METHOD FOR THE IDENTIFICATION OF CD4+ REGULATORY T-CELLS FOR USE IN THE TREATMENT OF INFLAMMATORY AND AUTOIMMUNE DISEASES

Abstract

The present invention relates to a method for identifying CD4.sup.+ Treg cells suitable for use as starting material in cellular immunotherapy, the method comprising i) analysing samples from target tissue A to identify CD4.sup.+ Treg cells with migratory in character between the diseased tissue, collecting lymphatics, peripheral blood, distinct tissue adjacent to the diseased target tissue A and/or distinct tissue that is not vicinal though has migratory Treg communication with target tissue A, v) analysing samples from peripheral blood, tissue C, to identify CD4.sup.+ Treg cells with migratory character and/or functional character where the Treg cells are also emigrant from target tissue A, vi) analysing sample(s) from tissue compartments A and/or B and C, that are analytically or physically depleted of emigrants from thymus and/or immigrants from peripheral blood to a lymph node, to restrict analyses to CD4.sup.+ Treg cells of target tissue A origin and/or tropism, to identify emigrant CD4.sup.+ Treg cell populations of target tissue A, to identify emigrant CD4.sup.+ Treg cell populations with propensity to immigrate to target tissue A, to identify a migratory and/or functional defect in the CD4.sup.+ Treg cell population identified as expressing migratory and/or functional elements specific for target tissue A in any of tissue A, B or C, and whereby a combination of surface or intracellular markers on CD4.sup.+ Treg cells is identified, which combination identifies which surface or intracellular markers should be present and which surface markers should not be present in CD4.sup.+ Treg cell populations suitable for use as starting material in cellular immunotherapy.

Claims

1-36. (canceled)

37. A method for identifying CD4.sup.+ Treg cells suitable for use in cellular immunotherapy, comprising (i) analyzing samples from diseased target tissue A to identify CD4.sup.+ Treg cells with migratory character between target tissue A and one or more of collecting lymphatics, peripheral blood, distinct tissue adjacent to target tissue A and/or distinct tissue that is not vicinal to but has migratory Treg communication with target tissue A, (ii) optionally, analyzing samples from target tissue A to identify CD4.sup.+ Treg cells with immunosuppressive function in target tissue A, (iii) optionally, analyzing samples from lymphatic tissue B to identify CD4.sup.+ Treg cells with migratory character between disease draining lymphatics and non-disease draining lymphatics of diseased or non-diseased target tissue A, (iv) optionally, analyzing samples from lymphatic tissue B to identify CD4.sup.+ Treg cells with immunosuppressive function in tissue B that are emigrant from target tissue A, (v) analyzing samples from peripheral blood (tissue C) to identify CD4.sup.+ Treg cells with migratory character and/or immunosuppressive function that are emigrant from target tissue A, (vi) analyzing sample(s) from target tissue A A and/or tissue B and tissue C, that are analytically or physically depleted of emigrants from thymus and/or immigrants from peripheral blood to a lymph node, to restrict analyses to CD4.sup.+ Treg cells of target tissue A origin and/or tropism, to identify emigrant CD4.sup.+ Treg cell populations of target tissue A, to identify emigrant CD4.sup.+ Treg cell populations with propensity to immigrate to target tissue A, to identify a migratory and/or functional defect in the CD4.sup.+ Treg cell population identified as expressing migratory and/or functional elements specific for target tissue A in any of tissue A, B or C, whereby a combination of surface or intracellular markers on CD4.sup.+ Treg cells is identified, which combination identifies which surface or intracellular markers should be present and which surface markers should not be present in CD4.sup.+ Treg cell populations suitable for use in cellular immunotherapy.

38. A method according to claim 37, wherein the sample from target tissue A is selected from solid tissues, interstitial fluids of solid tissue, oedemic or inflammatory fluids of diseased tissue regions, and tissues represented in a fluid phase.

39. A method according to claim 37, wherein the sample from target tissue A is selected from one or more of the following: (i) epithelial mucosal surfaces for investigation of intra-epithelial cell populations as collected by mucosal scrapings or lavage sampling, or fractionation of biopsy/resection specimens, (ii) sub-epithleial surfaces as collected by biopsy or resection, (iii) stroma of solid tissues as collected by biopsy or resection, (iv) parenchyma of solid tissues as collected by biopsy or resection, (v) endothelial and endothelial-vicinal tissues as collected by resection, (vi) dermal layers as collected by cutaneous punch sampling, biopsy by incision or samples of tissues collected for grafting, (vii) interstitial fluids of solid tissues collected by passive fluid collection methods, (viii) synovial fluids of joint capsules or bursae as collected by active sampling methods, (iix) cerebrospinal fluids as collected by active sampling methods, (ix) oedemic or lymphedema fluids of solid tissues of bodily cavities collected by passive or active sampling methods, and (x) nervous tissues as collected by biopsy or recovery from resected tissues or limb amputation, (xi) skeletal muscle tissues as collected by biopsy or recovery from resected tissues or limb amputation.

40. A method according to claim 37, wherein the method includes step (iii) and the comparison is made either with (a) samples from a single subject suffering from an inflammatory or autoimmune disease or (b) samples from a subject suffering from an inflammatory or autoimmune disease and a healthy volunteer.

41. A method according to claim 37, wherein tissue B comprises lymph nodes directly draining target tissue A via collecting lymphatic vessels.

42. A method according to claim 37, wherein tissue B is selected from one or more of the following: (i) disease draining (sentinel) lymph nodes as sampled by resection and processing or by active sampling by puncture and fluid draw, (ii) lymph fluids of diseased target tissue A as collected by microsurgical access of collecting lymph vessel and installation of a cannula for passive fluid collection. (iii) distal lymph fluids communicating from disease draining lymph nodes as collected by surgical installation of a cannula for passive fluid collection of minor distal lymphatic vessels, or by active sampling of major distal lymphatic vessels.

43. A method according to claim 37, wherein one or more of the analyses is effected by single-cell analysis or by highly restricted cell population analyses.

44. A method according to claim 43, wherein the single-cell analysis or highly restricted cell population analyses comprises one or more of the following: (i) flow cytometric methods detecting surface antigen expression, (ii) flow cytometric methods detecting intracellular antigen expression, (iii) flow cytometric methods detecting transcript or genomic parameters by in situ probe hybridisation techniques, and (iv) flow cytometric analyses of enzyme function or metabolite abundance.

45. A method according to claim 44, wherein the single-cell analysis or highly restricted cell population analyses comprises flow cytometric methods detecting surface antigen expression.

46. A method according to claim 44, wherein assayed parameters on analyte cell populations identified by analytical inclusion/exclusion markers include one or more of: (i) abundance of cell surface expressed somatically invariant protein antigens, (ii) abundance of surface expression of somatically rearranged protein antigens, (iii) enzyme activity or metabolite abundance by fluorometric/colorimetric linked conversion assays, (iv) abundance of intracellular expressed protein antigens, and/or (v) transcript abundance or genomic rearrangement detection by in situ probe hybridisation.

47. A method according to claim 37, further comprising a purification or enrichment step.

48. A method according to claim 47, wherein the purification or enrichment step comprises one or more of: (i) flow cytometric purification of single cells for submission to downstream analytical workflows, (ii) flow cytometric purification of highly restricted cell populations and subpopulations for submission to downstream analytical workflows, (iii) substrate immunoaffinity enrichment of highly restricted cell populations and subpopulations for submission to downstream analytical workflows, and/or (iv) substrate immunoaffinity enrichment of highly restricted cell populations and subpopulations, coupled with flow cytometric purification of single cells and/or highly restricted cell populations, for submission to downstream analytical workflows.

49. A method according to claim 48, further comprising analysing one or more of the following: (i) protein abundance by immuno-blotting or other immuno-detection methods, (ii) coding transcript abundance or presence by quantitative or qualitative PCR methods, (iii) coding transcript abundance or presence sequencing or resequencing methods, (iv) non-coding transcript abundance or presence by PCR methods, (v) analyses of somatic genomic rearrangements and anomalous genomic rearrangement by PCR methods, (vi) analyses of somatic genomic rearrangements and anomalous genomic rearrangement by direct sequencing or resequencing methods, (vii) analysis of protein or metabolite secretion by immunodetection, enzyme-linked assay or direct spectroscopic methods, and/or (viii) analysis of cellular function by mixed cell reactions.

50. A method according to claim 37, wherein the method identifies X and Y type markers that enable further definition of target tissue A specific X type emigrant or tropic migratory CD4.sup.+ Tregs or Y type regulatory functional CD4.sup.+ Tregs, respectively, for identification of target tissue A CD4.sup.+ Treg cells.

51. A method according to claim 37, wherein the method identifies X and Y type markers that enable further definition of target tissue A specific X type emigrant or tropic migratory CD4.sup.+ Tregs or Y type regulatory functional CD4.sup.+ Tregs, respectively, and wherein the method further comprises using the identified X and/or Y type markers as inclusion or exclusion criteria for reanalysis by a method according to claim 37, to identify further X and/or Y type markers for identification of tissue A Treg cells or subtypes of Treg cells.

52. A method for obtaining a CD4.sup.+ Treg cell population for use in cellular immunotherapy, comprising subjecting peripheral blood from a patient suffering from an inflammatory or an autoimmune disease to single-cell analysis, and separating from the blood CD4.sup.+ Treg cells having signatures that: (i) identify that the cells are CD4.sup.+ regulatory T-cells, (ii) identify that the regulatory T-cells are tissue type tropic that can migrate to the diseased area, (iii) optionally, identify that the Treg cells are diseased tissue tropic homing cells that can localize in the diseased tissue, (iv) identify that the regulatory T-cells are antigen-experienced emigrant cells that originated from target tissue A, (v) optionally, identify that the regulatory T-cells are capable of being retained in the diseased tissue after administration to a subject, and optionally one or more X-signatures and/or Y-signatures, wherein X is a signature indicating that the CD4.sup.+ Tregs can localize, have emigrated from, or are marked for preferential retention in the diseased area and Y is a signature indicating immunosuppressive regulatory function or restriction of inflammatory function.

53. A method according to claim 52, wherein the separating comprises applying analytical filters to (i) exclude cells that gain access to lymph nodes via HEV, and/or (ii) exclude cells that are recent thymic emigrants.

54. A method according to claim 53, wherein the excluded cells that gain access to lymph nodes via HEV are CD62L.sup.+ cells.

55. A method according to claim 53, wherein the excluded cells that are recent thymic emigrants are selected from CCR9.sup.+CD45RA.sup.+, CCR9.sup.+CCR7.sup.+, CCD9.sup.+CD62L.sup.+, and CCR9.sup.+CD45RO.sup. cells.

56. A method according to claim 53, wherein the excluded cells are CCR9+CCR7+CD62L+CD45RA+CD45RO cells.

57. A method according to claim 52, further comprising identifying CD4.sup.+ Treg cells that are emigrant and immigrant cells such as integrin-type or other adhesion molecules associated with Target-A tissue adhesion and transmigration through tissue-integral vasculature.

58. A method according to claim 52, wherein the obtained CD4.sup.+ Treg cells have specific signatures that (i) identify that the cells are CD4.sup.+ regulatory T-cells, (ii) identify that the regulatory T-cells are tissue type tropic that can migrate to the diseased area, (iii) optionally, identify that the Treg cells are diseased tissue tropic homing cells that can localize in the diseased tissue, (iv) identify that the regulatory T-cells are antigen-experienced emigrant cells that originated from target tissue A, (v) optionally, identify that the regulatory T-cells are capable of being retained in the diseased tissue after administration to a subject, and optionally one or more X-signatures and/or Y-signatures.

59. A method according to claim 52, wherein the signature (i) is selected from CD4.sup.+CD25.sup.hi, CD4.sup.+CD25.sup.hiCD127.sup.lo, and CD4.sup.+Y.sub.n, where n is an integer of 1 or more.

60. A method according to claim 52, wherein the signature (ii) is for a gastrointestinal mucosa and is selected from 47.sup.+ and 4.sup.+7.sup.+.

61. A method according to claim 52, wherein the signature (iii) is for localization in the small bowel and is CCR9.sup.+, optionally in combination with one or more X signatures.

62. A method according to claim 53, wherein the signature (iv) is for antigen-experienced cells and is selected from CD62L.sup. and/or CD38.sup.+ and/or 4.sup.+E.sup.+7.sup.high, and/or one or more X signatures.

63. A method according to claim 52, wherein an X-signature is selected from any of (a) CD26.sup., CD97.sup., CD143.sup., CD195.sup., CD278.sup.+, (b) CD61.sup., CD63.sup., CD146.sup., CD183.sup., CD197.sup.+, CD200.sup.+, CD244.sup., (c) CD20.sup., CD130.sup.+, and CD166.sup..

64. A method according to claim 52, wherein a Y-signature is selected from any of (g) CD21.sup., CD35.sup., CD73.sup., CD122.sup.+, CLIP.sup.+l , CD120b.sup.+, (h) CD6.sup., CD39.sup.+, CD50.sup.+, CD109.sup.+, CD226.sup., CD243.sup., CD268.sup.+, CD274.sup., CD210.sup.+, (j) CD49c.sup.+, CD53.sup.+, CD84.sup., CD95.sup.+, and CD107a.sup..

