Therapy for Multiple Sclerosis

Related Applications

This application claims benefit from Australian provisional patent application No. 2007906261 entitled "Therapy for Multiple Sclerosis" filed 15 November 2007, the entire contents of which is incorporated herein by reference. Technical Field

The present invention relates to methods for the treatment and/or inhibition of relapses in multiple sclerosis, and to methods of generating autologous T reg cells for use in such methods of treatment. Background of the Invention

Multiple sclerosis (MS) is a devastating neurodegenerative disease that affects approximately 1,100,000 people worldwide, particularly young adults. It is the most common demyelinating disease of the central nervous system, resulting in sclerotic plaques and axonal damage, and yet its etiology remains unknown.

One of the reasons underlying the lack of progress in thoroughly characterizing and therefore treating MS is the marked variability and unpredictability in clinical progression. Neurological signs associated with MS encompass a wide array of symptoms including limb weakness, compromised motor and cognitive function, sensory impairment, bladder disorders, sexual dysfunction, fatigue, ataxia, deafness and dementia.

Despite such variation in symptoms, the progression of several clinical courses has been classified. The majority of patients with MS follow a relapsing-remitting course in the early stages of the disease, characterised by increased severity of existing symptoms and the appearance of new symptoms, followed by variable periods of total or partial recovery. Relapsing-remitting MS (RRMS) may be inactive for several years between distinct attacks. However, most patients with RRMS ultimately enter a secondary chronic progressive phase, characterised by progressive disability and classified as secondary progressive MS (SPMS). This disease state may also involve relapses, thereby known as relapsing progressive MS (RPMS). While both SPMS and RPMS are pre-empted by RRMS, a further distinct classification of the disease involves a gradual worsening of symptom severity over time without initial intermittent relapses. This form of MS, known as primary chronic

progressive MS, is variously referred to as either chronic progressive MS (CPMS) or primary progressive MS (PPMS). This form of the disease affects about 10% of patients. Such diversity in MS progression is thought to be due at least in part to the wide array of risk factors that are suspected to cause the disease. These include genetic, immunologic and environmental factors such as infectious viruses and bacteria.

An understanding of MS etiology has been sought through the identification of genes that are differentially expressed in MS patients when compared with healthy individuals, hi this regard, gene microarrays have been used to compare post-mortem transcription from MS plaque types (acute verses chronic) and plaque regions (active verses inactive). Microarrays have also been used to examine peripheral blood mononucleocytes in RRMS patients verses controls, from patients both with and without interferon-β treatment, and from CNS cells in stages of experimental allergic encephalomyelitis (EAE) in mice, an animal model of MS (Lock et al. 2002 and 2003). This work has produced a number of expected results, including the finding that pro- inflammatory, proliferation genes are up-regulated and anti-inflammatory, anti-apoptotic genes are down-regulated. Such studies have also indicated potential novel targets for therapeutic application such as osteopontin. However, many genes that have been identified as differentially regulated in MS patients compared with healthy individuals remain of unknown significance in MS development. As yet, these microarray studies, together with genome wide analyses, have only identified CD 127 as affecting MS susceptibility and/or progression in a variety of populations of MS subjects.

Linkage and association studies have been able to identify several factors which are associated with risk of MS. In relation to genetic factors, it has been demonstrated that identical twins have a 30% chance of developing MS if one twin is affected, with fraternal twins and siblings and children of probands having a 1-2% chance; this compares with a prevalence of MS in the normal population of about 0.1%. The genes responsible for this heritability have been sought by linkage and association studies, and through candidate gene analysis. The MHC Class II DRB 1501 allele confers a 3-4 fold relative risk in most populations, and other associated genes have been identified with a much lower risk, but the full genetic basis for MS remains unexplained, despite extensive genomic screens.

Recent studies have confirmed that the Interleukin 7 Receptor α chain (IL7Rα) has allelic and functional association with multiple sclerosis (Booth et al, 2005; Gregory et al,

2007 and McKay et al., 2007). These studies identified four common haplotypes of the IL 7 R gene which were found to be associated with susceptibility to MS. Of these, one haplotype (Haplotype 2) was considered to be under-transmitted in MS subjects and accordingly may be protective, while other haplotypes (Haplotypes 1, 3 and 4) were associated with increased risk. These haplotypes could be differentiated by the expression of an allele at rs6897932, with the expression of a "C" allele demonstrating a twofold increase in the expression of a soluble, non-functional form of IL7R by peripheral blood mononuclear cells (presumably with a concomitant decrease in the expression of functional membrane-bound IL7R) and which was associated with an increased risk for MS when compared to the expression of the "T" allele. McKay et al. 2007 considered that this hypothesis was consistent with their previous observations that IL7Rα expression in blood cells was reduced in individuals with primary progressive multiple sclerosis.

The potential involvement of the IL7R gene in the development of MS is consistent with the role of the gene product in two different receptors, one for IL7 and one for thymic stromal lymphopoietin (TSLP) signalling pathways. Gregory et al., (supra) for instance, speculated that because of a possible decrease in expression of IL7R on the surface of T cells which results from the expression of soluble IL7Rα, T cells may be less responsive to IL7 ligand. Booth et al. 2005 (supra) suggested that lower expression of IL7R on the surface of T reg cells may reduce T reg survival when IL7 ligand is limiting. Summary of the Invention

The present inventors have now found that dendritic, cells from subjects expressing an "MS susceptibility" CD 127 haplotype produce a much higher proportion of soluble IL7Rα than multiple types of T cells. This identification suggests a greater role for dendritic cells in the causation of MS than previously identified, and allows for alternative approaches for therapeutic intervention of MS which may be applicable not only in circumstances in which the subject expresses a MS susceptibility CDl 27 haplotype, but also in MS arising from other genetic or environmental causes.

Accordingly, in a first aspect there is provided a method for ameliorating multiple sclerosis in a subject, comprising generating autologous T reg cells ex vivo by incubating autologous cells of T cell lineage with TSLP-stimulated autologous cells of dendritic cell lineage and administering the autologous T reg cells to the subject.

In certain embodiments, the autologous cells of T cell lineage are autologous thymocytes, autologous circulating naϊve T cells or autologous T reg progenitor cells.

In certain embodiments the cells of dendritic cell lineage are thymic dendritic cells or TSLP-stimulated autologous peripheral dendritic cells.

