CD8+ REGULATORY T-CELLS FOR USE IN THE TREATMENT OF INFLAMMATORY DISORDERS OF THE HUMAN GASTROINTESTINAL TRACT

Abstract

The present invention relates to composition comprising an isolated CD8.sup.+ Treg cell population, wherein the Treg cells have signatures for i) identifying that the T-cells are CD8.sup.+ regulatory Tcells, ii) identifying that the Treg cells are tissue type tropic, i.e they can migrate to the diseased tissue, iii) optionally identifying that the Treg cells are tropic with respect to the diseased tissue, i.e. they are homing cells, iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the target tissue, and v) optionally identifying that the Treg cells are capable of being retained in the target tissue and optionally one or more X-signatures and/or one or more Y-signatures.

Claims

1-31. (canceled)

32. An isolated CD8.sup.+ Treg cell population, wherein the Treg population consists of cells having signatures for (i) identifying that the T-cells are CD8.sup.+ regulatory Tcells, (ii) identifying that the Treg cells are mucosal tissue type tropic that can migrate to the diseased mucosal tissue, (iii) optionally, identifying that the Treg cells are homing cells tropic with respect to the diseased mucosal tissue, (iv) identifying that the Treg cells are emigrant cells that originate from the target mucosal tissue, and (v) optionally, identifying that the Treg cells are capable of being retained in the target mucosal tissue and optionally having one or more X-signatures and/or one or more Y-signatures, wherein X is a signature indicating that the CD8.sup.+ Tregs can localize, have emigrated from, or are marked for preferential retention in the specific part of the gastrointestinal tract that is diseased, and Y is a signature indicating immunosuppressive regulatory function, wherein the isolated CD8.sup.+ Treg cell population has T-cell receptor clonal diversity restricted to clonotypes specific for antigens present in the mucosal tissue types to which the selected signatures relate.

33. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the diseased tissue originates from inflamed tissue or tissue subject to an autoimmune disease.

34. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the signature (i) is selected from CD8.sup.+CD122.sup.+, CD8.sup.+Y.sub.n, and CD8.sup.+CD122.sup.+Y.sub.n, wherein n indicates that one or more Y signatures may be present.

35. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the signature (ii) is for a gastrointestinal mucosa and is selected from 47.sup.+, 4.sup.+7.sup.+, 47.sup.+X.sub.n and 4.sup.+7.sup.+X.sub.n, wherein n indicates that one or more X signatures may be present.

36. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the signature (iii) is for localization in the small bowel and is selected from CCR9.sup.+ and CCR9.sup.+X.sub.n, wherein n indicates that one or more X signatures may be present.

37. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the signature (iv) is for antigen-experienced cells and is selected from CD62L.sup., X.sub.n, Y.sub.p, X.sub.nY.sub.p, CD62L.sup.X.sub.n, CD62L.sup.Y.sub.p, CD62L.sup.X.sub.nY.sub.p, wherein n and p indicate that one or more X signatures and/or one or more Y signatures, respectively, may be present.

38. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the signature (v) is 4.sup.+E.sup.+7.sup.hi.

39. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the CD8.sup.+ Treg cell population comprises at least one of the following signatures: CD8.sup.+47.sup.+CD62L.sup. CD8.sup.+47.sup.+CD62L.sup. CD8.sup.+47.sup.+CD62L.sup.CCR9.sup.+ CD8.sup.+4.sup.+7.sup.+CD62L.sup.CCR9.sup.+ CD8.sup.+47.sup.hiE.sup.+CD62L.sup. CD8.sup.+4.sup.+7.sup.hiE.sup.+CD62L.sup. CD8.sup.+CD62L.sup.47.sup.highE.sup.+CCR9.sup.+ CD8.sup.+CD62L.sup.47.sup.highE.sup.+CCR9.sup.+.

40. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the CD8.sup.+ Treg cell population comprises at least one of the following signatures: CD8.sup.+47.sup.+CD62L.sup.X/Y CD8.sup.+4.sup.+7.sup.+CD62L.sup.X/Y CD8.sup.+47.sup.+CD62L.sup.CCR9.sup.+X/Y CD8.sup.+4.sup.7.sup.+CD62L.sup.CCR9.sup.+X/Y CD8.sup.+47.sup.hiE.sup.+CD62L.sup.X/Y CD8.sup.+4.sup.+7.sup.hiE.sup.+CD62L.sup.X/Y CD8.sup.+CD62L.sup.47.sup.highE.sup.+CCR9.sup.+X/Y and CD8.sup.+CD62L.sup.4.sup.+7.sup.highE.sup.+CCR9.sup.+X/Y wherein X/Y means that at least one X, at least one Y, and/or at least one X and at least one Y is present, wherein X may be X.sup.+ or X.sup., and Y may be Y.sup.+ or Y.sup..

41. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the CD8.sup.+ Treg cell population comprises at least one of the following signatures: CD8.sup.+47.sup.+CD62L.sup.X CD8.sup.+4.sup.+7.sup.+CD62L.sup.X CD8.sup.+47.sup.+CD62L.sup.CCR9.sup.+X CD8.sup.+4.sup.+7.sup.+CD62L.sup.CCR9.sup.+X CD8.sup.+47.sup.hiE.sup.+CD62L.sup.X CD8.sup.+4.sup.+7.sup.hiE.sup.+CD62L.sup.X CD8.sup.+CD62L.sup.47.sup.highE.sup.+CCR9.sup.+X CD8.sup.+CD62L.sup.47.sup.highE.sup.+CCR9.sup.+X CD8.sup.+47.sup.+CD62L.sup.Y CD8.sup.+4.sup.+7.sup.+CD62L.sup.Y CD8.sup.+47.sup.+CD62L.sup.CCR9.sup.+Y CD8.sup.+4.sup.+7.sup.+CD62L.sup.CCR9.sup.+Y CD8.sup.+47.sup.hiE.sup.+CD62L.sup.Y CD8.sup.+4.sup.+7.sup.hiE.sup.+CD62L.sup.Y CD8.sup.+CD62L.sup.47.sup.highE.sup.+CCR9.sup.+Y CD8.sup.+CD62L.sup.4.sup.+7.sup.highE.sup.+CCR9.sup.+Y CD8.sup.+47.sup.+CD62L.sup.XY CD8.sup.+4.sup.+7.sup.+CD62L.sup.XY CD8.sup.+47.sup.+CD62L.sup.CCR9.sup.+XY CD8.sup.+4.sup.+7.sup.+CD62L.sup.CCR9.sup.+XY CD8.sup.+47.sup.hiE.sup.+CD62L.sup.XY CD8.sup.+4.sup.+7.sup.hiE.sup.+CD62L.sup.XY CD8.sup.+CD62L.sup.4.sup.+7.sup.highE.sup.+CCR9.sup.+XY and CD8.sup.+CD62L.sup.4.sup.+7.sup.highE.sup.+CCR9.sup.+XY wherein X may be X.sup.+ or X.sup., and Y may be Y.sup.+ or Y.sup..

42. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the one or more X signatures is at least one selected from any of (a) CD49d.sup.+, CD54.sup.+, CD99.sup., CD99R.sup.+, CD166.sup.+, (b) CD49a.sup., CD49c.sup., CD49f.sup., CD102.sup., CD165.sup.+, CDw328.sup., CDw329.sup., (c) CD37.sup., CD38.sup., and CD49e.sup..

43. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the one or more Y signatures is at least one selected from any of (d) CD25.sup.+, CD58.sup.+, CD73.sup.+, CD95.sup.+, CD105.sup.+, CD107a.sup.+, CD107b.sup.+, CD122.sup.+, CD244.sup.+, CD268.sup.+, CD274.sup.+, (e) CD31.sup., CD35.sup.+, CD39.sup.+, CD41a.sup.+, CD63.sup.+, CD85.sup., CD88.sup.+, CD97.sup.+, CD108.sup.+, CD120b.sup.+, CD127.sup.+, CD130.sup., CD132.sup.+, CD151.sup.+, CD210.sup.+, CD221.sup., CD226.sup.+, CD335.sup., CD336.sup., EGF-R.sup., (f) CD126.sup., CD150.sup.+, CD161.sup.+, CD195.sup.+, CD200.sup., and CD279.sup.+.

44. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the population of CD8.sup.+ Treg cells with signatures (iv) identifying the Treg cells as emigrant cells that originated from the target mucosal tissue does not contain recent thymic emigrant CD8.sup.+ Treg cells characterized by the presence of one or more of CD62L.sup.+, CCR9.sup.+CD45RA.sup.+, CCR9.sup.+CCR7.sup.+, CCD9.sup.+CD62L.sup.+, CCR9.sup.+CD45RO.sup. and CCR9.sup.+CCR7.sup.+CD62L.sup.+CD45RA.sup.+CD45RO.sup..

45. An isolated CD8.sup.+ Treg cell population according to claim 32, wherein the CD8.sup.+ Treg cells further are CD38.sup.+, CD69.sup.+ and/or CD44.sup.+ to denote recent activation.

46. A composition comprising an isolated CD8.sup.+ Treg cell population according to claim 32, wherein the cells are suspended in a saline-based solution having a physiological pH, further comprising an additive to promote cell survival, stability and/or cryopreservation.