65. A method according to claim 52, wherein the CD4.sup.+ Treg cells are CD62L.sup.+, CCR9+CD45RA+, CCR9+CCR7+, CCD9+CD62L+, CCR9+CD45RO and/or CCR9+CCR7+CD62L+CD45RA+CD45RO.

66. A method according to claim 52, wherein CD4.sup.+ Treg cells are CD38+, CD69+ or CD44+ to denote recent activation.

67. A method according to claim 52 further comprising at least one of purifying, isolating, culturing or enriching cells.

Description

LEGENDS TO FIGURES

[0582] FIG. 1. CD4.sup.+FOXP3.sup.+ T.sub.regs isolated from human intestinal LP carry higher levels of CCR9 than CD4.sup.+FOXP3.sup. T.sub.eff counterparts. (A) Single cell suspensions were prepared from LP dissected from histologically normal small and large bowel, resected from a representative CD patient with ileoceacal disease. Cells were stained for CD4, FOXP3, 7 and CCR9, then analysed by flow cytometry. Lymphocytes were gated for CD4.sup.+, expressed as CD4vsFOXP3 dotplots (left hand panels), and Tregs defined as CD4.sup.+FOXP3.sup.+ with CD4.sup.+FOXP3.sup. defined as effector T-cells. Overlaid histograms of CD4.sup.+FOXP3.sup.+ and CD4.sup.+FOXP3.sup. populations are presented for CCR9 and 7 signal intensities relative to singly unstained controls. (middle panels) and for CCR9 intensity in the CD4.sup.+7.sup.hiFOXP3.sup.+ in addition to CD4.sup.+7.sup.hiFOXP3.sup. populations (right hand panels). Histogram gates in (A) were used to quantify percentage of positive cells (n=3), presented in (B).

[0583] FIG. 2. CD4.sup.+FOXP3.sup.+ Tregs isolated from human MLN draining small bowel carry higher levels of CCR9 than CD4.sup.+FOXP3.sup. T.sub.eff counterparts. (A) Single cell suspensions were prepared from MLNs draining the small bowel, resected from a CD patient with ileoceacal disease. Cells were stained for CD4, FOXP3, 7 and CCR9, then analysed by flow cytometry. Lymphocytes were gated for CD4.sup.+, expressed as CD4vsFOXP3 dotplots (left hand panel), and Tregs defined as CD4.sup.+FOXP3.sup.+ with CD4.sup.+FOXP3.sup. defined as effector T-cells. Overlaid histograms of CD4.sup.+FOXP3.sup.+ and CD4.sup.+FOXP3.sup. populations are presented for CCR9 and 7 signal intensities relative to singly unstained controls (right hand panels). Histogram gates in (A) were used to quantify percentage of positive cells (n=3), presented in (B).

[0584] FIG. 3. CD4.sup.+CD25.sup.hiCD127.sup.loFOXP3.sup.+ Tregs have higher CCR9 expression than CD4.sup.+CD25.sup.loCD127.sup.hiFOXP3.sup. non-Tregs in MLN draining the small bowel. A) Single cell suspensions were prepared from small bowel draining MLNs resected from a CD patient and stained for CD4, CD25, CD127, FOXP3 and CCR9. CD4.sup.+CD25.sup.hiCD127.sup.lo Treg and CD4.sup.+CD25.sup.loCD127.sup.hi effector T-cell populations were gated and overlaid as histograms of FOXP3 intensity (top panel). Histogram gates were used to select FOXP3.sup.+ and FOXP3.sup. populations and expressed as overlaid histograms of CCR9 intensity for CD4.sup.+CD25.sup.hiCD127.sup.loFOXP3.sup.+ Treg and CD4.sup.+CD25.sup.loCD127.sup.hiFOXP3.sup. effector populations (bottom panel). CCR9 histogram gate was used to quantify results (n=3) in (B).

[0585] FIG. 4. CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiFOXP3.sup.+ Tregs have higher CCR9 expression than CD4.sup.+CD25.sup.loCD127.sup.h7.sup.hiFOXP3.sup. non-Tregs in peripheral blood. PBMCs were prepared from healthy controls and stained for CD4, CD25, CD127, FOXP3, 7 and CCR9. CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hi Treg and CD4.sup.+CD25.sup.loCD127.sup.hi7.sup.hi effector T-cell populations were gated and overlaid as overlaid histograms of FOXP3 intensity (top panel). Histogram gates were used to select FOXP3.sup.+ and FOXP3.sup. populations and expressed as overlaid histograms of CCR9 intensity for CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiFOXP3.sup.+ Treg and CD4.sup.+CD25.sup.loCD127.sup.hi7.sup.hiFOXP3.sup. T.sub.eff populations (bottom panel). CCR9 histogram gate was used to quantify results (n=3) in (B).

[0586] FIG. 5. CD4.sup.+CD38.sup.+CD62L.sup. mucosal-educated FOXP3.sup.+ T.sub.regs in peripheral circulation carry higher CCR9 marking than FOXP3.sup. T.sub.effs and CD patients have diminished overall CCR9 marking on mucosal-educated T-cells. (A) PBMCs prepared from healthy controls (HC) were stained for CD4, FOXP3, CD38, CD62L, 7 and CCR9 then analysed by flow cytometry. Lymphocytes were gated for CD4.sup.+, expressed as CD38vsCD62L dot plots and divided into quadrants (left hand panel). Histograms of each quadrant are displayed for signal intensity of 7, CCR9 and CCR9 after further gating on 7.sup.hi (upper panels). Histogram gates were used to quantify signal intensities in each quadrant (n=7, lower panels). (B-D) PBMCs isolated from HC (n=7) and small bowel CD patients (n=6) were analysed as in (A). (B) Percentage of FOXP3.sup.+ (upper panel) and CCR9.sup.+ (lower panel) cells among total CD4 cells. (C) Percentage of FOXP3.sup.+ (upper panel) and CCR9.sup.+ (lower panel) cells among gated CD4.sup.+CD38.sup.+CD62L.sup.7.sup.hi cells. (D) Percentage of CCR9.sup.+ cells among gated CD4.sup.+CD38.sup.+CD62L.sup.7.sup.hi further gated on FOXP3.sup.+ and FOXP3.sup. cells.

[0587] FIG. 6. T-cells of CD38.sup.+CD62L.sup. phenotype predominate in the LP of the small and large bowel. Single cell suspensions prepared from LP (top panels) and MLN (bottom panels) of large bowel (left panels) and small bowel (right panels) were stained for CD4, C38 and CD62L. CD4.sup.+ cells were expressed as CD38vsCD62L dotplots. Values in upper left quadrant represent percentage of CD38+CD62LCD4 cells in this quadrant (n=1). Small bowel LP and MLN were from the ileum, large bowel LP and MLN were from the right colon resected from patient undergoing surgery for colorectal cancer.

[0588] FIG. 7. CD4.sup.+4.sup.hi7.sup.hi mucosal-tropic FOXP3.sup.+ Tregs in peripheral circulation carry higher CCR9 marking than FOXP3.sup. T.sub.effs and CD patients have diminished CCR9 marking on mucosal-tropic T-cells (A) PBMCs prepared from HC were stained for CD4, FOXP3, 7, 4 and CCR9 then analysed by flow cytometry. Lymphocytes were gated for CD4.sup.+, expressed as 7vs4 dot plots and gated for 4.sup.hi7.sup.hi, 4.sup.hi7.sup.lo, 4.sup.lo7.sup.lo (left panel), and displayed as overlaid histograms of CCR9 intensity (right panel). (B) Quantified CCR9 positivity among HC (n=7), for analysis as performed in (A). (C-D) PBMCs isolated from HC (n=7) and CD patients (n=4) were analysed as in (A). (C) Percentage of FOXP3.sup.+ cells among gated CD4.sup.+4.sup.hi7.sup.hi cells. (D) Percentage of CCR9.sup.+ cells among gated CD4.sup.+4.sup.hi7.sup.hi cells, further gated on FOXP3.sup.+ and FOXP3.sup. cells.

[0589] FIG. 8. The majority of CD4.sup.+CCR9.sup.+ T-cells in peripheral circulation carry 4.sup.hi7.sup.hi expression. (A) PBMCs prepared from healthy controls and stained for CD4, 4, 7 and CCR9. CD4.sup.+ cells were expressed as CD4vsCCR9 dotplots and CD4.sup.+CCR9.sup.+ and CD4.sup.+CCR9.sup. populations subsequently expressed as 7vs4 dotplots. The percentage of cells inside the 4.sup.hi7.sup.hi gate is quantified (n=7) in (B).

[0590] FIG. 9. No difference in total numbers of CD4.sup.+4.sup.hi7.sup.hi T-cells in peripheral circulation of CD patients and healthy controls. PBMCs prepared from healthy controls (HC) and CD patients were stained for CD4, FOXP3, 7, 4 and CCR9 then analysed by flow cytometry. Lymphocytes were gated for CD4.sup.+ and expressed as 7vs4 dotplots. The percentage of CD4+ cells inside the 4.sup.hi7.sup.hi is shown for HC (n=7) and CD patients (n=4).

[0591] FIG. 10. MACS 2-Step enrichment of MLN Tregs. Total lymphocytes recovered from MLN of patient with CD were processed using Miltenyi Regulatory T Cell Isolation Kit II and an autoMACSpro instrument. Input cells are displayed in the far left plot, gated on CD4, and expressed as CD25vsFOXP3. CD25.sup.hiFOXP3.sup.+ cells are enriched in the CD4.sup.+CD127.sup. flow-through of the first negative selection step, then compared to CD4.sup.+CD127.sup.+ positively selected eluate from the same step (middle panels). Further enrichment of CD25.sup.hiFOXP3.sup.+ cells to approximately 80% is observed in the positive selection eluate of the second MACS step.

[0592] FIG. 11. MACS enrichment and FACS purification of Tregs from MLN. Total lymphocytes recovered from MLN of patient with CD were processed using Miltenyi CD4+ T Cell Isolation Kit II and an autoMACSpro instrument. Cells were immediately labeled with CD4, CD25 and CD127 antibodies and run on FACSaria II instrument. Ungated events are displayed as SSCvsCD4 to assess enrichment (A). CD4-gated events in A) are displayed in the left panel of B). CD25.sup.hiCD127.sup.lo and CD25.sup.loCD127.sup.hi gates of the resulting plot represent sorting-gates (left panel). A small amount of resulting sorted populations were immediately re-acquired on the FACSaria and displayed in the same plot format (right panels) to assess purity. A portion of the resulting cell population was fixed/permeabilised and stained for FOXP3. Cells were acquired on a FACScalibur instrument, and displayed as FOXP3 histograms of CD25.sup.loCD127.sup.hi and CD25.sup.hiCD127.sup.lo populations (C).

[0593] FIG. 12. Viable cell numbers of MACS-enriched MLN Tregs over 18 days of ex vivo expansion. Total lymphocytes recovered from MLN of patient with CD were processed using Miltenyi Regulatory T Cell Isolation Kit II and an autoMACSpro instrument. Resulting CD4.sup.+CD25.sup.loCD127.sup.hi cells were rested overnight before being activated for 24 hrs with antiCD3/CD28 magnetic microbeads. After 24 hours of activation IL2 (100 IU.mL) was added in the presence or absence of Rapamycin. Magnetic beads were removed after 24 hrs further culture, and IL2 and rapamycin were refreshed every 24 to 48 hrs while maintaining cell density below 310.sup.6.mL by addition of fresh media and culture vessel exchanges. All cells were restimulated with antiCD3/CD28 magnetic microbeads for 24 hrs starting on day 12. Treg100 group was cultured without rapamycin supplement for the entire 18 day experiment, where Treg100 Rapa groups was cultured with rapamycin for the entire 18 days.

[0594] FIG. 13. CD25FOXP3 expression on MACS-enriched MLN Tregs on days 4, 12 and 18 of 18-day of ex vivo expansion. MLN Treg expansion from FIG. 12 analysed at days 4, 12 and 18 for expression of CD25 and FOXP3. Cells were recovered for counting viable population of each day, and a small aliquot stained for CD25 and FOXP3. Data was acquired on a FACScalibur instrument. Dead cells are excluded by Live/Dead fixable viability stain.

[0595] FIG. 14. Homing receptor pattern on MACS-enriched MLN Tregs after 18-days of ex vivo expansion. MLN Treg expansion from FIG. 12 analysed at day 18 for expression of beta7 and alpha4 integrins and CCR9 expression. Cells were recovered for experimental manipulation, and a small aliquot stained for beta7 and alpha4 integrins and CCR9. Data was acquired on a FACScalibur instrument. Dead cells are excluded by Live/Dead fixable viability stain.

[0596] FIG. 15. Homing recpetor expression and repatterning on MACS-enriched and FACS-purified MLN Tregs after 4-days of ex vivo expansion. Total lymphocytes recovered from MLN of patient with CD were processed using Miltenyi CD4+ T Cell Isolation Kit II and an autoMACSpro instrument. Cells were immediately labeled with CD4, CD25 and CD127 antibodies and sorted on FACSaria II instrument for CD4.sup.+CD25.sup.hiCD127.sup.lo cells. Resulting purified population of Tregs was rested overnight before 24 hr activation with antiCD3/CD28 magnetic microbeads. After 24 hours IL2 and all other indicated stimuli were added, and cultures left for a further 72 hrs. all-trans retinoic acid (RA) was added an 1 nM final concentration, IL2 at 100 IU.mL and microbeads at a ratio of 1:1 beads to cells. TGF in A) was added at 5 ng.mL. Rapamycin was added at 100 nM final concentration. Cells were recovered in 5-volumes of FACS-buffer, washed briefly, and stained for beta7integrin and CCR9 before analysis on FACScalibur instrument.