5 In one embodiment, the T reg cells are generated by incubating autologous T cell lineage thymocytes with autologous TSLP-stimulated thymic dendritic cells.

In one embodiment, the T reg cells are CD4 ⁺ CD25 ⁺ Foxp3 ⁺ T reg cells. In another embodiment, the T reg cells are CD4 ⁺ CD127 ^low CD25 ^high Foxp3 ⁺ T reg cells.

In one embodiment, the method of ameliorating multiple sclerosis comprises I ₀ delaying relapse or reducing the severity of a relapse of multiple sclerosis in a subject.

In certain embodiments the subject with MS is not heterozygous or homozygous for CD 127 Haplotype 2. In certain embodiments the subject with MS is heterozygous or homozygous for CD 127 Haplotype 1 or 4.

In one embodiment the dendritic cells are exposed to any one or more autoantigen is selected from the group consisting of myelin basic protein, myelin associated glycoprotein, myelin oligodendrocyte glyprotein, proteolipid protein and alpha-beta- crystallin. The autoantigen may be exposed to the dendritic cells prior to TSLP stimulation and/or during TSLP stimulation and/or following TSLP stimulation of the dendritic cells.

₂₀ Also provided is a method of generating T reg cells from a subject suffering from multiple sclerosis, the method comprising incubating ex vivo autologous T cells from the subject with TSLP-stimulated cells of dendritic cell lineage.

In certain embodiments, the T reg cells are autoantigen-specific T reg cells. In certain embodiments, the cells of dendritic cell lineage are autologous thymic dendritic 2 ₅ cells or TSLP-stimulated autologous peripheral dendritic cells.

Also provided is the use of TSLP, and optionally an autoantigen, for the ex vivo generation of T reg cells for alleviating multiple sclerosis, wherein the autoantigen is selected from the group consisting of any one or more of myelin basic protein, myelin associated glycoprotein, myelin oligodendrocyte glycoprotein, proteolipid protein and 3Q alpha-beta-crystallin. Abbreviations

CNS central nervous system

CPMS chronic progressive MS

DCs Dendritic cells

EAE experimental allergic encephalomyelitis

Foxp3 Forkhead box P3 iDCs inflammatory dendritic cells IDO indolamine-2,3-dioygenase

IL7 Interleukin 7

IL7R Interleukin 7 receptor gene, which encodes IL7Rα polypeptide

IL7R Interleukin 7 receptor polypeptide (a dimer of CD 127 and cytokine receptor common γ polypeptide) IL7Rα Interleukin 7 receptor α chain polypeptide (also known as CD 127)

LPS lipopolysaccharide

MAG Myelin-associated glycoprotein

MBP Myelin Basic Protein

MOG Myelin oligodendrocyte glycoprotein MS Multiple Sclerosis

PBMC peripheral blood mononuclear cells

PLP Proteolipid protein

PPMS primary progressive MS qRT-PCR quantitative real time polymerase chain reaction RRMS Relapsing-remitting MS

T reg Regulatory T cells

SNP single nucleotide polymorphism

SPMS secondary progressive MS

TSLP Thymic stromal lymphopoietin TSLPR Thymic stromal lymphopoietin receptor (a heterodimer of CD 127 and cytokine receptor-like factor 2)

Definitions

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

It should be noted that, as used in the present specification, the singular forms "a",

"an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus,

for example, reference to "an autoantigen" includes a single autoantigen, as well as two or more autoantigens and so forth.

As used herein the term "ameliorating multiple sclerosis" refers to any one or more of preventing, delaying, slowing or reversing the progression of the pathology and/or one or more symptoms of multiple sclerosis or preventing or delaying the establishment of multiple sclerosis in a human or non-human animal subject. Thus, in certain embodiments ameliorating multiple sclerosis is intended to encompass interfering with the progression of multiple sclerosis in a subject diagnosed or suspected as having multiple sclerosis. In certain embodiments ameliorating multiple sclerosis is intended to encompass preventing, interfering with or delaying the relapse of multiple sclerosis in a subject who has previously suffered from multiple sclerosis but who is currently in remission.

"Multiple sclerosis" is intended to encompass conditions which fall within the recognised diagnostic criteria described in Table 3 of McDonald et al., (2001), the entire contents of which is incorporated herein by reference. Briefly, these diagnostic criteria rely upon one or a combination of clinical identification of at least one MS episode, and where there are multiple episodes their dissemination over time, and the pathophysiological identification of at least one causative lesion, and where there are multiple lesions their dissemination over space. Also amongst the range of clinical conditions intended to be encompassed by this definition are any one or more of relapse remitting multiple sclerosis, secondary progressive multiple sclerosis, relapsing progressive multiple sclerosis, chronic progressive multiple sclerosis and primary progressive multiple sclerosis.

In particular embodiments, the term "multiple sclerosis" is intended to exclude subjects with CD127 ^low multiple sclerosis. A subject exhibiting CD127 ^low multiple sclerosis may be diagnosed with multiple sclerosis according to the criteria of McDonald et al., (200\)(supra). Subjects with "CD127 ^low MS" represent a small sub-population of all MS subjects, in which there is an overall decrease in CD 127 mRNA expression in whole blood cells when compared to the level of expression in a normal population. The expression of CD 127 mRNA in whole blood of an individual may be determined using quantitative real-time PCR as described in Booth et al., 2005. The level of expression of CD 127 mRNA in whole blood of a normal population may be determined by determining

the average level of expression of CD 127 in whole blood from at least 20 subjects with no clinical signs of MS (McKay et al, 2007 (supra))..

As used herein the term "CD127 ^low " in respect of a cell refers to the under- expression of CD 127 (including the underexpression of the combination of both soluble and membrane bound iso forms of CD 127), where such under-expression is relative to any one of (i) a larger cellular population in which the cell is located, for example where a particular cell population exhibits a consistently lesser level of CDl 27 than other cells present in the cell population when examined for example, by flow cytometry techniques, or (ii) a basal level of measured expression of CD 127 expression on cells of the same type within the general population, or in a control sample of non-MS sufferers. Techniques for determining the level of expression of membrane bound and/or soluble CD 127 are known in the art. For example, flow cytometry techniques utilising an antibody to CD 127 (Immunotech) for assessing the level of CD 127 expression on peripheral blood, buffy coat cells, thymus or lymph node mononuclear cells are described in Seddiki et al. (2006), the entire contents of which is incorporated herein by reference. Levels of soluble CD 127 may be readily determined using quantitative immunoaffinity techniques, such as ELISA.