47. A pharmaceutical composition comprising an isolated CD4.sup.+ Treg cell population according to claim 32.

48. A method of treating an inflammatory or autoimmune disease, comprising administering a pharmaceutical composition according to claim 47 to a subject in need thereof.

49. The method of claim 48, wherein the inflammatory or autoimmune disease is selected from Crohn's disease and ulcerative colitis.

50. The method of claim 48, wherein the inflammatory or autoimmune disease is selected from primary scelrosing cholangitis and acute celiac disease.

51. A method for treating a patient suffering from an inflammatory or autoimmune disease of the gastrointestinal tract, comprising (a) obtaining Treg cells according to claim 32 from a patient suffering from the disease, (b) expanding the Treg cell population in vitro, (c) optionally, re-patterning the expanded Treg cells to obtain Treg cells that have signatures as defined in claim 32, wherein the signatures resulting from (b) or (c) are for (i) identifying that the Treg cells are mucosal tissue type tropic, (ii) optionally, identifying that the Treg cells are diseased mucosal tissue tropic, (ii) identifying that the Treg cells are emigrant cells that originated from the target mucosal tissue, and (iv) optionally, identifying that the Treg cells are capable of retention in the target mucosal tissue, (d) administering the Treg cells obtained from (b) or (c) to the patient.

52. A method according to claim 51, wherein the Treg cell population is obtained from peripheral blood of the patient.

53. A method for obtaining an isolated CD8.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 CD8.sup.+ Treg cells having signatures for (i) identifying that the T-cells are CD8.sup.+ regulatory Tcells, (ii) identifying that the Treg cells are mucosal tissue type tropic that can migrate to the diseased mucosal tissue, (iii) optionally, identifying that the Treg cells are homing cells tropic with respect to the diseased mucosal tissue, (iv) identifying that the Treg cells are emigrant cells that originate from the target mucosal tissue, and (v) optionally, identifying that the Treg cells are capable of being retained in the target mucosal tissue and optionally having one or more X-signatures and/or one or more Y-signatures, wherein X is a signature indicating that the CD8.sup.+ Tregs can localize, have emigrated from, or are marked for preferential retention in the specific part of the gastrointestinal tract that is diseased, and Y is a signature indicating immunosuppressive regulatory function, wherein the isolated CD8.sup.+ Treg cell population has T-cell receptor clonal diversity restricted to clonotypes specific for antigens present in the mucosal tissue types to which the selected signatures relate.

54. A method according to claim 53, 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.

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

56. A method according to claim 54, 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.

57. A method according to claim 54, wherein the excluded cells are CCR9.sup.+CCR7.sup.+CD62L.sup.+CD45RA.sup.+CD45RO.sup. cells.

58. A method according to claim 53, further comprising identifying CD8.sup.+ Treg cells that are emigrant and immigrant cells such as integrin-type or other adhesion molecules associated with target tissue adhesion and transmigration through tissue-integral vasculature.

59. A kit containing antibodies against (i) CD8.sup.+ and/or CD122.sup.+, (ii) 4, 4.sup.+ and/or 7.sup.+, (iii) optionally, CCR9.sup.+, (iv) CD62L.sup., and (v) optionally, 4.sup.+E.sup.+7.sup.hi.

60. A kit according to claim 59, further comprising one or more antibodies against one or more X signatures selected from any of (a) CD49d, CD54, CD99, CD99R, CD166, (b) CD49a, CD49c, CD49f, CD102, CD165, CDw328, CDw329, (c) CD37, CD38, and CD49e.

61. A kit according to claim 59, further comprising one or more antibodies against one or more Y signatures selected from any of (d) CD25, CD58, CD73, CD95, CD105, CD107a, CD107b, CD122, CD244, CD268, CD274, (e) CD31, CD35, CD39, CD41a, CD63, CD85, CD88, CD97, CD108, CD120b, CD127, CD130, CD132, CD151, CD210, CD221, CD226, CD335, CD336, EGF-R, (f) CD66, CD126, CD150, CD161, CD195, CD200, and CD279.

62. A kit according to claim 59, further comprising one or more antibodies against one or more of CD38, CD69, CD44, CD45, CCR7, and CD45RO.

Description

LEGENDS TO FIGURES

[0146] FIG. 1. CD8.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 CD8 lymphocytes expressed as beta7 vs alpha4 integrin dot plots. Each gate defining alpha4+beta7++, alpha4+beta7+, alpha4-beta7- and alpha4+beta7 are redisplayed as CD103 vs CCR9 contour plots in B), C), D) and E), respectively.

[0147] FIG. 2. CD8.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 CD8 lymphocytes expressed as CD8 vs CD103 dot plots. Each gate defining CD8+CD103 and CD8+CD103+ are redisplayed as beta7 vs alpha4 integrin dotplots in B) and C), respectively.