[0597] FIG. 16. 47 receptor pattern on MACS-enriched MLN Tregs after 18-days of ex vivo expansion and 6-days of receptor repatterning stimulation. MLN Treg expansion from FIGS. 12 and 14 were recovered and aliquoted at 200 k cells per well of a 96-well U-bottom plate in 150 uL medium. Cells were stimulated with 100 IU.mL IL2, and indicated stimuli. Control was unstimulated other than IL2. TGF-beta concentration was 12.5 ng.mL. IL10 concentration was 1 ng.mL. Cell medium and stimuli were refreshed at days 2 and 4 by pipetting 50 L of media SN, and resuspending the entire well with 50 L fresh supplemented media. On day-6 cells were recovered in 5-volumes of FACS-buffer, washed briefly and stained for beta7 and alpha4 integrins, and CCR9, before analysis on FACScalibur instrument. Dead cells are excluded by Live/Dead fixable viability stain.

[0598] FIG. 17. 47 receptor pattern on MACS-enriched MLN Tregs after 18-days of ex vivo expansion and 6-days of receptor repatterning stimulation. Analysis from FIG. 16 represented as beta7integrin vs CCR9.

[0599] FIG. 18. Day-18 expanded SLN Tregs suppress fresh-thawed CD4 Teff proliferation. Cryopreserved total SLN cells were thawed on Day 15 of Treg expansion and cultured for 48 hours in the presence of IL2. On day 17 Teff cells were sorted as CD4+CD127hiCD25lo and stimulated overnight with CD3/CD28 dynal beads. On day 18 expanded Treg cells were harvested and mixed at indicated ratios with bead-depleted and CFSE labelled Teff cells. CFSE fluorescence was assessed by flow cytometry 4 days after initiation of assay with FOXP3 counterstaining, where histogram show FOXP3 cells.

[0600] FIG. 19. CD4.sup.+CD25.sup.hiFOXP3.sup.+ Tregs and CD4.sup.+CD25.sup.loFOXP3.sup. Teffs have altered CD38.sup.+CD62L.sup. distribution in inflamed tissues of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease were immediately stained with the indicated antibodies. All plots display CD38 vs CD62L of CD4.sup.+CD25.sup.hiFOXP3.sup.+ Tregs (right panels) and CD4.sup.+CD25.sup.loFOXP3.sup. Teffs (left panels).

[0601] FIG. 20. CD4.sup.+CD25.sup.hiFOXP3.sup.+ Tregs and CD4.sup.+CD25.sup.loFOXP3.sup. Teffs have altered CD103 and CCR9 expression in inflamed tissues of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease were immediately stained with the indicated antibodies. All plots display CD103 vs CCR9 of CD4.sup.+CD25.sup.hiFOXP3.sup.+ Tregs (right panels) and CD4.sup.+CD25.sup.loFOXP3.sup. Teffs (left panels).

[0602] FIG. 21. CD4.sup.+CD25.sup.hiFOXP3.sup.+CD38.sup.+CD62L.sup. Tregs have altered CD103 and CCR9 expression in inflamed tissues of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease were immediately stained with the indicated antibodies. All plots display CD103 vs CCR9 of CD4.sup.+CD25.sup.hiFOXP3.sup.+CD38.sup.+CD62L.sup. Tregs.

[0603] FIG. 22. Diminished representation of CD45.sup.+CD11c.sup.hiCD80.sup.+HLA-DR.sup.hiCD103.sup.+ DCs in inflamed MLN of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease were immediately stained with the indicated antibodies. All plots display HLA-DR vs CD103 of CD45.sup.+CD11c.sup.hiCD80.sup.+ cells.

[0604] FIG. 23. CCR9 enrichment in CD4.sup.+CD38.sup.+CD62L.sup.4.sup.+7.sup.+ T-cells in the peripheral blood. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Displays gating strategy flow with arrows. B) and C) display total CD4+ PBMCs as reference.

[0605] FIG. 24. CCR9 enrichment in CD4.sup.+FOXP3.sup.+CD25.sup.hiCD38.sup.+CD62L.sup.4.sup.+7.sup.+ T-cells in the peripheral blood. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Displays gating strategy flow with arrows. Left panels display FOXP3.sup.CD25.sup.lo Teffs and right panels display FOXP3.sup.+CD25.sup.hi Tregs.

[0606] FIG. 25. CD4.sup.+4.sup.+7.sup.high T-cells in the peripheral blood are enriched for CD103 and CCR9 expression. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD4 lymphocytes expressed as beta7 vs alpha4 integrin dot plots. Each gate defining alpha4+beta7++, alpha4+beta7+, alpha4beta7 and alpha4+beta7 are redisplayed as CD103 vs CCR9 contour plots in B), C), D) and E), respectively.

[0607] FIG. 26. CD4.sup.+CD103.sup.+ T-cells in peripheral circulation are highly enriched for 4.sup.+7.sup.high expressing T-cells. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD4 lymphocytes expressed as CD4 vs CD103 dot plots. Each gate defining CD4+CD103 and CD4+CD103+ is redisplayed as beta7 vs alpha4 integrin contour plots in B) and C), respectively.

[0608] FIG. 27. CD4.sup.+7.sup.highCD103.sup.+ T-cells in peripheral circulation are enriched for 4.sup.+CCR9 expressing T-cells. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD4 lymphocytes expressed as beta7integrin vs CD103 dot plots. Each gate defining beta7+CD103 and beta7++CD103+ is redisplayed as alpha4 integrin vs CCR9 contour plots in B) and C), respectively.

[0609] FIG. 28. CD4.sup.+4.sup.+7.sup.highCD103.sup.+CCR9.sup.+ T-cells in peripheral circulation contain an enriched proportion of CD38.sup.+CD62L.sup. mucosal emigrants. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD4 lymphocytes expressed as CD103 vs CCR9 dot plots. B) Total CD4 lymphocytes expressed as B7 vs a4 dot plots. C) Total CD4 lymphocytes expressed as CD38 vs CD62L dot plots. D) to F) gates of single and double positive CD103 and CCR9 cells in A) redisplayed as B7 vs a4 (top panels) and CD38 vs CD62L (bottom panels) dot plots.

[0610] FIG. 29. CD4.sup.+CD25.sup.hiFoxP3.sup.+CD38.sup.+CD62L.sup.CCR9.sup.+ T-cells do not express CD45RA. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD4+ lymphocytes expressed as FoxP3 vs CD25 dot plot. B) and C) display CD4.sup.+CD25.sup.hiFoxP3.sup.+ cells as CD38 vs CD62L and CCR9 vs CD45RA dot plots, respectively. D) to G) display CCR9 vs CD45RA contour plots of gated populations from B) as indicated.

[0611] FIG. 30. CD4.sup.+CD25.sup.hiFoxP3.sup.+CD38.sup.+CD62L.sup. T-cells do not contain CD45RA/CCR7 double positives but CD4.sup.+CD25.sup.hiFoxP3.sup.+CD62L.sup.+ T-cells are enriched for CD45RA/CCR7 double positive nave cells. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD4.sup.+ lymphocytes displayed as FoxP3 vs CD25 dot plot. B) and C) display CD4.sup.+CD25.sup.hiFoxP3.sup.+ cells as CD38 vs CD62L and CCR7 vs CD45RA dot plots, respectively. D) to G) display CCR7 vs CD45RA contour plots of gated populations from B) as indicated.

[0612] FIG. 31. T-cell migratory-type surface markers correlated with CD4.sup.+CD62L.sup.CCR9.sup.+ mucosal T-cells. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry in a high throughput screen. A) Total CD4.sup.+ lymphocytes displayed as CD62L vs CCR9 dot plot. B) displays CD4.sup.+CD62L.sup.CCR9.sup. cells as SSC vs 7 contour plot. C) and D) display SSC vs CD195 contour plots of CD4.sup.+CD62L.sup.CCR9.sup.7.sup.+ cells and CD4.sup.+CD62L.sup.CCR9.sup.+ cells, respectively. E) Summary of ranked preferable migratory markers of X.sup.+/X.sup. condition for identification of small bowel tropic T-cells.

[0613] FIG. 32. T-cell functional-type surface markers correlated with CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+ mucosal T-cells. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry in a high throughput screen. A) Total CD4.sup.+4.sup.+7.sup.+ lymphocytes displayed as CD127 vs CD25 dot plot. B) and C) show SSC vs CD39 contour plots of CD4.sup.+4.sup.+7.sup.+CD25.sup.loCD127.sup.hi cells and CD4.sup.+4.sup.+7.sup.+CD25.sup.hiCD127.sup.lo cells, respectively. D) Summary of ranked preferable functional markers of Y.sup.+/Y.sup. condition for identification of regulatory mucosal T-cells. Markers noted with hi in parenthesis indicate that the population with high expression of the indicated marker is of interest, indicating that both low and negative expression populations also exist.

[0614] FIG. 33. Purification of a distinct subset of CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiCCR9.sup.+ Tregs from peripheral blood.

[0615] PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and purified by fluorescent-activated cell sorting (FACS). Pseudocolor plots from A) to C) show lymphocytes gated from total PBMC in A) and subsequent sub-gates for single cells in B) and CD4+ cells in C). Plot D) redisplays total CD4+ lymphocytes as Beta7integrin vs CCR9. The sub-population Beta7integrin.sup.HiCCR9.sup.+ defined in plot D is re-displayed in plot E) as CD127 vs CD25 to define the sub-population CD25.sup.HiCD127.sup.lo of T-regs. Plot F) and G) show the enrichment of the rare Beta7integrin.sup.hiCCR9.sup.+CD25.sup.hiCD127.sup.lo population of CD4 cells sorted according to the gating strategy outlined in plots A) to E) and re-analyzed by flow cytometry for the degree of sort purity. Panels H) and I) shows a parallel analyses of B7.sup.hiCCR9.sup.+ cells with CD38 vs CD62L dotplots of panel I) being the daugthers of populations gated in panel H).

[0616] FIG. 34. Expansion of sorted CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiCCR9.sup.+ Tregs in culture. The subset of CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiCCR9.sup.+ T-cell purified by FACS was cultured over several days. The proliferation curve displays the expansion of a starting pool of 5000 sorted CD4.sup.+CD25.sup.hiCD127.sup.loCCR9.sup.+Beta7.sup.Hi T-cell reaching almost 3 billons cells over 20 days of culture.

[0617] FIG. 35. Peripheral CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+CCR9.sup.+ T-cells have skewed V usage compared to CD4.sup.+CD25.sup.hiCD127.sup.lo cells. PBMC recovered from blood of three healthy donors over ficoll were immediately labelled with CD4, CD25, CD127, CD38, CD62L, CD49d, 7 and CCR9 antibodies in addition to panels of V-specific antibodies and analysed by flow cytometry. Coverage of donor C12 (top panel) was CD4.sup.+CD25.sup.hiCD127.sup.lo 75% and CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+CCR9.sup.+ 95%. Coverage of donor C6 (middle panel) was CD4.sup.+CD25.sup.hiCD127.sup.lo 72% and CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+CCR9.sup.+ 77%. Coverage of donor C26 (bottom panel) was CD4.sup.+CD25.sup.hiCD127.sup.lo 77% and CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+CCR9.sup.+ 93%.

[0618] FIG. 36. Investigational model for identification of diseased tissue-specific Treg. Model of investigational elements for identification of Treg cells originating from tissue Target (A), migrating through lymphatics (B, B and B), and subsequently migrating through peripheral blood (C) to recirculate to tissue of origin (A). Alternate route of recirculation is to a secondary tissue of interest (X). Tissues A, B, B, B, C and X are considered major sampling points for investigation. Major migratory processes involving recirculation of Tregs back to tissue of origin are defined for target tissue emigration (), and immigration (). Emigration from secondary tissue of interest (X) is defined as X, and immigration X. Migration between main target tissue A, and communicating tissue X, is defined as migration process . Migration processes , , X, X and are considered as definable by Treg migratory marker expression, as applied to Tregs recovered from sampled tissues A, B, B, B, C and X. Major sources of analytical noise are those cells collected in all compartments that bare migratory marker expression for migratory processes and . and migratory process markers are considered negative selection criteria for investigation of Treg cells of A or X origin, with , , X, X or migratory behaviour.

[0619] FIG. 37. CD103.sup.+ and/or CCR9.sup.+CD4.sup.+ T-cells are present in large bowel LP at low frequency compared to small bowel LP. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease were immediately stained with the indicated antibodies. Plots display CD103 vs CCR9 of CD4.sup.+ gated cells.