As used herein the term "CD 127" refers to the polypeptide CD 127, otherwise known as IL7Rα-chain, or its precursors or derivatives thereof and, unless the context identifies otherwise, includes both soluble and membrane-bound isoforms of IL7Rα- chain.

As used herein the term "CD132" refers to the polypeptide CD132, otherwise known as the common γ-chain.

As used herein the terms "IL7R" and "IL7 receptor" refer to the IL7 receptor heterodimeric protein complex, comprising CD 127 (otherwise known as the IL7Rα- chain) and CD 132 (otherwise known as the common γ-chain).

As used herein the term "soluble" as it pertains to a IL7R or TSLPR receptor refers to a receptor or part thereof which comprises an isoform of CD 127 which is not membrane bound and which is therefore unable to initiate signal transduction as a result of ligand binding. Typically, but not exclusively, a soluble form of IL7R will retain the ability to bind the ligand IL7. Typically, but not exclusively, a soluble form of TSLPR will retain the ability to bind the ligand TSLP.

As used herein the term "TSLP" refers to thymic stromal lymphopoietin. Also encompassed within the scope of the invention are homologues or mimetics of TSLP which possess qualitative biological activity in common with the full-length mature activated TSLP, such as the ability to bind and activate the TSLPR. The amino acid sequence of mature TSLP is presented herein as SEQ ID NO:6.

As used herein the terms "TSLPR" and "TSLP receptor" refer to the TSLP receptor heterodimeric protein complex, which comprises a IL7Rα-chain and the TSLPR chain.

As used herein the term "T cell lineage cell" refers to a cell which expresses the T cell receptor or which is capable of differentiating into a cell which expresses the T cell receptor. As used herein, a "T cell lineage cell" is capable of differentiation into a T reg cell. Non-limiting examples of T cell lineage cells include thymocytes, T reg progenitor cells and naive T cells. Brief Description of the Figures and Sequence Listing

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures.

Figure 1. Schematic presentation of the CD127 gene (IL7R) showing SNP composition of the haplotypes described herein. The haplotypes are tagged by the first three 5' untranslated region SNPs. The haplotypes illustrated from top to bottom were designated numbers 1 to 4. The haplotype 2 (shaded) has been internationally validated as being protective for MS in general. Light grey boxes indicate the position of exons, with the exon number provided below each exon. The -504 and exon 8 SNPs both tag the haplotype 4, the -449 and exon 2 SNPs both tag haplotype 1. From 5' to 3' the SNPs are: rs7718919, rsl 1567685, rsl 1567686, rsl494558, rsl494555, rs6897932, and rs3194051.

Figure 2. Bar chart illustrating the ratio of full length and soluble CD 127 isoforms expressed in whole blood by MS or control individuals expressing different haplotypes associated with multiple sclerosis. A "carrier" is heterozygous or homozygous for the particular haplotype identified by the characteristic SNPs which are listed beneath each column. A "non-carrier" does not express the particular haplotype listed below the column. Figure 3. (A) Bar chart illustrating the ratio of expression of CD 127 mRNA (all isoforms) by haplotypes 2 and 1 in peripheral blood mononuclear cells (PBMC), T cells and inflammatory dendritic cells (iDCs) from heterozygous Hap2/Hap 1 individuals as determined by an allele extension assay. Similar results were obtained for other

haplotype comparisons (see Table 1).

(B) Bar chart illustrating the ratio of expression of full-length (FL) to soluble (S) CD127 mRNA transcripts in LPS-activated monocyte-derived dendritic cells from homozygous individuals, as determined by PCR followed by gel separation of PCR fragments using an Agilent bioanalyser. The ratio of full length to soluble isoform transcripts is reduced in individuals homozygous for a MS susceptibility CD 127 haplotype (Hapl) when compared to the protective haplotype (Hap 2).

Figure 4. (A) Bar chart illustrating the level of expression of soluble CD 127 polypeptide by cultured dendritic cells at rest and following LPS activation from individuals homozygous for haplotype 1 or 2, as measured by ELISA. Dendritic cells from an individual with the MS-susceptibility haplotype (Hap 1) produced proportionally more soluble CD 127 following activation when compared to dendritic cells from an individual having the MS protective haplotype (Hap 2).

(B) Dot plot illustrating levels of soluble CDl 27 (sCD127) polypeptide measured by ELISA from the serum of individuals who were either homozygous or heterozygous for CDl 27 haplotype 2 (right hand column) or who did not express CD 127 haplotype 2 (left hand column). This graph, in combination with the results with the results presented in Figures 2 and 3, demonstrates that individuals expressing CD 127 haplotype 2 not only produce less of the mRNA isoform encoding soluble CD 127 but also produce less sCD127 polypeptide in vivo.

Figure 5. Bar chart illustrating the responsiveness of dendritic cells to an administration of TSLP as measured by the expression of Indoleamine-2,3-dioxygenase (IDO), determined by qRT-PCR. IDO is a marker of TSLP signaling and may be used to measure the ability of dendritic cells to respond to this cytokine and so increase the generation of T reg cells. The responsiveness of dendritic cells from homozygous individuals homozygous for CD 127 Haplotype 1 was considerably reduced when compared to dendritic cells from an individual homozygous for Haplotype 2. Dendritic cells from an individual expressing Haplotype 4 were intermediate in their responses to TSLP. Figure 6. Relative expression of cDNA encoding full length (thick arrow) and soluble (thin arrow) isoforms in inflammatory dendritic cells from individuals homozygous for a sensitivity haplotype, Haplotype 1 (top), heterozygous for Haplotypes 1 and 2 (middle) and homozygous for a protective haplotype, Haplotype 2 (bottom).

cDNA was measured by PCR amplification and capillary gel electrophoresis. Different plots in each row represent the results from different individuals.

The amino acid sequence set forth in SEQ ID NO:1 is the amino acid sequence of human IL-7. The nucleotide sequence set forth in SEQ ID NO:2 is the polynucleotide sequence of the cDNA encoding human IL-7.

The nucleotide sequence set forth in SEQ ID NO:3 is the polynucleotide sequence of the cDNA encoding human CD 127.

