓΪ3ίΓ cg07751669 C9orf84 j 15840 j chromosome 9 open reading frame 84 1

I 1318 og03812676 AIP 9049 j aryf hydrocarbon receptor interacting protein i 1319 cg19215266 CREM j 1390 i cAMP responsive element modulator j j 1320 cg06401414 E1F2C2 I 27161 i eukaryotic translation initiation factor 2C, 2 i 1321 cg07175582 LYPLAL1 j Ϊ270Ϊ8 i iysophosp o!ipase-irke Ϊ

i 1352 cg06520331 NA NA I NA

i 1323 cg11465404 NA I N.A i NA

i Dnai (Hsp40) homolog, subfamiiy C, member 5

1324 cg07197831 DNAJC5G I 285126

j gamma

j 1325 cg_.06861115 L36G I 56300 i inferteuKiri 36, gamma

! 1326 cg24417845 NA j NA j NA

j ksirin homology domain containing, family G 1327 cg07533239 PLE HG6 j i plec

55200

i {with RhoGef domain) member 6

1 1328 eg 19628600 FOXP1 j 27086 j forkhead box P

j 1329 cg10668121 ARHGAP25 j 9938 i Rho GTPase activating protein 25

j 330 cg17445097 PHF12 } 57649 i PHD finger protein 12

1 1331 cg06501716 C22orf33 I 128977 j chromosome 22 open reading frame 39 j j 1332 cg0274¾35S A RD11 j 29123 i ankyrin repeat domain 11 ! holocarboxylase synthetase ( biotin-(proprionyl-CoA-

I 333 cg21871091 HLCS j I

3141

I carboxylase (ATP-hydrofyaing)) ligase) i 1334 cg04450052 NA ^~~j^~NA ^"TNA

i 1335 cg18025409 TNF SFS [ 360 { tumor necrosis factor receptor superfamily, member j

20!

21)

i ProbdD SYMBOL I ENTRE2ID i OES C

i glutamate receptor, tonotropic, delta 2 (Grid2)

2277 cg07281318 GRID2IP J 392862 I interacting protein

! 2278 cgl 7245726 MA I NA ί NA

! 2279 cg10483055 TSPAN5 I 10098 jjetraspartin 5

1 22SQ cg05582310 EVC [ 2121 j Ellis van Creveld syndrome

i 2281 cg13694466 SEPHS1 22929 j se!enophosphate synthetase 1

j 2282 cg15057323 KIF2SB j 55083 i klrsesin family member 268

j 2283 cg20737500 CR P1 j 1400 I collapsin response mediator protein 1 i 2284 cg19479086 RNF145 j 153830 j ring finger protein 145

I 2285 cg02376282 MOB2 j 81532 j MOB kinase activator 2

I 2286 cgl4Di 1S?1 AN 3 j 288 j ankyrin 3, node of anvier {ankyrin G) i 2287 eg10547781 C20orf196 i 149840 i chromosome 20 open reading frame 196

! 2288 cg27264018 CACUL1 143384 i CDK2-a$soctated. cullin domain 1

I 2289 cg07019638 EHD1 10938 i EH -domain containing 1

I 2290 cgQ5966431 CAM I A1 j 23281 i calmoduli binding transcription activator 1

! 2291 cg19979108 SH3PXD2B I 285590 j SH3 and PX domains 2B

j 2292 Gg00479463 NA j NA ! NA

i 2293 cg03319315 PAOX j 198743 j poiyamine oxidase (exo-l*i4-amino)

I 2294 eg 18247179 C22orf39 j 128977 I chromosome 22 open reading frame 39

! 2295 cg08293636 NA 1 NA i NA

! 2296 cg13138S54 NA j NA I NA

I 2297 cgO8365609 NA NA NA

2298 cg04462662 ZDHHC7 j _5§625 I zinc finger, DHHC-type containing 7

I -1,3-N^«

2299 cg17253709 B3GNT2 10878 j UDP-GlcNAc:betaGal beta

I acetylglucosaminyitransferase 2

j 2300 cg00808S45 MKL2 I 57496 i MKL'myocardin-lifce 2

j 2301 cg07547765 HGXB7 3217 j homeobo B7

! 2302 cg223213i8 NA j NA i NA

j 2303 cg03788239 C3orf19 j 51244 i chromosome 3 open reading frame 9 i 2304 cg22482700 WLS j 79971 ! vmtless homolog (DrosophJa)

j 2305 cg25771113 NA NA j NA

i leucine rich repeat and fibronectin type III domain

2306 cg26910511 LRF 1 57622

j containing 1

Γ2 0 ^" gO¾7W ~C3orm I 51066 j chromosome 3 open reading frame 32 j 2306 cg05208056 NC 2 i NCK adaptor protein 2