[0620] Based on experiments, the inventors have made the following observations: CD4 Tregs in the human peripheral blood may be identified and analytically and/or physically enriched through small-bowel tropic cell surface marker sets, and these CD4 Treg cells are strongly diminished in numbers within the inflamed tissues of CD patients. In additional the mechanistic basis of this immunological defect in CD patients is proposed to embody a numerical deficiency in CD103.sup.+ DC in inflamed tissues of CD patients. Mucosal emigrant and mucosal tropic Tregs as defined by the presented marker sets are considered as therapeutic candidates for the management of CD and other IBDs. Cells of the various identified compositions can be non-invasively recovered from peripheral blood preparations an expanded in vitro. Preparing targeted Treg subpopulations in this manner is proposed to restrict TCR clonal diversity to clonotypes specific for tissue-associated antigens. This supported by a skewed V usage within the mucosal CD4+ Treg populations observed in peripheral circulation, when compared to non-mucosal Treg populations. These findings yield a practical method for prospecting any diseased target tissue, though particularly mucosal and skin tissues, to identify cells that are emigrant from said diseased tissue. Preparing targeted Treg subpopulations in this manner is proposed to restrict TCR clonal diversity to clonotypes specific for tissue-associated antigens, making these Treg fractions most suitable for starting materials for manufacture of cellular immunotherapies to treat inflammatory and autoimmune disorders of any tissue under investigation. This method also allows consideration of primary diseased tissue and the establishment of secondary inflammatory and autoimmune conditions in tissue with migratory lymphocyte communication, and offers a framework that would allow treatment of a disease in such a communicating tissue through treatment of the primary diseased tissue.

[0621] Firstly the inventors addressed whether CD4.sup.+FOXP3.sup.+ T.sub.regs in the human gut displayed higher CCR9 expression than CD4.sup.+FOXP3.sup. counterparts. FIG. 1A shows flow cytometry analyses of single cell suspensions prepared from relatively proximal healthy tissue of the small intestine of a representative CD patient. CD4.sup.+FOXP3.sup.+ cells show greater CCR9 expression relative to CD4.sup.+FOXP3.sup. Teffs in both small and large bowel LP (mucosal lamina propria). CCR9 marking of CD4.sup.+FOXP3.sup.+ cells is greater overall in the small bowel LP, consistent with the regional differences in CCR9 marking reported previously (Papadakis et al. Gastroenterology 2001, 121, pp 246-254). The CD4.sup.+FOXP3.sup.+ population also carried a greater proportion of cells with integrin 7.sup.hi phenotype, particularly in the small bowel LP. Cells with a CD4.sup.+FOXP3.sup.+7.sup.hi phenotype almost exclusively expressed CCR9 (FIG. 1B). The preferential marking of CD4.sup.+FOXP3.sup.+ cells was also reflected in the mesenteric lymph nodes (MLNs) draining the small bowel (FIG. 2). Tregs more stringently gated on CD4.sup.+CD127.sup.loCD25.sup.hiFOXP3.sup.+ phenotype(9), similarly have higher CCR9 expression than Teffs with CD4.sup.+CD127.sup.hiCD25.sup.loFOXP3.sup. phenotype in small bowel MLN (FIG. 3); and in peripheral blood after further analytical enrichment by gating on 7.sup.+ (FIG. 4). These results are consistent with a stronger CCR9-dependent small-bowel tropism of Tregs compared to Teff counterparts from human gut. Moreover, direct parallels can be drawn with recent results regarding CCR9 expression on mouse T-cells, and the CCR9-dependency of oral tolerance (Wermers et al. Gastroenterology 2011, 140 (5), pp. 526-1535, and Cassani et al. Gastroenterology 2011, 141 pp 2109-2118).

[0622] These observations reveal some interesting features of the co-expression of markers in the small and large bowel mucosal lamina propria (LP), and by association the MLN draining these tissues. Specifically, the presence of a 7-high population dominantly in small bowel is suggestive of the presence of and additional integrin pairing within this small bowel population. At the time it was speculated that this could be the E7 pairing, which is addressed and confirmed herein.

[0623] Strikingly, the 7-high population in LP has the almost exclusive CCR9 marking in the healthy tissue analysed here. Thus 7-high and its integrin co-markers represent a proxy CCR9 marker at least in these tissues. This has implications for the understanding of fundamental migration mechanisms of T-cells in humans, but also provides analytical and therapeutic possibilities as mentioned herein.

[0624] Mucosal-Educated FOXP3.sup.+ Tregs in Peripheral Circulation Carry Higher CCR9 Marking than FOXP3.sup. Teffs and CD Patients have Diminished Overall CCR9 Marking on Mucosal-Educated T-Cells

[0625] The inventors excluded that preferential CCR9 marking was only occurring on resident Tregs in the LP and MLN, by investigating CCR9 expression of both mucosal-educated and mucosal-tropic Tregs in peripheral blood. A CD38.sup.+CD62L.sup. phenotype was used to enrich T-cells educated within the intestinal mucosa in flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) and CD patients with small bowel disease. Observations confirmed the enrichment of 7 and CCR9 marking on CD38.sup.+CD62L.sup. T-cells, and showed that the CD38.sup.+CD62L.sup.7.sup.+ subpopulation is further enriched for CCR9 positivity (FIG. 5A). Furthermore, T-cells of CD38.sup.+CD62L.sup. phenotype predominate in both the human small and large bowel LP (FIG. 6). CD4.sup.+FOXP3.sup.+ Treg numbers in circulation are not significantly diminished in CD patients compared to healthy controls as previously reported, although trend in this direction (FIG. 5B). There is no significant difference in the total number of mucosal-educated CD4.sup.+CD38.sup.+CD62L.sup. Tregs between CD patients and healthy controls, while overall CCR9 positivity in this sub-population is strikingly reduced in CD patients (FIG. 5C). Preferential CCR9 marking of CD4.sup.+FOXP3.sup.+ T.sub.regs over CD4.sup.+FOXP3.sup. effectors is observed in CD38.sup.+CD62L.sup.7.sup.+ mucosal emigrants present in peripheral circulation of healthy controls (HC). This preferential CCR9 marking of Tregs is maintained in CD patients, however the overall numbers of CCR9.sup.+ Tregs and non-Tregs are diminished in the mucosal-educated sub-population relative to healthy controls (FIG. 5D).

[0626] There are two intriguing aspects to this directed analysis of peripheral recent mucosal emigrants. The overall numbers of Tregs in peripheral circulation is trending towards a deficiency in CD compared to healthy controls. However, when we look only at the recent emigrants that are also likely destined for recirculation to mucosa (CD38.sup.+CD62L.sup.7.sup.+), then we actually observe a trend towards increased numbers of Tregs. In addition these recent emigrant and recirculating T-cells have a marked reduction in CCR9 expression. Taken together, these data suggest that CD is not a disease defined by a deficiency of Tregs per se, but a deficiency in their ability to recirculate to the small bowel. This could also be construed as a strong retention of CCR9-carrying T-cells in the inflamed bowel of CD patients.

[0627] It is possible that the Treg population under the strong inflammatory conditions in the inflamed lamina propria is undergoing atrophy. The general inflammatory state would also draw more nave Tregs from peripheral circulation, decreasing the apparent incidence of total Tregs in the periphery. It appears that T-cells can still be exported from the inflamed mucosa-associated lymphoid tissues (i.e. MLN), though lack the important chemokine receptor trigger (CCR9) to facilitate re-import to the inflamed mucosa.

[0628] CD4.sup.+4.sup.+7.sup.+ Mucosal-Tropic FOXP3.sup.+ Tregs in Peripheral Circulation Carry Higher CCR9 Marking than FOXP3.sup. T.sub.effs and CD Patients have Diminished CCR9 Marking on Mucosal-Tropic T-Cells

[0629] In addition to investigating CCR9 expression on mucosal-educated T-cells having emigrated to peripheral circulation, the total pool of mucosal-tropic T-cells in peripheral blood was examined through analytical enrichment of 4.sup.+7.sup.+ expressing CD4.sup.+ T-cells. We show that T-cells with this 4.sup.+7.sup.+ phenotype in peripheral circulation are highly enriched for CCR9 positivity (FIG. 7A-B); indeed on average greater than 75% of all CCR9.sup.+ cells in peripheral circulation carry 4.sup.+7.sup.+ expression (FIG. 8). Surprisingly, even though the total number of CD4.sup.+4.sup.+7.sup.+ T-cells is similar in healthy controls and CD patients (FIG. 9), CD4.sup.+FOXP3.sup.+ Treg numbers are relatively increased in this mucosal-tropic subpopulation in the peripheral blood of CD patients (FIG. 7C). The CD4.sup.+FOXP3.sup.+ Tregs in the peripheral mucosal-tropic population carry significantly higher CCR9 marking when compared to CD4.sup.+FOXP3.sup. Teffs in both healthy controls and CD patients, while overall CCR9 positivity is diminished in CD patients (FIG. 7D). These results strongly suggest that there are relatively more Tregs in the peripheral circulation of CD patients with the capacity to traffic to mucosal tissues, but these mucosal-tropic cells lack small-bowel specificity due to reduced levels of CCR9 expression. Explanations may be either a defect in CCR9 imprinting in the LP/MLN of CD patients, or a strong migration of CCR9-expressing T-cell subclasses to the inflamed small bowel mucosa in CD patients.

[0630] This data for the total pool of mucosal-tropic T-cells is in agreement with the data above regarding recent mucosal emigrants, revealing a striking defect in CCR9 expression on Tregs from CD patients. More interestingly, while the total numbers of mucosal-tropic T-cells are similar, the percentage of Tregs is actually higher. This is in contrast with the proportion of Tregs among all CD4 cells in circulation, and to the concept that CD is a disease defined by a deficiency in Treg populations. This data further supports the notion that CD is defined by Tregs being unable to recirculate to the small bowel mucosaand indeed there is an apparent excess of mucosal-targeted Tregs available.

[0631] Treg Purification

[0632] Two different purification strategies were used to purify Treg populations from mesenteric lymph nodes of patients, a full MACS enrichment, or a MACS pre-enrichment followed by FACS purification. The recovered populations are highly enriched in cells with the desired characteristics. That is, of character CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+CCR9.sup.+, simply owing to their MLN origin (see FIGS. 1, 2, 3 and 6). Both of these enrichment and purification types were used to study Treg cell growth, homing receptor pattern stability and dynamics, and functionality, ex vivo. The enrichment/purification methods used in the present context are for illustrative purposes due to limiting yields for larger scale preparative experiments from peripheral blood. Purification of target populations from peripheral blood is presented below. Other suitable methods enrichment and purification may also be applied.

[0633] MACS 2-Step Enrichment of Tregs from MLN

[0634] For research-graded preparations of Tregs from MLN and other tissues Miltenyi MACS approaches have primarily been used. FIG. 10 presents purification data using a Regulatory T Cell Isolation Kit II and an autoMACSpro instrument. This procedure uses a negative affinity selection on non-CD4-expressing and CD127-expressing cells. A subsequent positive affinity selection purifies the final CD4.sup.+CD127.sup.loCD25.sup.hi population based on CD25 labelling.

[0635] MACS Enrichment and FACS Purification of Tregs from MLN

[0636] A second means of Treg purification from MLN material is a 2-step approach using Miltenyi MACS enrichment of total CD4+ cells, then FACS purification on various parameters including CD25 and CD127 (FIG. 11). The pre-enrichment was routinely conducted due to limitations in both FACS instrumentation and biological reagent resources at the time this work was conducted.

[0637] Treg Expansion

[0638] The expansion of Tcells ex vivo utilises standardised methods that rely on stimulation of three core T-cell activating inputs. The minimal signals that a Tcell requires to activate and proliferate are stimulation of the TCR (aka CD3), co-stimulation via CD28 receptor, and a secondary mitogenic stimulation via Interleukin-2 receptor (IL2R). Standardized approaches use antibodies to stimulate both CD3 and CD28. Recombinant IL2 is used for the mitogenic input. Various modifications of the general method are used with regard to the vehicle, dosage and duration of CD3 and CD28 stimulation, in addition to the subsequent IL2 input. A general method is described in detail in the methods section, but it is believed that the T-cells can be expanded by generic methods. FIG. 12 shows typical ex vivo expansion curve of MACS-purified Tregs from patient MLN, where approximately 120- to 150-fold population expansion is achieved over 18 days of culture. Rapamycin was included in selected cultures to assess impact on Treg growth and culture stability. Rapamycin is routinely used to limit the growth of Teff subclasses in human Treg cultures, and thus polarise to a Treg phenotype over time. Three different Rapamycin dosing strategies were used here, where it was present for the entire 18 days, or just the first 8 or 15 days of expansion. In our hands Rapamycin strongly inhibited Treg growth (FIG. 12), and did not promote Treg characteristic stability (FIG. 13). The reason for this may be the MLN source of the cells, where we expect a bulk to be induced/experience Tregs, and not nave Treg precursors isolated from peripheral circulation as investigated by others. In addition to the basic CD25FOXP3 parameters presented in FIG. 13, we monitored CD127 levels, which mirrored the CD25FOXP3 population. Moreover, we expanded and manipulated Teff populations in parallel to ensure accurate comparison of what were likely to be stable Treg cells (not shown). These complimentary analyses suggest that the CD25FOXP3 expression was indeed indicative of a true Treg phenotype, and not a sole result of strong activating stimuli.