The nucleotide sequence set forth in SEQ ID NO:4 is the polynucleotide sequence of the cDN A encoding human CD 132.

The nucleotide sequence set forth in SEQ ID NO:5 is the polynucleotide sequence of the cDNA encoding human TSLP receptor chain.

The amino acid sequence set forth in SEQ ID NO: 6 is the amino acid sequence of human TSLP. The nucleotide sequence set forth in SEQ ID NO:7 is the polynucleotide sequence of the cDNA encoding human TSLP.

The amino acid sequence set forth in SEQ ID NO: 8 is the amino acid sequence of the soluble isoform of human CD 127.

The nucleotide sequences set forth in SEQ ID NOS:9 and 10 are polynucleotide sequences of primers for CD 127 amplification.

The nucleotide sequences set forth in SEQ ID NOS: 11 and 12 are polynucleotide sequences of primers for CD 127 amplification.

The nucleotide sequences set forth in SEQ ID NOS: 13, 14 and 15 are polynucleotide sequences of primers for CD 127 amplification. Detailed Description

The TSLP receptor complex is a heterodimeric protein composed of a TSLPR chain and CD 127. The dimer complex provides a high affinity receptor for TSLP which is able to activate the intracellular transcription factor STAT5. This heterodimeric complex is expressed primarily but not exclusively on monocytes and myeloid-derived dendritic cells, and is thought to play a role in allergy and inflammation. The ligand for the TSLP receptor is TSLP, a haematopoietic protein that is expressed in the stroma of the thymus, the heart, liver and prostate. TSLP, similarly to IL7, induces phosphorylation of STAT3 and STAT5 upon binding to its receptor, but unlike IL7 and its receptor uses kinases other

than the JAKs for activation.

Although TSLP is known to influence lymphocyte development, TSLP ligand knockout mice appear to display normal T cell and B cell development. Evidence from TSLPR knockout animals suggests that TSLP may be involved in the survival and development of CD4 ⁺ T cells in mice. TSLP signaling is required for T reg cell development in mice. Recent studies in humans have demonstrated that TSLP expressed in Hassall's corpuscles in the thymic medulla activates thymic dendritic cells to positively select high affinity T reg cells in the thymus.

The unequivocal genetic association of haplotype 2 of CDl 27 with MS demonstrates that MS pathogenesis is affected by this gene. The present inventors have now identified that dendritic cells from individuals with certain CD 127 haplotypes associated with multiple sclerosis (Haplotypes 1 and 4) express an increased level of soluble CD127 isoforms, relative to a known protective haplotype (Haplotype 2). The inventors have demonstrated that a consequence of this is that CD 127 haplotype 2 individuals have an increased sensitivity to TSLP. The differential expression of the soluble, non-signalling form of CD 127 in MS sensitive haplotypes correlates with a decreased ability of the dendritic cells to respond to TSLP. Without wishing to be bound by any proposed mechanism, it is proposed that the increased level of expression of soluble CD 127 isoforms leads to a decreased expression of functional, membrane bound CD 127 on the surface of dendritic cells, and thus a decrease in signaling-capable TSLPR complex as well as increased competition with the membrane bound receptor for the ligand, and that this decrease interferes with the ability of dendritic cells in the thymus to generate self-reactive T reg cells. Thus, individuals expressing the haplotypes associated with an increased risk of MS appear to have a decreased ability to generate the auto- antigen specific T reg cells which are needed to prevent the formation of and progression of MS.

The inventors have proposed that the microenvironment in which the differential expression of soluble and membrane bound forms was maximal is the one most likely to be important in pathogenesis. By testing many T cell microenvironments and comparing with activated DCs (Table 1) the inventors have identified that the variation in sensitivity to ligand due to CD 127 haplotype variation is greatest in DCs (Fig. 6). DCs respond to TSLP, but not to IL7. This points to different sensitivity to TSLP signaling as the basis for the protection or sensitivity conferred by haplotypes in MS. Because the role of TSLP

in T reg development could affect MS pathogenesis, and the demonstrated potential of T reg cells in the amelioration of autoimmune diseases in animal models, it is concluded that T reg therapy based on TSLP treatment of leukocytes is a suitable therapy for MS. The genetic association of IL7R (CD 127) with MS indicates it affects MS pathogenesis, and it is suggested that the deficiency in T reg cells is the basis of this pathogenic effect. This T reg cell deficiency may also be reached through other genetic and environmental factors, so it is proposed that the addition of regulatory T cells will ameliorate the disease in those with susceptibility haplotypes, as well as those who have protective CD 127, but insufficient control of self-reactive T cells through these other genetic and environmental factors. By obtaining a population of autologous T cell lineage cells, such as thymocytes or naϊve T cells, and a population of dendritic cells from a subject and combining them ex vivo in the presence of supplemented TSLP, it is possible to overcome the deficit of TSLPR on the surface of the dendritic cells by providing excess TSLP, and thus generate the expansion and differentiation of T reg cells. The autologous T reg cells thus generated may then be administered to the subject in order to normalise autoimmune responses in the subject, and thereby ameliorate multiple sclerosis in the subject.

Thus, in a first aspect there is provided a method for ameliorating multiple sclerosis in a subject, comprising generating autologous T reg cells ex vivo by incubating autologous cells of T cell lineage, such as autologous thymocytes or autologous circulating naϊve T cells, with TSLP-stimulated autologous dendritic cell lineage cells, such as TSLP-stimulated thymic dendritic cells or TSLP-stimulated autologous peripheral dendritic cells, and administering the autologous T reg cells to the subject.

The autologous cells of T cell lineage may be CD4 ⁺ CD8 ^" CD25 ^" thymocytes. The autologous cells of T cell lineage may be T helper cell/T reg cell progenitors, which are known to be able to differentiate into T reg cells under appropriate stimuli. The autologous cells of T cell lineage may be naϊve, circulating T cells which are able to differentiate into T reg cells. In a particular embodiment, the T reg cells are generated by incubating autologous T cell lineage thymocytes with TSLP-stimulated thymic dendritic cells.

The T reg cells which are generated may be CD4 ⁺ CD25 ⁺ Foxp3 ⁺ T reg cells. In another embodiment, the T reg cells are CD4 ⁺ CD127 ^low CD25 ^high Foxp3 ⁺ T reg cells.