[ 2309 cg2?961852 RHEB j 6009 j Ras homolog enriched in brain

j 2310 cg10400081 NA NA ί NA

j 2311 eg 16264807 MTWR4 J 9110 i myotubu!arin related protein 4

! 2312 cg20728419 USP47 j 55031 i libiquitin specific peptidase 47

j 2313 cg18026626 LOC91450 j §1450 j uncharacteiized LOC91450

j 2314 cg15464148 LPAR5 j 57121 i iysophosphalidic acid receptor 5

j microtubule-associated protein, RP/EB family,

2315 cg1180Q794 MAPRE1 j 22919

i member 1 TABLE 12

Primers for bisulfite sequencing and qPCR

TABLE 13

1 nTreg F0XP3 Gene Description Chr, Start Bid

Meth. Binding human immunodeficiency virus

HIVEP3 chrl 42384310 42384564 Hyper C iP-chip

[ type i enhancer binding protein 3

j tumor necrosis factor receptor

TNFRSF9 chrl 8001027 8001309 Hypo

1 s perfamsiy, member 9

f ΑΪΜ2 j absent in melanoma 2 ctir1 159046773 j 159047163 Hypo j ChiP-chip

TABLE 14

CD

(Z

on

CD m

cn

I

m

73

CZ

—

m

σ>

73

o

:

(Z

(Z

on

CD m

cn

1

m

73

(Z

—

m

r

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

—

m

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

—

m

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m

m 4

73

(Z

I- m

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

—

m

σ> TABLE 15

73

O

:

(Z

u i

< 8

(Z

on

CD m

cn

I

m

73

(Z

I- m

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m t m

73

(Z

I- m

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

I- m

σ>

73

O

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

—

m

σ>

73 O

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

—

m

r

σ>

73

o

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

I

m

ro

σ>

73

o

:

(Z

(Z

on

CD m

cn

I

m

73

(Z

—

m

σ>

73

O

:

(Z

CD

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on

CD m

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I

m

73

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I

m

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73

o

:

(Z

4

BIBLIOGRAPHY

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Aryee JvO et al (2011) Biosiatisfics 12:197-210

Bailey TL et al (1994) P c Int Confhitell Syst Mol Biol ;2:28-36

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Claims

CLAIMS:

1. A method for identifying the level of T-regulatory cell activity in a test biological sample comprising immune cells, the method comprising screening a test sample for the test methylation profiles of a pre-selected genetic locus/loci selected from the list consisting of:

(i) at least one genetic locus or at least two genetic loci defined i one or more of Table 1 to Table 5;

(ii) at least one genetic locus or at least two genetic loci defined in one or more of Table 6 to Table 0: and

(iii) at least two genetic loci defined i one or more of Table I to Table 5, and one or more of Table 6 t Table 10; and determining the degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the le vel of T-regulatory cell activity in the sample.

2. The method of claim 1 , wherein (i) comprises at least one genetic locus or at least two genetic loci defined in one or more of Table 1 and Table 2.

3. The method of claim 1 or 2, wherein (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

4. The method of any one of claims 1 to 3, wherein at least 4 to at least 10 or at least 10 to at least 100 preselected genetic loci are screened,

5. The method of any one of claims 1 to 4, wherein the sample is derived from a human subject,

6. The method of any one of claim 1 to 5, wherein the test sample is enriched for C.D4+ T cells.

7. The method of any one of claim 1 to 6, wherein the methylation profile of the reference sample is derived from naive CD45RA+ CD45 Q- CD25- T ceils,

8. The method of any one of claim 1 to 6, wherein the methylation profile of the reference sam e is derived from CD45RA+ CD45RO- CD25+ T-regalatory cells.