[0639] Despite a natural enrichment for cells of mucosal emigrant and mucosal-tropic characteristics in our MACS-enriched Treg cultures (refer FIGS. 2 to 8), we found that after 18 days of expansion the resultant T-regs were almost devoid of small-bowel tropic homing receptor expression (FIG. 14). In vivo, these receptors are imprinted by tolerogenic stimuli provided directly by DCs. In the absence of such inputs to maintain receptor expression, it is clear why we observe little or no receptor expression after 18 days of culture

[0640] This absence of gut-tropic homing receptors presents a caveat for our method of therapy. Namely, we select the Treg populations based on their inherent homing characteristics, however, once expanded in vitro by generic methods, they lack the qualities required to traffic back to the diseased bowel on re-infusion to the patient. It has been established, mainly using mouse tissues, that T-cells can be forced to express small-bowel homing receptors including 4 and 7 intergrins, and CCR9.

[0641] In the following a simple set of observations outlines the considerations and method behind homing receptor repatterning after expansion.

[0642] Treg Receptor Re-Patterning

[0643] To determine the culture parameters required for receptor repatterning on ex vivo expanded Tregs, we first used short term (4 days) growth of Treg cells in the presence of absence of the tolerogenic stimuli ATRA (all-trans retinoic acid, also referred to as RA in some figures), and transforming growth factor-beta (TGF or TGF-beta). In addition, we assessed the impact of Rapamycin on the expression of homing receptors of the short-term, and on the tolerogenic induction of homing recptors.

[0644] FIG. 15a shows 4-day expansions of MACS-enriched and FACS-purified Tregs in the presence of the indicated stimuli. The top left panel shows cells activated and grown in a generic manner without further stimulation. These cells retain significant levels of 7 intergrin and CCR9 co-expression. Strikingly, the addition of Rapamycin (RAPA) strongly attenuates the expression of 7 intergrin and CCR9 (bottom left panel). Addition of TGF/ATRA to these cultures strongly induces 7 intergrin, but not CCR9 (top right). Rapamycin attenuates CCR9 expression, but not 7 intergrin (bottom right).

[0645] FIG. 15b demonstrates that increasing concentrations of TGF on a background of ATRA stimulation can almost completely polarise Tregs towards a 7.sup.+CCR9.sup.+ phenotype. Rapamycin addition almost completely abrogates CCR9 positivity in this system.

[0646] These experiments establish the basic parameters of receptor repatterning, in as much that we now appreciate that any such approach should be done in the absence or Rapamycin, and with minimal ATRA and TGF stimuli. FIG. 16 shows day-18 expanded MACS-enriched Tregs that have been stimulated for a further 6 days with combinations of three tolerogenic stimuli. These stimuli were part of a much larger titration, and are presented in this focused context for clarity. FIG. 16a shows that the lack of 47 expression on Tregs after 18-days of expansion (refer FIG. 14) persists after 6 further days of culture without additional stimuli. ATRA alone is sufficient to induce 47 expression in a dose-dependent manner (FIG. 16c,d). IL10 is sufficient to weakly induce 7 but not 4 integrin expression (FIG. 16e), an effect that appears to be anergised by TGF (FIG. 16g). The most effective stimuli for 47 induction were found to be low doses ATRA in combination with IL10, or IL10 and TGF (FIG. 16h, i).

[0647] The expression of CCR9 on these stimulated cells was further assessed in parallel, which is displayed against 7 expression in FIG. 17. Several important interactions of the stimuli can be observed when considered in context with the above 47 data. First, while TGF alone is insufficient to induce 47 expression (FIG. 16b), it is a moderately effective inducer of CCR9 expression at the dosage presented (FIG. 17b). While IL10 is a strong inducer of CCR9 expression ((FIG. 17e), TGF and IL10 appear to have an anergistic effect when provided as co-stimuli (FIG. 17g). ATRA alone is a moderate inducer of CCR9 expression (FIG. 17c,d), an effect that may be marginally enhanced in combination with IL10 (FIG. 17h). However, all three tolerogenic stimuli in combination have a strong polarising effect on CCR9 expression, while maintaining a correct 47 pattern (FIG. 17i) and (FIG. 16i).

[0648] Overall, conditions were established with which to repattern the correct 4.sup.+7.sup.+CCR9.sup.+ homing characteristics on ex vivo expanded Tregs. A combination of low-dose ATRA, TGF and IL10 is sufficient to achieve correct homing receptor patterns with acceptable efficiency.

[0649] Treg Functionality

[0650] The standard manner in which to test the immunosuppressive functionality of Tregs in vitro is a mixed culture assay. Part of Treg immunosuppressive function is to suppress Teff cell division, we thus measure the degree of Teff cell division in the presence of Treg cells. In our setup we used partially purified Teffs from MLN to test the functionality of ex vivo expanded Tregs from the same patient. Teffs are labeled with carboxyfluorescein succinimidyl ester (CFSE), which is a strongly fluorescent compound that is taken up by the Teff cells. Rapid crossing of the plasma membrane is facilitated by the succinimidyl group, which is subsequently cleaved by intrinsic cellular esterase activity, ensuring retention of the fluorescent carboxyfluorescein in the cell. On cell division, the diffuse carboxyfluorescein is partitioned approximately equally between the two resulting cells. Therefore, one can monitor the cell division of Teff cells in vitro in the presence of (unlabeled) Tregs by monitoring step-wise decrease in Teff fluorescence intensity.

[0651] FIG. 18 shows the suppression of freshly purified Teff cells by 18-day expanded autologous Tregs. This demonstrates the suppressive capacity of expanded Treg cells in this system.

[0652] Mucosal Emigrant and Homing Markers on Small Bowel Tregs in Inflamed CD Patient Tissues

[0653] The experimental data presented above regarding purification and expansion of Tregs utilised disease-draining SLN as Treg source material. It is reasoned that SLN will be highly enriched in Tregs with relevant TCR clonotypes, considering their physical disease and/or tissue association. Moreover, SLN and MLN in general will be highly enriched for the tropic and emigrant populations that are of general interest for therapeutic purposes, with their observed deficiencies in peripheral blood. While the SLN are indeed highly enriched for these populations when compared to the peripheral blood compartment, close analysis of patient material provides further insights into the observed peripheral deficiency of CCR9-expressing Tregs.

[0654] FIG. 19 shows the contour plots of CD38 vs CD62L of total CD4+FOXP3CD25lo Teffs (left panels) and CD4+FOXP3+CD25hi Tregs (right panels) recovered from resected tissues of a representative CD patient with ileocaecal disease. Normal MLN in the surgical field, two SLN (1 and 2), inflamed lamina propria (LP) and normal LP is presented from top to bottom. When observing both Tregs and Teffs from normal MLN one can see the expected CD38 positivity and CD62L negativity that correlates with mucosal emigrant populations. This population is more strongly represented in the Treg cells. In both SLNs analysed, the proportion of CD62L+ cells, that is likely to represent direct MLN immigrants from peripheral circulation, is notably increased in both Treg and Teff populations. This may either be due to increased direct immigration to the SLNs compared to healthy MLN, or relatively diminished trafficking of cells from the LP to the SLN. In the four bottommost panels we can observe significant CD62L+ cell numbers in the inflamed LP, but not adjacent healthy tissue. Again, this could reflect a strong and aberrant immigration of cells from circulation, and/or a poor patterning of correct receptors of the T-cells within the LP.

[0655] To test whether correct patterning was being achieved on T-cells in inflamed and adjacent healthy CD tissue, CD103 and CCR9 expression was assessed. FIG. 20 shows CD103 vs CCR9 contour plots of total CD4+FOXP3CD25lo Teffs (left panels) and CD4+FOXP3+CD25hi Tregs (right panels) recovered from resected tissues of a representative CD patient with ileocaecal disease from tissues as above. In normal MLN one can immediately observe the fundamental difference between Teffs (left) and Tregs (right), in that Tregs in the MLN carry very high levels of CD103 and CCR9 and Teffs do not. This could indicate that Tregs are more attuned to trafficking from LP to MLN, and/or they are more likely to be patterned in this way by MLN DCs. The latter is consistent with the tolerogenic function of CD103+ DCs that pattern both CCR9 and CD103 expression. The second striking contrast that may be observed here is the complete loss of both CD103 and CCR9 expression on cells in disease-draining SLN. Moreover, a similar though less dramatic decrease in CD103 and CCR9 expression can be observed in the inflamed LP compared to the healthy tissue. Interestingly, it is the Teff cells that are most strongly expressing CD103 and CCR9 in the normal LP, not the Tregs as in the normal MLN. This may indicate that Teffs are more likely to be retained in the LP than the Tregs, and thus express high levels of the retention integrin CD103. This is consistent overall with the balance of Tregs and Teffs in these two compartments.

[0656] To ensure that the observed loss of CD103 and CCR9 expression was not simply due to the dilution of these cells by immigrants from circulation (see FIG. 19), CD38.sup.+CD62L.sup. Tregs were analysed in the same compartments as FIG. 20. FIG. 21 shows a similar analysis of Tregs as in FIG. 20, though a further analytical enrichment of CD38.sup.+CD62L.sup. was used. These data demonstrate that there appears to be a deficiency in CD103 and CCR9 expression on Tregs in the inflamed tissues, despite expression of a mucosal phenotype (CD38.sup.+CD62L.sup.). One can also note the relative enrichment in the expression of CD103 and CCR9 (FIG. 21) when compared to the right hand panels of FIG. 20.

[0657] The data presented above collectively suggest a defect in the CD103 and CCR9 patterning of T-cells within inflamed tissues of CD patients. The DC subset that is responsible for patterning this receptor expression on T-cells are known to be a CD103+ DC subset. The possibility of a numerical deficiency in this CD103+ DC population was tested as a possible cause of CCR9 and CD103 deficiency. FIG. 22 shows analysis of CD45.sup.+CD11c.sup.hiCD80.sup.+HLA-DR.sup.hiCD103.sup.+ DC in the healthy MLN and disease-draining SLN of a CD patient. Strikingly, there is huge numerical deficiency in the CD103+ subset of HLA-DR.sup.hi DC cells in the SLN. Sufficient cell numbers could not be recovered for a reliable analysis of inflamed and normal LP from this patient. However, limited analyses show a similar trend (not shown).

[0658] Identification of Mucosal Emigrant and Small Bowel Tropic Tregs in Peripheral Blood

[0659] Aside from the obvious practical limitations of harvesting Tcell material from SLN for therapeutic applications, and the strong CCR9 expression defect observed in some patients even within inflamed tissue, the recovery of mucosal emigrant and small bowel tropic Tcells (Tregs?) directly from peripheral blood is attractive. In FIGS. 5 and 7, these populations were treated somewhat separately. While mucosal emigrants were defined as CD38.sup.+CD62L.sup.7.sup.+, this does not fully embody a fully pure candidate for sorting of this target population from peripheral blood. FIG. 23a shows the full gating strategy used for analytical identification of mucosal emigrant and small bowel tropic Tcells in peripheral blood. Full enrichment is achieved by the addition of alpha4 integrin to the staining panel, allowing for exclusion of 7.sup.+ cells that are 4 negative, a major contaminant using just 7+ criteria. An antibody for staining of a shared 47 epitope was not available for this study, though the two dimensional plot of these parameters reveals novel relationships as described in subsequent sections. FIGS. 23b and 23c show the staining of total PBMCs for the enriched parameters, demonstrating the strong enrichment of desired cell populations.

[0660] FIG. 24 displays the use of the gating strategy to analytically enrich Teffs and Tregs for comparison. Here FOXP3 vs CD25 is used to identify fixed cells. Exchanging FOXP3 for CD127 can be used to target viable cells. The very high enrichment of the target 4.sup.+7.sup.+CCR9.sup.+ small bowel tropic population can be seen within the total CD4 T-cell population (refer FIG. 23), and compared to the Teff population.

[0661] Addition of CD103 Mucosal Retention Marker Identifies Peripheral T-Cell Subsets

[0662] In the above data was presented that indicated two distinct populations of T-cells with regard to their positive expression of 7integrin, a 7.sup.+ and a 7.sup.hi population. It was shown that the 7.sup.hi population in both the small and large bowel LP displayed almost 100% CCR9 positivity, in healthy tissue. The quantitative difference in 7 expression is clearly due to the co-expression of a second integrin pair, in this case E (CD103). The majority of 7.sup.+ cells were presumed to be cells expressing solely the 47 pair, while 7.sup.hi cells express higher levels of 7 owing to the fact that they require additional 7 to pair with E, suggesting 7.sup.hi cells express both the 47 and E7 integrin pairs. The significance of this is that 47 is thought to be required for migration into mucosal tissues, while E7 is required for retention.

[0663] Given that E7 is regarded as a retention marker for mucosal T-cells, it is obvious why we observe high levels 7.sup.hi cells in mucosal tissues. What was not clear is why we observed a significantly greater proportion of 7.sup.hi cells in the small bowel, when compared to the large bowel (FIG. 1). An explanation comes from co-expression of CCR9 on 7.sup.hi cells. It is known that both E and CCR9 are strongly induced by a similar set of stimuli, including ATRA and TGFbeta, which are likely to be provided by CD103.sup.+ DCs in the LP and MLN environments. Cells recovered from the bowel LP thus represent cells that are being environmentally imprinted with 47 and E7 integrin pairs and CCR9. Interestingly, CD103 is often cited as being of higher expression on CD4 Tregs, and has been proposed to be a defining marker of a subset of CD8 Tregs. This relationship was investigated in peripheral blood. Although CD103/E is considered a mucosal retention marker, one does indeed observe CD103 positive cells in the peripheral blood. This is because the retention of E7-expressing cells within mucosal tissue is likely to be directed by short-range homing. That is, cells that emigrate from the local mucosal tissues into blood stream will dominantly and selectively re-enter mucosa when expressing E7 due to binding of cognate receptors (E-cadherin) on local HEVs.