Cells of dendritic cell lineage are derived from hemapoietic bone marrow progenitor cells. Dendritic cells may mature directly from bone marrow progenitors, or from monocytes. Dendritic cell lineage cells include, but are not necessarily limited to immature dendritic cells, mature dendritic cells and monocytes, such as Type II monocytes. As used herein, the term dendritic cell or cells of dendritic cell lineage encompasses those cells which can activate T cell lineage cells, such as thymocytes, or naϊve T cells or T reg precursor cells to become T reg cells, and in particular those cells which can present antigen to the thymocytes, or naϊve T cells or T reg precursor cells to become antigen-specific T reg cells. T cell lineage thymocytes and thymic dendritic cells may be prepared from sampled thymic tissue according to the methods described by Watanabe et al. (2004) or Watanabe et al. (2005), the entire contents of which are incorporated herein by reference. Thymic tissue may be obtained by needle biopsy using standard procedures for thymic biopsy, or by surgical biopsy. Cell suspensions may be generated from the thymic tissue using techniques as described in Watanabe 2005 {supra). For example, a single cell suspension may be generated using tissue digestion techniques such as enzymic digestion, and mononuclear cells are isolated by Ficoll centrifugation. T cell lineage thymocytes (CD4 ⁺ , CD25 ^" ) may be obtained by negative depletion using a mixture of mouse monoclonal antibodies to the lineage markers CDl Ic, CD14, CD15, CD20, CD56 and CD235a, followed by removal of murine antibody-bound cells using goat-anti-mouse coated magnetic beads. CD4 ⁺ , CD8 ^" CD25 ^" T cell lineage thymocytes then may be isolated using magnetic beads.

Thymic dendritic cells may be isolated by negative depletion, followed by fluorescence activated cell sorting of CDl Ic ⁺ lineage negative CD4 ⁺ cells. Alternatively, naϊve circulating T cells expressing CD4 ⁺ CD45RA ⁺ CCR7 ⁺ may be isolated by flow cytometry techniques from peripheral blood buffy coat cells or lymphoid organs according to methods described in Watanabe et al., (2004) (supra).

Peripheral dendritic cells expressing CDl Ic ⁺ may be isolated from peripheral blood buffy coat cells or lymphoid tissue using techniques identified in Watanabe et al., (2004) (supra). TSLP-stimulated circulating dendritic cells may be used to generate T reg cells if the dendritic cells behave in a thymic dendritic cell like manner, such as expressing appropriate levels of CD80 and CD86 and being able to stimulate the generation of T reg cells rather than ThI or Th2 effector or memory cells.

Monocyte-derived dendritic cells can be generated in vitro from PBMCs. Plating of PBMCs in a tissue culture flask permits adherence of monocytes. Treatment of these monocytes with IL-4 and granulocyte-macrophage colony stimulating factor leads to differentiation to immature dendritic cells (iDCs) in about a week. Subsequent treatment with tumor necrosis factor alpha (TNFa) further differentiates the iDCs into mature dendritic cells. In particular embodiments immature dendritic cellsare used in the methods described herein.

TSLP stimulation of dendritic cells may be achieved by incubation of the dendritic cells in culture for at least 24 hours with recombinant human TSLP (for example using at least approximately lOng/ml, more preferably 15ng/ml human recombinant TSLP, Cell Sciences, Cat No. CRT702B). As the dendritic cells are treated ex vivo, an excess of TSLP may be used to ensure maximal stimulation of these cells, thus at least partially overcoming a possible deficit in the level of signalling full-length TSLP receptors expressed on the dendritic cell surface. The degree of stimulation of the dendritic cells may be assessed by the increased expression of surface markers characteristic of activated dendritic cells, such as any one or more of DC-LAMP, HLA-DR, CD209, CD80 and CD86. Ideally the TSLP-stimulated dendritic cells will be stimulated sufficiently to match the level of expression of these markers by dendritic cells from a subject who is heterozygous or homozygous for CD 127 haplotype 2. While the "priming" of dendritic cells with TSLP would usually be carried out prior to co-culture with autologous T cell lineage cells, the dendritic cells may be exposed to TSLP at the same as being co-cultured with the T cell lineage cells.

In certain embodiments it may be advantageous to provide one or more MS autoantigens to the dendritic cells prior to and/or at the same time as the dendritic cells are co-cultured with the T cell lineage cells, in order to stimulate the generation of autoantigen-specific T reg cells, instead of a polyclonal expansion of T regs. In particular, autoantigens to which an immune response has been mounted in the subject with MS could be used to direct the generation of autoantigen-specific T reg cells. Typical antigens to which an autoimmune response is observed in MS include myelin basic protein, myelin associated glycoprotein, myelin oligodendrocyte protein, proteolipid protein and alpha-beta-crystallin. Recombinant forms of these proteins or proteins isolated from the subject may be used to generate autoantigen-specific T reg cells.

The expansion and differentiation of the T cell lineage cells to T reg cells is achieved by co-culture of the T cell lineage cells with the TSLP -primed dendritic cells. If required, additional techniques to expand T reg cell numbers, such as methods described in Peters et al., (2008), may be used to produce sufficient T reg cells for one or more administrations.

The generation of T reg cells may be monitored by sampling the cells generated and assessing the expression of CD4 ⁺ , CD8 ^" , CD127 ^low CD25 ⁺ Foxp3 ⁺ . The T reg cells generated by incubation with TSLP-treated dendritic cells may be CD4 ⁺ , CD8\ CD25 ⁺ Foxp3 ⁺ T reg cells. T reg cells generated ex vivo may be readily isolated from the dendritic cells and other non-T reg cells, for example using the methods described in Peters et al., (2008) (supra). Antigen specific T reg cells may be isolated from a T reg cell population by affinity techniques available in the art (see for example Koenen and Joosten 2006).

It will be understood that more than one round of co-culture may be used on the same T cell lineage cell population, such as a thymocyte or naive T cell sample, to continue to expand the population of T reg cells generated. T reg cells generated in each round of co-culture may be removed, and stored frozen or in culture until a sufficient population of T regs is available for administration to the subject.