9. The method of any one of claim 1 to 6, wherein the methylation profile of the reference sample is derived from CD45RA+ CD45RO- CD25^high T-regulatory cells.

10. The method of any one of claim .1 to 9, wherein at least 40%, or at least 50%, or at least 60%, or at least 70% or at least 80% or at least 90% of individual nuclear genomes present in the test sample are screened in order to determine the number, proportion or activity of T-regulatory cells in the test sample.

11. A method for determining whether or not a subject has or is at risk of developing an autoimmune condition or of developing a type 1 allergic condition, the method comprising screening a test sample comprising immune cells from a subject for the test methylation profiles of a pre-seleeted genetic region selected from one of the following groups:

(i) at least one genetic loc us defined in one or more of Table 1 to Table 5;

(ii) at least one genetic locus defined in one or more of Tab le 6 to Tabl e 10; and

(iii) at least two genetic loci defined in one or more of Table 1 to Table 5 and one or more of Table 6 to Table 10; and determining the degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample.

12. A method of treatment or prophylaxis of a subject, the method comprising screening a test biological sample comprising immune cells for the test methylation profiles of a preselected genetic region selected from the list consisting of:

(i) at least one genetic locus or at least two genetic loci defined in one or more of Table 1 to Table 5;

(ii) at least one genetic locus or at least two genetic loci defined in one or more of Table 6 to Table 1 ; and

(iii) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; determining tire degree of concordance between the test methylation profiles and a reference methylation profile, wherein the degree of concordance identifies the level of T-regulatory cell activity in the sample, and then providing therapeutic and/or behavioural modification of the subject.

13. A use of a panel or array of oligonucleotides specific to a pre-selected genetic region selected from the list consisting of:

(i) at least one genetic locus or at least two genetic loci defined in one or more of Table I to Table 5;

(ii) at least one genetic locus or at least two genetic loci defined in one or more of Table 6 to Table 10; and

(in) at least two genetic loci defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 1.0;

in the manufacture of a kit or solid support for identifying the level of T-regulatory cell activity in a biological sample, tissue or subject, or the presence, risk, state, classification or progression of immune system dysregulatkm such as found in an autoimmune or type 1 allergic condition i a subject.

14. A method of screening for an agent which modulates immune cell function, said method comprising screening an immune cell for a degree of concordance compared to a reference sample in the methylation profile of a pre-selected genetic region selected from the list consisting of:

(iii) at least two genetic loci that are defined in one or more of Table 1 to Table 5, and one or more of Table 6 to Table 10; and optionally

(iv) at least one genetic locus/loci defined in Table .15 representing differentially methylated loci after activation of naive CD4+ T-cells; in the presence or absence of an agent to be tested wherein tire agent is selected if it induces a change in the degree of concordance between the methyiation profile of the immune cell and the reference sample methyiation profile,

15. The method of any one of claim 11 to 14, wherein (i) comprises at least one genetic locus or at least two genetic loci de ined in one or more of Table 1 and Table 2.

16. The method of any one of claim 1 1 to 15, wherein (ii) comprises at least two genetic loci defined in one or more of Tables 6 and Table 7.

17. The method of any one of claims 1 1 to 16. wherein at least 4 to at least 10 or at least 10 to at least 100 preselected genetic loci are screened.

18. The method of any one of claims 1 1 to 17, wherein the subject is a human subject.

19. The method of any one of claim I I to 18, wherein the test sample is enriched for CD4+ T cells.

20. The method of any one of claim 1 1 to 19, wherein the methyiation profile of the reference sample is derived from naive CD45 A+ CD45RO- CD25- T ceils,

21. The method of any one of claim 11 to 19, wherein the methyiation profile of the reference sample is derived from CD45RA+ CD45RO- CD25+ T-regu!atory cells.

22. The method of any one of claim 11 to 19, wherein the methyiation profile of the reference sample is derived from CD45RA+ CD45RO- CD25^higb T-regulatory cells.

23. The method of any one of claims 1 to 22, wherein tire at least one locus is or includes a TIGIT locus.

24. The method of any one of claims 1 to 22, wherein the at least one locus is or includes a MIR21 locus.