[0664] FIG. 25a presents an analysis of blood from a healthy donor where CD4 cells are gated and displayed as 7 vs 4 dotplots, as in FIGS. 7 and 8. Our expectation is that CD4 cells with a 4.sup.+7.sup.hi phenotype will be highly enriched for CD103, and naturally CCR9 as a strongly co-expressed marker (note, figures designate 7.sup.hi as B7++). Cells within gates presented in FIG. 25a are displayed as CD103 vs CCR9 contour plots in FIG. 25b to FIG. 25e. As anticipated, the 4.sup.+7.sup.hi population is highly enriched for CD103, with some 80% of all cells expressing CD103. This population is also highly enriched for CCR9 expression (FIG. 25b).

[0665] To confirm and expand the relationship between CD103 expression and the expression of 4 and 7 integrins, one can treat the same data in a differing manner. FIG. 26a simply shows gated CD4 cells as a CD4 vs CD103 dotplot. From here, total gated CD103 and CD103+ cells are displayed a 7 vs 4 dotplots in FIGS. 26b and 26c respectively. The negative population appears as a standard pool of CD4 T-cells with regard to 7 and 4 expression, although strikingly lack the expression of a 4.sup.+7.sup.hi population (FIG. 26b). In contrast the CD103+ population is highly enriched for the 4.sup.+7.sup.hi (FIG. 26bc, and compare FIG. 25a). We can also observe the enrichment of cells of another rare population, those that carry 7 expression, but lack 4. The tissue origin of these cells that likely express the E7 pair in the absence of 47, is unclear.

[0666] Finally, this data can be used to visualise the quite clear expression of CD103 on the 7.sup.hi population (FIG. 27a). This CD4.sup.+7.sup.hiCD103.sup.+ population is highly enriched for 4.sup.+CCR9.sup.+ cells (FIGS. 27b and 27c).

[0667] Overall, these simple analyses demonstrate an analytical enrichment strategy for identifying CD4.sup.+4.sup.+7.sup.hiE.sup.+CCR9.sup.+ cells in the peripheral blood. These cells are likely to represent mucosal emigrants with a very strong propensity to recirculate to the small bowel. Therefore one could predict the CD4.sup.+4.sup.+7.sup.hiE.sup.+CCR9.sup.+ population to be highly represented in the CD38.sup.+CD62L.sup. population. This is confirmed in the analyses presented in FIG. 28. Panels B) and C) display the now familiar parameters of total CD4 T-cells in a 7 vs 4 dotplot and CD38 vs CD62L dotplot, respectively, while panel A) displays CD103 vs CCR9 dotplot of total CD4 T-cells. Single positive CD103 cells are redisplayed in panel D), CD103 CCR9 double positives in panel E), and CCR9 single positives in panel F). These populations reveal the expected 4 and 7 staining consistent with above analyses. Both of the CD103 CCR9 double positive and CCR9 single positives show an enrichment of the CD38.sup.+CD62L.sup. mucosal emigrant population. CCR9 single positives are also enriched for CD38.sup.+CD62L.sup.+, and are very likely to represent recent thymic emigrants.

[0668] In summary, the preliminary identification of CD4.sup.+4.sup.+7.sup.hiE.sup.+CCR9.sup.+ population in peripheral blood, which is likely to represent mucosal emigrants with a strong propensity to recirculate to the small bowel, presents a further means to identify Treg cells based on homing receptor patterns for adoptive immunotherapy. Coupled to Treg markers and the CD38CD62L marker sets, we are able to identify the signatures described with therapeutic potential in Crohn's disease, in particular the following two overlapping subsets of Tregs. [0669] 1) CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.highE.sup.+CCR9.sup.+ [0670] 2) CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+

[0671] The significance of the CD103+CD4 Treg population is underscored by the recent work defining the role of CCR9 in establishment of oral tolerance. A new theory suggests that dominant recirculation of iTregs from the LP back to the LP is required for establishment of oral tolerance in a CCR9-dependent manner. Thus, CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.highE.sup.+CCR9.sup.+ Tregs could represent a Treg population that makes a major contribution to intestinal homeostasis, despite their low numbers in the periphery.

[0672] To further confirm both thymic emigrant nature of CD4.sup.+CD38.sup.+CD62L.sup.+ cells, and indeed the expected antigen-experienced nature of CD4.sup.+CD38.sup.+CD62L.sup. cells, the expression of CCR7 and CD45RA was analysed on these subpopulations. Firstly, FOXP3.sup.+ Tregs gated for CD38.sup.+CD62L.sup.+ were most highly enriched fro CD45RA expression (FIG. 29e), supporting their enrichment of nave cells. In contrast, CD38.sup.+CD62L.sup. cells expressed little CD45RA, and were enriched from CCR9 expression (FIG. 29d). The recent thymic emigrant nature of CD4.sup.+CD25.sup.hiCD127.sup.loCD38.sup.+CD62L.sup.+CCR9.sup.+ cells was further confirmed by the high enrichment of CCR7 expression within this population (FIG. 30e).

[0673] In order to more define the recent mucosal emigrant population of Treg cells of the small bowel, a high throughput screen was conducted using CD4.sup.+CD62L.sup.CCR9.sup.+ as the test population (small bowel emigrant and tropic) and CD4.sup.+CD62L.sup.CCR9.sup.7.sup.+ as the generally mucosal-tropic reference population. FIG. 31 shows an example of different adhesion molecule expression in the CD4.sup.+CD62L.sup.CCR9.sup.+ population in comparison to the CD4.sup.+CD62L.sup.CCR9.sup.7.sup.+ population that is targeted to mucosal tissues in general (FIG. 31A to D). In this example, CD195 (CCR5) is almost absent in the CD4.sup.+CD62L.sup.CCR9.sup.+ population. It is thus anticipated that CD195 may be used as a marker of preferred condition X, with which to select for mucosal emigrant, immigrant and educated CD4+ Treg cells with small bowel tropism. The table presented in FIG. 31E summarises other migratory-type markers associated with the CD4.sup.+CD62L.sup.CCR9.sup.+ population. The markers positively correlated are of condition X+ and the markers negatively correlated are of condition X. In the preferred aspect markers denoted X+ are used as a positive selection marker and markers denoted X are used as a negative selection marker for the purification of mucosal emigrant, immigrant and educated CD4+ Treg cells with small bowel tropism. Each marker in this table is also assigned a class, where class 1 represents a strong association with the CD4.sup.+CD62L.sup.CCR9.sup.+ population and high functional significance. Class 2 represents a strong association with the CD4.sup.+CD62L.sup.CCR9.sup.+ population or high functional significance. Class 3 represents weak association and/or uncertain functional significance.

[0674] The aforementioned markers relate to tissue localisation, emigration, immigration and retention. In a similar experiment a high throughput screen was conducted to identify functional markers that are enriched within mucosal-tropic Treg populations when contrasted against mucosal-tropic cells that are non-Treg in nature. Analyses of cells with CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+ character revealed strong enrichment of surface markers that denote regulatory function, and a restriction of markers that generally denote proinflammatory functions.

[0675] FIG. 32 shows an example of a functional marker, CD39 (ENTPD1), which is a putative immunosuppressive element on the surface of T-cells, and which is enriched in the CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+ population. It is thus anticipated that CD39 may be used as a marker of preferred condition Y+, with which to select for Treg cells within mucosal emigrant, immigrant and educated CD4+ T-cell populations. The table presented in FIG. 32D summarises other functional-type markers associated with the CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+ population. The markers positively correlated are of condition Y+, and largely represent entities with putative immunosuppressive activities, where in the preferred aspect they are used as a positive selection marker for the purification of Treg cells from mucosal emigrant, immigrant and educated CD4.sup.+ T-cell populations. The markers negatively correlated are of condition Y, and largely represent entities with putative pro-inflammatory activities, where in the preferred aspect they are used as a negative selection marker for the purification of Treg cells from mucosal emigrant, immigrant and educated CD4.sup.+ T-cell populations. Each marker in this table is also assigned a class, where class 1 represents a strong association with the CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+ population and high functional significance. Class 2 represents a strong association with the CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+ population or high functional significance. Class 3 represents weak association and/or uncertain functional significance.

[0676] In order to assess the feasibility of recovering the rare mucosal emigrant and tropic CD4.sup.+ Tregs from peripheral circulation, CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiCCR9.sup.+ Tregs were sorted from peripheral blood at high purity; PBMCs from healthy donors were labelled and sorted on the basis of these defined markers (FIG. 33). FIG. 33a to e show the basic gating strategy of FACS-based purification of these cells, and FIGS. 33f and g displays achieved purity of greater than 95%. FIGS. 33h and i show that CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiCCR9.sup.+ target cells are largely of antigen experienced and recent activation character.

[0677] As proof of concept that, CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.hiCCR9.sup.+ purified from peripheral blood of could be expanded as a therapeutic population, cells purified by FACS as described in FIG. 33 were expanded with recombinant stimuli in vitro. FIG. 34 displays a representative growth curve of such an expansion.

[0678] Finally, to test the hypothesis that mucosal emigrant CD4.sup.+ Tregs in peripheral circulation are in some way clonally restricted due to their activated, emigrant and recirculating nature, an assessment of vp usage among CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+CD38.sup.+CD62L.sup.CCR9.sup.+ in peripheral circulation was conducted with total peripheral Tregs (CD4.sup.+CD25.sup.hiCD127.sup.lo) as reference. (FIG. 35). Across three healthy donors, the usage of V segments was markedly different between CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+CD38.sup.+CD62L.sup.CCR9.sup.+ and the total pool of CD4.sup.+CD25.sup.hiCD127.sup.lo lymphocytes. This indirectly supports the proposal that CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+CD38.sup.+CD62L.sup.CCR9.sup.+ mucosal emigrant Tregs are activated against a restricted set of antigens in the mucosa, and exported for recirculation in order to support regional and/or systemic tolerance.

[0679] From the case study above, a method for identification of Treg cells originating from any diseased tissue of interest is elucidated. The major elements of this model are defined in FIG. 36. Cells from the tissue of interest, Target tissue (A), are exported through lymphatics (B, B and B), and subsequently migrate through peripheral blood (C) to recirculate to tissue of origin (A). Alternate route of recirculation is to a secondary tissue of interest (X). Tissues A, B, B, B, C and X are considered major sampling points for investigation. Major migratory processes involving recirculation of Tregs back to tissue of origin are defined for target tissue emigration (), and immigration (). Emigration from secondary tissue of interest (X) is defined as X, and immigration X. Migration between main target tissue A, and communicating tissue X, is defined as migration process . Migration processes , , X, X and are considered as definable by Treg migratory marker expression, as applied to Tregs recovered from sampled tissues A, B, B, B, C and X. Major sources of analytical noise are those cells collected in all compartments that bare migratory marker expression for migratory processes and . and migratory process markers are considered negative selection criteria for investigation of Treg cells of A or X origin, with , , X, X or migratory behaviour.

[0680] FIG. 37 shows that CD4.sup.+ cells with CCR9.sup.+CD103.sup.+ characteristics, denoting small bowel tropism, are present in the LP of the large bowel. This is also apparent in lymph nodes draining these distinct tissues, which suggests that colonic Tregs may be locally imprinted, in small numbers, to recirculate to the small bowel. This is suggestive that a process of emigration, but X immigration (as defined above) could be an active process, considering tissue A as colon, and tissue X as small bowel in this example.