The administration of ex vivo generated T reg cells to a subject with MS will typically be an intravenous administration. Typically from between 10 ³ to 10 ⁶ T reg cells will be administered in a single administration. Following administration the subject will be monitored clinically for signs of resolution of clinical symptoms and/or lesions. If necessary to maintain alleviation of MS, additional administration(s) of T reg cells may be made from 1-6 months after the previous administration. Methods for autologous cell transfer including the isolation, in vitro treatment and re-introduction of cells are known to those skilled in the art (see, for example, Homann and von Herrath (2004) and Weber et al. (2007), the disclosures of which are incorporated herein by reference).

Those skilled in the art will appreciate that the methods of treatment disclosed herein may be used in isolation or in combination with other treatments. The skilled addressee will understand "combination" to mean that the methods disclosed herein may be used in conjunction with other methods, for instance as part of a combination therapy together with alternative methods or compositions for the treatment of MS.

The response of the subject to the administration of T reg cells to a subject with or at risk of MS may be monitored by monitoring of the clinical condition of the subject, including but not limited to monitoring of the period of remission and time to relapse of MS, monitoring of the size and distribution of MS lesions by Standard methods such MRI, and by monitoring the number and distribution of T reg cells and/or autoantigen specific effector T cells in secondary lymphoid organs. Polypeptides and Polynucleotides

As described above the methods and compositions of the embodiments of the invention typically involve the use of TSLP. The amino acid sequence of the human TSLP protein is shown in SEQ ID NO:6 (GenBank Accession No. AY037115), and the polynucleotide coding sequence of the human TSLP is shown in SEQ ID NO:7 (GenBank Accession No. AY037115). Polypeptides other than full length TSLP may also be used, provided that they have the ability to activate the TSLPR complex expressed on dendritic cells. According to embodiments of the invention, the polypeptides may comprise the amino acid sequences as set forth in the sequence listing. Alternatively the polypeptide may be encoded by a polynucleotide which displays sufficient sequence identity to hybridize to the TSLP polynucleotide sequence as set forth in the sequence listing. In alternative embodiments, the polypeptide may share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the TSLP polypeptide sequence as set forth in the sequence listing.

According to embodiments of the invention, the disclosed polynucleotides may have the nucleotide sequences as set forth in the sequence listing or display sufficient sequence identity thereto to hybridize to the nucleotide sequences as set forth in the sequence listing under stringent hybridization conditions.

Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that

lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, or less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 6O ⁰ C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C, and a wash in IX to 2X SSC (2OX SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55 ⁰ C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 600C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

In alternative embodiments, the nucleotide sequence of the polynucleotide may share at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequences as set forth in the sequence listing. Within the scope of the terms "protein", "polypeptide" and "polynucleotide" as used herein are fragments and variants thereof.

The term "fragment" refers to a contiguous nucleic acid or polypeptide sequence that encodes a constituent or is a constituent of a full-length protein or gene. In terms of the polypeptide the fragment possesses qualitative biological activity in common with the full-length protein.

The term "variant" as used herein refers to substantially similar sequences. Generally, nucleic acid sequence variants encode polypeptides which possess qualitative biological activity in common. Generally, polypeptide sequence variants also possess qualitative biological activity in common. Further, these polypeptide sequence variants may share at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

Further, a variant polypeptide may include analogues, wherein the term "analogue" means a polypeptide which is a derivative of the disclosed polypeptides, which derivative comprises addition, deletion or substitution of one or more amino acids, such that the

polypeptide retains substantially the same function as the native polypeptide from which it is derived. The term "conservative amino acid substitution" refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (GIu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.

Typically, in therapeutic applications, the treatment would be for the duration of the disease state. A dosage regime for T reg cells may be determined by a clinician by monitoring the progression or receding of symptoms of MS, and by quantifying the change in severity of the autoimmune response responsible for the pathology in MS.

Those skilled in the art will appreciate that the T reg cells may be administered as part of a combination therapy approach to the treatment of MS, employing the methods disclosed herein in conjunction with other therapeutic approaches to MS treatment. For such combination therapies, each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired therapeutic effect. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. Alternatively, the components may be formulated together in a single dosage unit as a combination product. Suitable agents which may be used in combination with the compositions of the present invention will be known to those of ordinary skill in the art. For example, the current main therapies for MS include interferon-β and glatiramer acetate (formerly called Copolymer- 1 or COP-I), with many other therapies used to relieve the various symptoms of MS. In addition, monoclonal antibodies have been developed which target MS-associated antigens. The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention. Examples General Methods Subjects Peripheral blood was collected from 34 subjects with primary progressive MS, 20 subjects with Relapsing Remitting MS, and 45 healthy controls. Blood was collected into PAX (Qiagen) vacutainer tubes and RNA extracted according to the manufacturer's instructions.

All subjects were diagnosed with definite MS by Poser criteria. The McDonald criteria for the diagnosis of Primary Progressive MS were applied retrospectively. All subjects had other relevant conditions excluded. All Primary Progressive MS subjects had a clinical course which was progressive from onset with observed progressive deterioration over a period of >1 year and with prominent paraparesis and typical MRI abnormalities. CSF results were not available in all cases.

The three groups did not differ with respect to sex or ethnicity, with all subjects being of northern European origin. None of the MS patients received immunomodulatory therapy. The Primary Progressive MS patients were older (mean age 54) than the controls (mean age 48) and Relapsing Remitting MS patients (mean age 44). There were no age effects for any parameters compared.

Healthy controls were staff and their relatives from Westmead Hospital.

This study was approved by the Sydney West Area Health Service Human Research Ethics Committee (HREC2002/9/3.6(1425)), and subjects gave informed consent, qRT-PCR cDNA was prepared from patient and control samples of total mRNA from PAX tubes (Becton Dickinson, Qiagen) or from direct cell lysis (Cell to Signal, Ambion) and RNeasy RNA purification (Qiagen) using Superscript III according to the manufacturer's instructions (Invitrogen). Semi-quantitative RT-PCR was used for CD 127 expression analysis to provide a comparative measurement of mRNA from each haplotype in heterozygotes and to determine the ratio of full-length CD 127 isotype to soluble CD 127 isotype mRNA expression. These methods were carried out as described in Teutsch et al. (2003), the entire contents of which are incorporated herein by reference. CD 127 mRNA levels were assayed using Sybr green and primers spanning intron 7 of CD 127. Primer sequences were:

5'-CTGGAACATCTTTGTAAGAAACCAAG-S ' (SEQ ID NO:9) and

5'-TAGCTTGAATGTCATCCACCCT-S ' (SEQ ID NO: 10).