[0681] Experimental Material and Methods

[0682] Material

[0683] Fluorochrome-conjugated antibodies were obtained from BD Biosciences or BioLegend; CD4-FITC, CD4-PE/Cy7 (OKT4), CD25-APC (2A3, M-A251), CD25-PE/Cy7 (BC960, M-A251), CD38-BV421 (HIRT2), CD38-PE (HIT2), CD45RO-PerCP/Cy5.5 (UCHL1) CD49d-PE/Cy7 (9F10), CD62L-PE/Cy7 (DREG-56), CD127-PerCP/Cy5.5, CD127-PE (A019D5, HIL-7R-M21), FOXP3-PE (259D/C7), FOXP3-AlexaFluor647 (206D), integrin7-PerCP/Cy5.5, integrin7-FITC (FIB27) CD62L BV421 (DREG-55), CD4 BV510 (SK3), CD25 BV605 (2A3), CD1c PE (L161), CD3 FITC (HIT3a), CD3 PE-CF594 (UCHT1), CD3 APC-H7 (SK7), CD4 BV605 (RPA-T4), CD4 PerCP (SK3), CD4 APC (RPA-T4), CD4 APC-H7 (RPA-T4), CD8 BV510 (RPA-T8), CD8 BV605 (SK1), CD8 BV786 (RPA-T8), CD8 Alexa 488 (RPA-T8), CD8 PerCP-Cy5.5 (RPA-T8), CD8 PE (RPA-T8), CD8 PE-Cy7 (RPA-T8), CD8 APC-H7 (SK1), CD11a PE (HI111), CD11b BV510 (ICRF44), CD11b PE-Cy7 (ICRF44), CD11c BV421 (B-ly6), CD11c BV605 (B-ly6), CD11c PE (B-ly6), CD14 BV510 (MP9), CD14 BV711 (MP9), CD14 APC (M5E2), CD16 PerCP-Cy5.5 (3G8), CD16 PE (B73.1), CD18 BV421 (6,7), CD19 BV510 (SJ25C1), CD19 BV711 (SJ25C1), CD19 PE-Cy7 (SJ25C1), CD25 BV510 (M-A251), CD25 BV786 (M-A251), CD25 PerCP-Cy5.5 (M-A251), CD25 PE-Cy7 (M-A251), CD28 BV421 (CD28.2), CD28 BV605 (CD28.2), CD28 BV711 (CD28.2), CD28 FITC (CD28.2), CD28 PerCP-Cy5.5 (CD28.2), CD28 APC-H7 (CD28.2), CD29 BV510 (MAR4), CD29 PE (MAR4), CD29 APC (MAR4), CD31 BV605 (WM59), CD38 FITC (HIT2CD38), PE-CF594 (HIT2CD38), PE-Cy7 (HIT2CD38), Alexa700 (HIT2), CD38 APC-H7 (HB7), CD39 BV711 (T66), CD39 FITC (T66), CD45 BV605 (HI30), CD45 BV786 (HI30), CD45 FITC (HI30), CD45 PE (HI30), CD45 PE-Cy7 (HI30), CD45RA BV421 (HI100), CD45RA BV605 (HI100), CD45RA BV711 (HI100), CD45RA PerCP-Cy5.5 (HI100), CD45RA PE (HI100), CD45RO BV605 (UCHL1), CD45RO BV711 (UCHL1), CD45RO APC (UCHL1), CD49a PE (SR84), CD49b PE (12F1), CD49c PE (C3 II.1), CD49d BV510 (9F10), CD49d BV711 (9F10), CD49d PerCP-Cy5.5 (9F10), CD49d PE (9F10), CD49d PE-CF594 (9F10), CD49e PE (IIA1), CD49f PE (GoH3), CD56 BV510 (NCAM16.2), CD56 BV711 (NCAM16.2), CD62L BV510 (DREG-56), CD62L BV605 (DREG-56), CD69 BV605 (FN50), CD69 BV711 (FN50), CD69 PerCP-Cy5.5 (FN50), CD69 PE-Cy7 (FN50), CD73 BV605 (AD2), CD79a BV421 (HM47), CD79a PE (HM47), CD79a APC (HM47), CD79b PE (3A2-2E7), CD79b PE-Cy5 (CB3-1), CD80 BV605 (L307.4), CD80 PE (L307.4), CD80 PE-Cy7 (L307.4), CD80 APC (2D10), CD83 PerCP-Cy5.5 (HB15e), CD83 APC (HB15e), CD86 BV421 (2331), CD86 PerCP-Cy5.5 82331), CD86 APC (2331), CD103 BV711 (Ber-ACT8), CD103 FITC (Ber-ACT8), CD103 PE (Ber-ACT8), CD127 BV421 (HIL-7R-M21), CD127 BV605 (HIL-7R-M21), CD127 BV650 (HIL-7R-M21), CD127 BV711 (HIL-7R-M21), CD127 FITC (HIL-7R-M21), CD141 BV510 (1A4), CD141 PE (1A4), CD152 BV421 (BNI3), CD152 BV786 (BNI3), CD163 PerCP-Cy5.5 (GHI/61), CD192 BV421 (K036C2), CD196 BV421 (11A9), CD197 FITC (3D12), CD197 PerCP-Cy5.5 (150503), CD199 Alexa 488 (112509), CD199 FITC (112509), CD199 PE (112509), CD199 PE (L053E8), CD199 PE (248621), CD199 PE-Cy7 (L053E8), CD199 Alexa 647 (112509), CD199 Alexa 647 (L053E8), CD199 Alexa 647 (BL/CCR9), CD199 APC (112509), CD303 BV421 (201A), CD357 APC (621), Annexin V APC, 7 integrin BV421 (FIB504), 7 integrin BV605 (FIB504), 7 integrin PE (FIB504), 7 integrin APC (FIB504), CX3CR1 PerCP-Cy5.5 (2A9-1), FoxP3 Alexa 488 (259D/C7), Granzyme B BV421 (GB11), Granzyme B FITC (GB11), Granzyme B PE-CF594 (GB11), Helios PE (22F6), HLA-A2 PE-Cy7 (BB7.2), HLA-A,B,C PE-Cy5 (G46-2.6), HLA-E PE (3D12), HLA-G PE (87G), HLA-DM PE (MaP.DM1), HLA-DR PerCP-Cy5.5 (G46-6), HLA-DR PE-Cy7 (G46-6), HLA-DR APC (G46-6), HLA-DRB1, HLA-DR, DP, DQ FITC (T39), HLA-DR, DP, DQ Alexa 647 (T39), HLA-DQ FITC (Tu169), IFN-g Alexa 647 (4S.B3), IL-1b PE (AS10), IL-2 FITC (MQ1-17H12), IL-2 FITC (MQ1-17H12), IL-4 FITC (MP4-25D2), IL-10 APC (JES3-19F1), IL-12 FITC (C11.5), IL-17A PE (SCPL1362), IL-35 PE (B032F6), Ig light chain PE (G20-193), Light chain PE (JDC-12), IgM BV605 (G20-127), IgM FITC (G20-127), IgM FITC IgM PE-Cy5 (G20-127), Lineage cocktail FITC, Perforin BV421 (G9), Perforin Alexa 488 (G9), Syk FITC (4D10), Syk PY352 PE (17A/P-ZAP70), Syk PY352 PE-Cy7 (17A/P-ZAP70), Syk PY352 Alexa 647 (17A/P-ZAP70), TCR BV510 (T10B9.1A-31), TCR BV786 (T10B9.1A-31), TCR FITC (B1), TCR -1 FITC (11F2), TCR PE-CF594 (B1), TGF-b1 BV421 (TW4-9E7), TNF-a APC (MAb11), and unlabelled antibodies were obtained from BD Biosciecnes; CD1a (HI149), CD28 (L293), CD51/61 (23C6), CD1b (M-T101), CD29 (HUTS-21), CD53 (HI29), CD1d (CD1d42), CD30 (BerH8), CD54 (LB-2), CD2 (RPA-2.10), CD31 (WM59), CD55 (IA10), CD3 (HIT 3a), CD32 (FL18.26), CD56 (B159), CD4 (RPA-T4), CD33 (HIM3-4), CD57 (NK-1), CD4v4 (L120), CD34 (581), CD58 (1C3), CD5 (L17F12), CD35 (E11), CD59 (p282, H19), CD6 (M-T605), CD36 (CB38, NL07), CD61 (VI-PL2), CD7 (M-T701), CD37 (M-B371), CD62E (68-5H11), CD8a (SK1), CD38 (HIT 2), CD62L (Dreg 56), CD8b (2ST 8.5H7), CD39 (TU66), CD62P (AK-4), CD9 (M-L13), CD40 (5C3), CD63 (H5C6), CD10 (HI10a), CD41a (HIP8), CD64 (10.1), CD11a (G43-25B), CD41b (HIP2), CD66 (a,c,d,e) (B1.1/CD66), CD11b (D12), CD42a (ALMA.16), CD66b (G10F5), CD11c (B-ly 6), CD42b (HIP1), CD66f (IID10), CD13 (WM15), CD43 (1G10), CD69 (FN50), CD14 (M5E2), CD44 (G44-26), CD70 (Ki-24), CD15 (HI98), CD45 (HI30), CD71 (M-A712), CD15s (CSLEX1), CD45RA (HI100), CD72 (J4-117), CD16 (3G8), CD45RB (MT4), CD73 (AD2), CD18 (6.7), CD45RO (UCHL1), CD74 (M-B741), CD19 (HIB19), CD46 (E4.3), CD75 (LN1), CD20 (2H7), CD47 (B6H12), CD77 (5B5), CD21 (B-ly 4), CD48 (T U145), CD79b (CB3-1), CD22 (HIB22 CD49a SR84 CD80 L307.4 CD23 EBVCS-5 CD49b AK-7 CD81 JS-81), CD24 (ML5), CD49c (C3 II.1), CD83 (HB15e), CD25 (M-A251), CD49d (9F10), CD84 (2G7), CD26 (M-A261), CD49e (VC5), CD85 (GHI/75), CD27 (M-T271), CD50 (TU41), CD86 (2331, FUN-1), CD123 (9F5), CD172b (B4B6), CD87 (VIM5), CD124 (hIL4R-M57), CD177 (MEM-166), CD88 (D53-1473), CD126 (M5), CD178 (NOK-1), CD89 (A59), CD127 (hIL-7R-M21), CD180 (G28-8), CD90 (5E10), CD128b (6C6), CD181 (5A12), CD91 (A2MR-alpha 2), CD130 (AM64), CD183 (1C6/CXCR3), CDw93 (R139), CD134 (ACT35), CD184 (12G5), CD94 (HP-3D9), CD135 (4G8), CD193 (5E8), CD95 (DX2), CD137 (4B4-1), CD195 (2D7/CCR5), CD97 (VIM3b), CD137 (Ligand C65-485), CD196 (11A9), CD98 (UM7F8), CD138 (Mi15), CD197 (2H4), CD99 (TU12), CD140a (alpha R1), CD200 (MRC OX-104), CD99R (HIT 4), CD140b (28D4), CD205 (MG38), CD100 (A8), CD141 (1A4), CD206 (19.2), CD102 (CBR-1C2/2.1), CD142 (HTF-1), CD209 (DCN46), CD103 (Ber-ACT8), CD144 (55-7H1), CD220 (3B6/IR), CD105 (266), CD146 (P1H12), CD221 (3B7), CD106 (51-10C9), CD147 (HIM6), CD226 (DX11), CD107a (H4A3), CD150 (A12), CD227 (HMPV), CD107b (H4B4), CD151 (14A2.H1), CD229 (HLy9.1.25), CD108 (KS-2), CD152 (BNI3), CD231 (M3-3D9, SN1a), CD109 (TEA 2/16), CD153 (D2-1173), CD235a (GA-R2, HIR2), CD112 (R2.525), CD154 (TRAP1), CD243 (17F9), CD114 (LMM741), CD158a (HP-3E4), CD244 (2-69), CD116 (M5D12), CD158b (CH-L), CD255 (CARL-1), CD117 (Y B5.B8), CD161 (DX12), CD268 (11C1), CD118 (12D3), CD162 (KPL-1), CD271 (C40-1457), CD119 (GIR-208), CD163 (GHI/61), CD273 (MIH18), CD120a (MABTNFR1-A1), CD164 (N6B6), CD274 (MIH1), CD121a (HIL1R-M1), CD165 (SN2), CD275 (2D3/B7-H2), CD121b (MNC2), CD166 (3A6), CD278 (DX29), CD122 (Mik-beta 3), CD171 (5G3), CD279 (MIH4), fMLP receptor (5F1), Ms IgG2a IC (G155-178), CD282 (11G7), TCR (B1), Ms IgG2b IC (27-35), CD305 (DX26), HPC (BB9), Ms IgG3 IC (J606), CD309 (89106), HLA-A,B,C (G46-2.6), CD49f (GoH3), CD314 (1D11), HLA-A2 (BB7.2), CD104 (439-9B), CD321 (M.AB.F11), HLA-DQ (TU169), CD120b (hTNFR-M1), CDw327 (E20-1232), HLA-DR (G46-6, L243), CD132 (TUGh4), CDw328 (F023-420), HLA-DR, DP, DQ (TU39), CD201 (RCR-252), CDw329 (E10-286), Invariant NK T (6B11), CD210 (3F9), CD335 (9E2/NKp46), Disialoganglioside GD2 (14.G2a), CD212 (2B6/12beta 2), CD336 (P44-8.1), MIC A/B (6D4), CD267 (1A1-K21-M22), CD337 (P30-15), NKB1 (DX9), CD294 (BM16), CD338 (5D3), SSEA-1 (MC480), SSEA-3 (MC631), CD304 (Neu24.7), SSEA-4 (MC813-70), CLA (HECA-452), T CR (T10B9.1A-31), TRA-1-60 (TRA-1-60), Integrin 7 (FIB504), 2-microglobulin (TU99), TRA-1-81 (TRA-1-81), Rt IgM IC (R4-22), BLTR-1 (203/14F11), V 23 (AHUT 7), Rt IgG1 IC (R3-34), CLIP (CerCLIP), V 8 (JR2), Rt IgG2a IC (R35-95), CMRF-44 (CMRF44), CD326 (EBA-1), Rt IgG2b IC (A95-1), CMRF-56 (CMRF56), Ms IgM IC (G155-228), EGF Receptor (EGFR1), Ms IgG1 IC (MOPC-21) and Zombie NIR Fixable Viability Kit or BD Biosciences; CD4-PacificBlue (RPA-T4); collagenaseIV, DNaseI, DTT, EDTA and sodium azide from SigmaAldrich; FicollPaquePlus from GEHealthcare, RPMI media, BSA and FCS from Life Technologies; IOTest Beta Mark TCR V Kit from Beckman Coulter.

[0684] Patients and Tissue Preparation

[0685] All subjects gave their written informed consent under the Helsinki guidelines and local ethics committee. CD patients undergoing ileoceacal resection were recruited to the study. We collected small bowel (ileum) and large bowel (ceacum/ascending colon), including MLN draining these regions. Control samples were from colorectal cancer patients undergoing right-sided hemicolectomy. Intestinal lamina propria from the small and large bowel was separated via microdissection. The dissected lamina propria was minced into 1-2 mm pieces and single cell suspensions were prepared in RPMI 1640 containing 5% FBS, 50 g/ml gentamycin and 50 g/ml Penicillin/Streptomycin using the Medimachine with a 50 m Medicon (BD Biosciences). The cell suspension was filtered through a 70-m nylon mesh (BD Biosciences), centrifuged and the pellet resuspended in FACS buffer (PBS containing 2% FBS) for subsequent antibody staining.

[0686] Lymphocytes from MLN were isolated by mechanical disruption of lymph nodes after surrounding fat tissue was removed by dissection. The cell suspension was filtered through a 40-m nylon mesh (BD Biosciences), centrifuged and the pellet resuspended in FACS buffer for subsequent antibody staining.

[0687] Patients and Blood Preparation

[0688] All subjects gave their written informed consent under the Helsinki guidelines and local ethics committee. Healthy donors were recruited to the blood cohorts. Blood drawn into EDTA tubes was diluted 1:2 in PBS with 2 mM EDTA and PBMCs collected over a FicollPaquePlus density gradient by centrifugation. PBMCs were washed 3 times in wash buffer (PBS, 0.2% BSA, 5 mM EDTA) before immediate flow cytometry.

[0689] Direct Cell Purification by FACS

[0690] Extracellular antigens were stained in FACS buffer (PBS, 2% BSA) using appropriate combinations of fluorophore-conjugated antibodies (BioLegend and BD Biosciences). Specific cell populations were purified by fluorescence-activated cell sorting (FACS) using a BD Influx cell sorter with BD FACS Sortware (BD Biosciences) to acquire data. Final analyses utilized FlowJo software (Tree Star Inc.).

[0691] Expansion of Sorted Cell Populations

[0692] The sorted cell populations were expanded in OpTmizer media with 2 mM Glutamax (both Life Technologies) and either autologous or commercial human serum (Sigma) using MACS GMP ExpAct Treg Kit (Miltenyi Biotec) and in the presence of recombinant human IL-2 (Miltenyi Biotec).

[0693] Flow Cytometry

[0694] Zombie NIR Fixable Viability Kit (Biolegend) was used as a dead cell marker. Surface antigens were stained in FACS buffer (PBS containing 2% FBS) and intracellular FoxP3 was stained after fixation and permeabilization using the human FoxP3 buffer set (BD Biosciences). Cells were acquired using a LSRFortessa flow cytometer with Diva 8 software (BD Biosciences). Final analysis was performed using FlowJo 10 software (Tree Star Inc.).

[0695] Statistics

[0696] All data was expressed as meanSEM. Pair wise comparisons were two-tailed Mann-Whitney U-tests. Significance testing of multiple parameters was calculated with Kruskal-Wallis one-way ANOVA and Dunn's post-test of selected columns. A p value<0.05 was considered significant.

[0697] Also provided are

[0698] 1. Treg cells for use in the treatment of an inflammatory disease, the Treg cells have signatures for

[0699] i) identifying that the T-cells are regulatory Tcells,

[0700] ii) identifying that the Treg cells are tissue type tropic, i.e they can migrate to the diseased tissue,

[0701] iii) identifying that the Treg cells are tropic with respect to the diseased tissue, i.e. they are homing cells,

[0702] iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the target tissue, and/or

[0703] v) identifying that the Treg cells are retained in the target tissue,

[0704] wherein the Treg cells have the signatures i), ii) and iii) and optionally iv) and/or v), or the Treg cells have the signatures i), ii) and v) and optionally iii) and/or iv), or the Treg cells have the signatures i), iii) and optionally ii) and/or v).

[0705] 2. Tregs for use according to item 1, wherein the inflammatory disease is chronic obstructive pulmonary disease (COPD), atherosclerosis, osteoarthritis, IBD, ankylosing spondylitis or polymyalgia rheumatica.

[0706] 3. Tregs for use according to item 1 or 2, wherein the disease is COPD.

[0707] 4. Treg cells for use according to any of the preceding items, wherein the signatures for identifying that the T-cells are regulatory T-cells are CD4.sup.+CD25.sup.hiCD127.sup.lo, CD4.sup.+CD25.sup.hi, CD8.sup.+, or CD8.sup.+CD28.sup.+.

[0708] 5. Treg cells for use according to any of the preceding items, wherein the signature for identifying that the Treg cells can migrate to the diseased tissue such as the mucosal tissue is 47.sup.+ or 4.sup.+7.sup.+.

[0709] 6. Treg cells for use according to any of the preceding items, wherein the signature for identifying that the Treg cells can be retained in the diseased tissue such as the mucosal tissue is 47.sup.highE.sup.+ or 4.sup.+7.sup.highE.sup.+.

[0710] 7. Treg cells for use according to any of the preceding items 3-9, wherein the signatures for identifying that the Treg cells are target tissue tropic is CCR9.sup.+.

[0711] 8. Treg cells for use according to any of the preceding items, wherein the signatures for identifying that the Treg cells are educated cells (emigrants) is CD62L.sup.CD38.sup.+.

[0712] 9. Treg cells for use according to any of the preceding items, wherein the Treg cells comprise a signature selected from the following signatures:

[0713] CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+CCR9.sup.+,

[0714] CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.highE.sup.+CCR9.sup.+,

[0715] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+CCR9.sup.+

[0716] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+

[0717] CD4.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+

[0718] CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+X

[0719] CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.highE.sup.+X

[0720] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+X

[0721] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.highE.sup.+X

[0722] CD4.sup.+4.sup.+7.sup.highE.sup.+X

[0723] CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.+

[0724] CD4.sup.+CD25.sup.hiCD127.sup.lo4.sup.+7.sup.highE.sup.+

[0725] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.+

[0726] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+4.sup.+7.sup.highE.sup.+

[0727] CD4.sup.+4.sup.+7.sup.highE.sup.+, and

[0728] CD8.sup.+4.sup.+7.sup.+CCR9.sup.+

[0729] CD8.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+

[0730] CD8.sup.+CD28.sup.+4.sup.+7.sup.+CCR9.sup.+,

[0731] CD8.sup.+CD28.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+

[0732] CD8.sup.+4.sup.+7.sup.+X

[0733] CD8.sup.+4.sup.+7.sup.highE.sup.+X

[0734] CD8.sup.+CD28.sup.+4.sup.+7.sup.+X

[0735] CD8.sup.+CD28.sup.+4.sup.+7.sup.highE.sup.+X

[0736] CD8.sup.+4.sup.+7.sup.+

[0737] CD8.sup.+4.sup.+7.sup.highE.sup.+

[0738] CD8.sup.+CD28.sup.+4.sup.+7.sup.+

[0739] CD8.sup.+CD28.sup.+4.sup.+7.sup.highE.sup.+

[0740] wherein X is the signature relating to tropism of the diseased tissue, and X may be X.sup.+ or X.sup., 4.sup.+ may be substituted with 4, and any of the signatures may also comprise CD62L.sup.CD38.sup.+

[0741] 10. A method for treating a patient suffering from an inflammatory disease, the method comprises

[0742] a) isolating Treg cells defined in any one of items 1-9 from a tissue sample obtained from a patient suffering from the inflammatory disease,

[0743] b) expanding the Treg cells in vitro,

[0744] c) optionally re-patterning the expanded Treg cells to obtain Tregs that have signatures ii) and iii) and optionally iv) and/or v), or signatures for iii) and v) and optionally ii) and/or iv) or signatures for ii) and optionally iii), iv) and/or v), wherein the signatures is for

[0745] ii) identifying that the Treg cells are tissue type tropic,

[0746] iii) identifying that the Treg cells are diseased tissue tropic,

[0747] iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the target tissue, and/or

[0748] v) identifying that the Treg cells are retained in the target tissue,

[0749] d) administering the Treg cells obtained from b) or c) to the patient.

[0750] 11. A method according to item 10, wherein the expanded Treg cells from step b) or c) have features as defined in any one of items 1-9.

[0751] 12. A method according to item 10 or 11, wherein the tissue sample is from peripheral blood of the patient.

[0752] 13. A method for obtaining Treg cells as defined in any one of items 1-9, the method comprises

[0753] a) isolating Treg cells defined in any one of items 1-9 from a tissue sample obtained from a patient suffering from an inflammatory disease,

[0754] b) expanding the Treg cells in vitro,

[0755] c) optionally re-patterning the expanded Treg cells to obtain Tregs that have signatures signatures ii) and iii) and optionally iv) and/or v), or signatures for iii) and v) and optionally ii) and/or iv) or signatures for ii) and optionally iii), iv) and/or v), wherein the signatures is for

[0756] ii) identifying that the Treg cells are tissue type tropic,

[0757] iii) identifying that the Treg cells are diseased tissue tropic relating to the diseased tissue,

[0758] iv) identifying that the Treg cells are emigrant cells, i.e. the originates from the target tissue, and/or

[0759] v) identifying that the Treg cells are retained in the target tissue.

[0760] 14. A method according to item 13, wherein step a) comprises the recovery of mononuclear cells from patient tissue specimens, and labelling said pool of mononuclear cells with antibodies specific for appropriate markers; once labelled, cells are purified by immunoaffinity and/or flow cytometric sorting techniques to yield highly enriched or purified Treg populations of desired characteristics.

[0761] 15. A method according to any of items 13-14 wherein step b) comprises recombinant T-cell stimulation in the form of anti-CD3/anti-CD28 activating antibodies in combination with IL2, or alternatively the outgrowth of Treg populations on transgenic feeder cell populations, or irradiated autologous peripheral monocytes with IL2 supplementation.

[0762] 16. A method according to any of items 13-15, wherein step c) comprises the recombinant reactivation of expanded T-cell populations with anti-CD3/anti-CD28 activating antibodies and subsequent introduction of stimuli in precise combination. Stimuli include all-trans retinoic acid, Interleukin-10 and transforming growth factor-beta.

[0763] 17. A method for obtaining Treg cells as defined in any one of items 1-9, the method comprising

[0764] a) providing Treg cells comprising a signature selected from

[0765] CD4.sup.+CD25.sup.hiCD127.sup.lo,

[0766] CD4.sup.+CD25.sup.hiCD127.sup.lo7.sup.highE.sup.+,

[0767] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+ and

[0768] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+7.sup.highE.sup.+

[0769] CD8.sup.+,

[0770] CD8.sup.+7.sup.highE.sup.+,

[0771] CD8.sup.+CD28.sup.+,

[0772] CD8.sup.+CD28.sup.+7.sup.highE.sup.+,

[0773] and the above-mentioned signatures may further comprise the signature CD62L.sup.CD38.sup.+,

[0774] and

[0775] b) re-patterning the Treg cells to further comprise the signature 47.sup.+, 47.sup.+X or 47.sup.+CCR9.sup.+, 4.sup.+7.sup.+, 4.sup.+7.sup.+X or 4.sup.+7.sup.+CCR9.sup.+, wherein X is as defined herein before.

[0776] 18. A method according to item 17, wherein step b) comprises the recombinant reactivation of expanded T-cell populations with anti-CD3/anti-CD28 activating antibodies and subsequent introduction of stimuli including all-trans retinoic acid, Interleukin-10 and transforming growth factor-beta

[0777] 19. A method for obtaining Treg cells as defined in any one of items 1-9, the method comprising

[0778] a) providing Treg cells comprising a signature selected from

[0779] CD4.sup.+CD25.sup.hiCD127.sup.loCCR9.sup.+,

[0780] CD4.sup.+CD25.sup.hiCD127.sup.loE.sup.+7.sup.highCCR9.sup.+,

[0781] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+CCR9.sup.+ and

[0782] CD4.sup.+CD25.sup.hiCD127.sup.loCD62L.sup.CD38.sup.+E.sup.+7.sup.highCCR9.sup.+.

[0783] CD8.sup.+CCR9.sup.+,

[0784] CD8.sup.+7.sup.highE.sup.+CCR9.sup.+,

[0785] CD8.sup.+CD28.sup.+CCR9.sup.+,

[0786] CD8.sup.+CD28.sup.+7.sup.highE.sup.+CCR9.sup.+,

[0787] and the above-mentioned signatures may further comprise the signature CD62L.sup.CD38.sup.+,

[0788] and

[0789] b) re-patterning the Treg cells to further comprise the signature 47.sup.+ or 4.sup.+7.sup.+.

[0790] 20. A method according to item 19, wherein step b) comprises the recombinant reactivation of expanded T-cell populations with anti-CD3/anti-CD28 activating antibodies and subsequent introduction of stimuli including all-trans retinoic acid, Interleukin-10 and transforming growth factor-beta.

[0791] 21. A pharmaceutical composition comprising Treg cells as defined in any of items 1-9 dispersed in an aqueous medium.

[0792] 22. Treg cells as defined in any of items 1-9.