δCT was used to measure comparative amplification (Livak and Schmittgen (2001)), and normalised against starting material.

Genotyping

The -1085 (rs7718919), -504 (rsl 1567685) and -449 SNPs (rsl 1567686) were genotyped by using restriction enzymes, as described previously (Teutsch et al. 2003,

supra). The SNP -449 G (+ strand) was used to tag haplotype 1, while -504 C (+) tagged haplotype 4, -1085 T (+) tagged haplotype 3, and -1085 G, -504 T, -449 A (all + strand) tagged haplotype 2, as illustrated in Figure 1.

The PCR primers used to amplify cDNA samples were specific for CD 127 mRNA membrane-bound and soluble splice variants, for the CD 127 exon 8 amino acid residue 336 (aa336) alleles (Korte et al. (2000)) and for CD127 exon 2 amino acid residue 46 (aa46) alleles. The PCR primer sequences were:

CD127X2F: 5 '-TGGAGAAAGTGGCTATGCTCA-S ' (SEQ ID NO: 1 1) and

CD127X2R: 5'-CAACCTTCACACATATATTGCTC-S ' (SEQ ID NO:12). Exon 8 was measured from the exon 5-exon 8 amplicon used in the QRTPCR reaction.

The aa336 and aa46 alleles were in complete linkage disequilibrium with the promoter alleles at nucleotides -504 and -449, respectively. cDNA primer extension assays using the SNaPshot system (Applied Biosystems, Foster City, CA, USA) were designed, involving three SNaPshot extension primers. These primers were designed to distinguish the CD127 exon 8 aa336 [AJG) SNP allele, with sequence

5 '-AGCTCCAACTGCCCATCTGAGGATGTAGTC-S ' (SEQ ID NO: 13), and the exon 2 aa46 (C/T) SNP allele, with sequence

5'-GTGCTTTTGAGGACCCAGATGTCAACA-S ' (SEQ ID NO:14), and the CD 127 soluble isoform with sequence

5'-TCCAGAGATCAATAATAGCTCAGG-S' (SEQ ID NO: 15) in individuals with representative CD 127 genotypes. The soluble and full length isoforms were amplified using the QRTPCR primers.

All reactions were performed in triplicate and means and standard errors were obtained for each individual. For the CD127 aa46 and aa336 SNaPshot reactions, the ratio of fluorescence peak heights of each allele in heterozygotes was calculated.

SNaPshot reactions for aa46 and aa336 alleles were also performed in triplicate on representative control genomic DNA samples to correct for any biases in allelic amplification. The mean of the ratio of SNaPshot peaks was used as a correction factor by which all aa46 and aa336 SNaPshot cDNA ratios were divided. Mean cDNA ratios of expression were compared between MS patients and controls using the unpaired t-test

(Graph Pad Quick Calcs) to obtain /?-values. Mean cDNA ratios of expression were compared with genomic DNA ratios using the Mann-Whitney £/-test (SPSS Inc.,

Chicago, IL, USA). An isoform of CDl 27, in which exon 6 is spliced out, makes up about 10% of the message in whole blood of healthy controls. Relative expression of this isoform from the different haplotypes can be measured using an oligo at the exon 6 splice site, and comparing the expression levels of the 'G' corresponding to full length cDNA, or 'A' corresponding to the soluble isoform mRNA, as in the cDNA primer extension assay, for known genotypes. The fluorescence peak height ratios of CD 127 mRNA splice variants were calculated and mean cDNA ratios of expression were correlated with CD 127 promoter genotypes using the unpaired t-test.

Agilent DNA electrophoresis was also used to quantify full length and soluble isoforms.

Example 1 : Haplotype expression and CD127 isoform expression

The expression of full length and soluble isoforms of CD 127 mRNA was examined in total PBMCs, and in T cells and inflammatory dendritic cells isolated from or derived from PBMCs. PBMCs were obtained from whole blood by Fi coll separation. CD4 T cells were obtained from PBMCs by magnetic bead separation using standard T cell isolation kits (Miltenyi Biotech). Monocytes were isolated from PBMCs by column separation using anti-CD 14 magnetic beads (Miltenyi Biotech).

Dendritic cells were prepared according to the method of Abbas et al. (2005) the entire contents of which is incorporated by reference. Briefly, monocytes were cultured at 5 x 10 ⁶ /ml in X Vivo 15 media with IL-4 and GMCSF for 5 days, with replenishment of cytokines on day 3. After 5 days cells were stimulated with LPS (lμg/ml). Cells were harvested at 24 h for RNA extraction and supernatants were harvested at 24 and 48 hours for measurement of soluble CDl 27 (sCD127). The results of these experiments are presented in Figures 2 and 3 A.

Individuals expressing the sensitivity haplotypes Haplotype 1 and to a lesser extent Haplotype 4 exhibited an overall decrease in the ratio of full length to soluble CD 127 isoforms when compared to individuals not carrying this haplotype (Figure 2). In contrast, individuals expressing the protective haplotype Haplotype 2 demonstrated an increased ratio of full-length to soluble CD 127 isoforms. An increased level of expression of soluble isoforms of CD 127 correlates with an increased sensitivity to MS.

The cellular distribution of haplotype expression was examined in Haplotype 1 /Haplotype 2 heterozygotes (Figure 3A). While the T cells and PBMCs of

these heterozygote subjects expressed each haplotype in nearly equal proportions, inflammatory dendritic cells from these subjects tended to express an increased proportion of Haplotype 2.

The expression of CD 127 isoforms by inflammatory dendritic cells from homozygous Haplotype 1 or 2 individuals and heterozygous was examined (Figure 3B and Figure 6). Activated dendritic cells from subjects who were homozygous for haplotype 1 expressed a much greater proportion of soluble CD 127 isoform than subjects who were homozygous for the protective haplotype 2 (Figure 3B). The results of PCR amplification experiments also confirmed this observation (Figure 6). Inflammatory dendritic cells from Haplotype 1 homozygous individuals expressed almost equimolar amounts of soluble and full length CD 127 isoforms, while dendritic cells from Haplotype 2 homozygous individuals expressed only minor proportion of soluble CD 127 isoform. Heterozygous individuals expressed ratios which were intermediate between the homozygous expression patterns.

Example 2: Functional changes associated with Dendritic Cell CD127 isoform expression

The previous example identified the relative proportions of CD 127 isoform-specific message produced by cells. The following example describes functional changes to dendritic cells which are associated with this CD 127 isoform-specific expression.

Resting and inflammatory dendritic cells were prepared from monocytes cultures derived from Haplotype 1 or 2 homozygous individuals as described in Example 1. Levels of sCD127 secreted into the culture medium were determined by sandwich ELISA using plate-bound anti-CD 127 (MAB306, R&D Systems) to capture antigen, followed by detection with biotinylated anti-CD 127 (raised against a non-overlapping epitope, BAF306 R&D Systems) and streptavidin-HRP (Chemicon). ELISAs were developed using tetramethylbenzidine (Sigma- Aldrich). The results of these experiments are presented in Figure 4. While unstimulated cultures of cells from each haplotype released approximately equal quantities of soluble CD 127 isoform polypeptide into the culture medium, activation of the dendritic cells by LPS lead to a doubling in the quantity of soluble CD 127 polypeptide released by cells from a Haplotype 1 homozygous subject at 48 hours, but with only a minor increase in the level of soluble CD 127 polypeptide released from dendritic cells isolated from a Haplotype 2 homozygous subject.

The association of haplotype and the TSLP mediated regulation of IDO expression in PBMCs was examined. Peripheral blood was collected in EDTA and mononuclear cells (PBMCs) were isolated by Ficoll density gradient separation. PBMCs were stimulated with TSLP according to the method of Urashima et al. (2005). Briefly, PBMCs were cultured at 5 x 10 ⁵ /ml in X- Vivo 15 media (Lonza) in the presence or absence of TSLP (20ng/ml; R&D Systems) for 96 h.

Gene expression was measured from cells (1 x 10 ⁵ ) which were harvested and lysed (Cells-to-Signal Lysis buffer, Ambion), RNA extracted (RNeasy, Qiagen) and reverse transcribed (Superscript III, Invitrogen), and transcript levels determined by quantitative RT-PCR using specific TaqMan probe-primer mixes (Applied Biosystems).

The results of these experiments are presented in Figure 5. While cells from Haplotype 2 homozygotes responded strongly to TSLP stimulation, cells from the sensitive Haplotype 1 and 4 subjects exhibited a reduced ability to increase the expression of IDO on stimulatio with TSLP, and could be considered less responsive. As the response to TSLP, here measured by the production of IDO by dendritic cells, is an essential step in the guidance by dendritic cells of the differentiation of T reg cells, the deficit in IDO production in response to TSLP suggests that the generation of T regs may be impaired in these subjects.

Example 3: ILTRalpha mRNA Expression in different T cell types and Dendritic cells

The level of IL7Rα mRNA expression by a variety of T cell types and by activated dendritic cells was examined. The method of preparation of each T cell type is listed briefly after Table 1, and a reference to the full method of cell preparation is provided.

In each case, IL7Rα mRNA was measured by quantitative Real time PCR, with the relative haplotype expression determined by SNAPSHOT (as described in Booth et al., 2005). The ratio of the expressed full-length/soluble IL7Rα iso forms was determined using an Agilent Bioanalyser.

IL7Rα mRNA expression decreases greatly in all T cell models with activation, and increases dramatically in activated DCs. There was little difference in mRNA expression between haplotypes in T cells, but a much greater difference was evidenced in DCs. The vast majority of CD 127 mRNA in T cells expressing the gene encoded the membrane- bound form of CD 127, but in DCs from Haplotype 1 homozygote individuals the mRNA isoforms were equimolar (also see Fig. 6).

Table 1. IL7Rα mRNA expression

(Hap=Haplotype; Het=Heterozygous; FL/S=Ratio of Full Length to Soluble forms) *Compared to time 0 cells: CD4+CD45RA+ naϊve T cells for T cells; CD 14+ monocytes for DCs

The following describes the preparation of each cell type listed in Table 1.

Activation: CD4 ⁺ CD45RA ⁺ naϊve T cells were primed with beads coated with anti- CD3 and anti-CD28 (Trickett & Kwan, 2003) over 48 hours; Signal Strength: CD4+CD45RA+ naϊve T cells were stimulated with titrated anti-

CD3/anti-CD28 beads;

ThI and Th2: naϊve cells were stimulated with anti-CD3/anti-CD28 and polarised to ThI or Th2 phenotypes using IL-12 (30ng/ml) and anti-IL-4 (0.5ug/ml) for ThI cells for 2 days, or with anti-CD3/anti-CD28 stimulation with IL-4 (20ng/ml) and anti-IFNγ (2.5ug/ml) for Th2 cells for 2 days.

IL-2 (200 IU/ml) was added to the ThO subset and replenished every 2 to 3 days up to a period of 8 days. Similarly, IL-2 was added to the ThI and Th2 subsets and was replenished with the relevant cytokines and blocking antibodies during the entire culture period (Habertson et al, 2002).

T reg: naive cells were cultured in the presence of TGFβ (lOug/ml) for 4 days (Fantini et al, 2006)

ThI 7: naϊve cells were cultured with IL- lβ (10ng/ml), IL-6 (50ng/ml) and anti- IFNγ for three days. On Day 3, additional cytokines IL-2 (20 IU/ml) and IL-23 (20ng/ml) were added every 2 to 3 days for a total of 12 days (Bettelli et al, 2007).

Homeostasis:CD4+CD45RA+ naϊve T cells were cultured with IL-7 (10ng/ml), IL- 7/2 or IL-2 (200 IU/ml) for 7 days. The cells were replenished with the relevant cytokines every 2 to 3 days during the entire culture period (Jaleco et al, 2003).

Tolerance: CD4+CD45RA+ naϊve T cells were cultured for four days with a low concentration of anti-CD3 beads , rested for 2 days, then restimulated with variable concentrations of anti-CD 3 beads for 4 further days (0. lug/ml- 10 ug/ml) (Willems et «/.,1995)

Central Memory: Inflammatory DCs (see below) were cultured with naϊve T cells and non-specific antigen for 5 days, then with IL-2 for a further 5 days (Langenkamp et al, 2000)

Inflammatory DCs: CD 14+ monocytes from whole blood were cultured for 5 days in GMCSF, and IL4, then stimulated with LPS (Abbas et al, 2005).

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