[0148] FIG. 3. CD8.sup.+7.sup.highCD103.sup.+ T-cells in peripheral circulation are enriched for 4.sup.+CCR9.sup.+ expressing T-cells. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD8 lymphocytes expressed as beta7 integrin vs CD103 dot plots. Each gate defining beta7+CD103 and beta7+CD103+ are redisplayed as alpha4 integrin vs CCR9 contour plots in B) and C), respectively.

[0149] FIG. 4. Distinct subsets of CD8.sup.+7.sup.highCD103.sup.+ and CD8.sup.+7.sup.+CD103.sup.+ T-cells in peripheral circulation do not express cytotoxic markers. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD8 lymphocytes expressed as beta7-integrin vs CD103 pseudocolour plot or B) perforin vs Granzyme B dotplots. C) to F) display Perforin vs Granzyme B dotplots of gated populations from from A) as indicated.

[0150] FIG. 5. CD8.sup.+7.sup.highCD103.sup.+ T-cells but not CD8.sup.+7.sup.+CD103.sup.+ T-cells in peripheral circulation are highly enriched for CCR9 expression but neither express cytotoxic markers. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD8 lymphocytes expressed as beta7-integrin vs CD103 pseudocolour plot or B) CCR9 vs Granzyme B dotplots. C) to F) display CCR9 vs Granzyme B dotplots of gated populations from from A) as indicated.

[0151] FIG. 6. CD8.sup.+7.sup.highCD103.sup.+CCR9.sup.+ T-cells do not express CD62L but CD8.sup.+7.sup.+CD103.sup.CCR9.sup.+ T-cells are enriched for CD62L expression. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD8 lymphocytes expressed as beta7-integrin vs CD103 pseudocolour plot or B) CCR9 vs CD62L dotplots. C) to F) display CCR9 vs CD62L dotplots of gated populations from from A) as indicated.

[0152] FIG. 7. CD8.sup.+7.sup.highCD103.sup.+CCR9.sup.+ T-cells do not express CD45RA but CD8.sup.+7.sup.+CD103.sup.CCR9.sup.+ T-cells are enriched for CD45RA expression. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD8 lymphocytes expressed as beta7-integrin vs CD103 pseudocolour plot or B) CCR9 vs CD45RA dotplots. C) to F) display CCR9 vs CD45RA dotplots of gated populations from from A) as indicated.

[0153] FIG. 8. CD8.sup.+7.sup.highCD103.sup.+ and CD8.sup.+7.sup.+CD103.sup.+ T-cells do not contain CD45RA/CCR7 double positives but CD8.sup.+7.sup.+CD103.sup. T-cells are enriched for CD45R/CCR7 double positive nave population. PBMC recovered from healthy donor blood over ficoll were immediately labelled with indicated antibodies and analysed by flow cytometry. A) Total CD8 lymphocytes expressed as beta7-integrin vs CD103 pseudocolour plot or B) CCR7 vs CD45RA dotplots. C) to F) display CCR7 vs CD45RA dotplots of gated populations from from A) as indicated.

[0154] FIG. 9. Reduced relative numbers of peripheral CD8+CCR9+ T-cell populations in CD patients when compared to healthy controls. PBMCs prepared from healthy controls (HC) and CD patients were stained for CD8, CD103, 7, 4 CD62L and CCR9 then analysed by flow cytometry. A) Observed percentage of 4.sup.+7.sup.hi cells among all CD8.sup.+ lymphocytes. B) Observed percentage of CD103.sup.+ cells among all CD8.sup.+ lymphocytes. C) Observed percentage of CCR9.sup.+ cells among CD8.sup.+4.sup.+7.sup.hiCD103.sup.+ lymphocytes (left panel) and CD8.sup.+4.sup.+7.sup.+CD103.sup.+ lymphocytes (right panel). D) Observed percentage of CCR9.sup.+ cells among CD8.sup.+CD103.sup.+ lymphocytes (left panel) and CD8.sup.+CD103.sup. lymphocytes (right panel). E) Observed percentage of CD8.sup.+CD62L.sup.CCR9.sup.+ cells among all CD8.sup.+ lymphocytes (left panel) and CD8.sup.+CD62L.sup.+CCR9.sup.+ cells among all CD8.sup.+ lymphocytes (right panel).

[0155] FIG. 10. CD8.sup.+ GranzymeB.sup.+ T-cells are largely local CD62L.sup. character in intestinal tissues and do not change in relative abundance in inflamed tissues of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease and were immediately stained with the indicated antibodies. All plots display CD62L vs Granzyme B contour plots of CD8.sup.+ gated cells.

[0156] FIG. 11. CD8.sup.+CD103.sup.+ T-cells are largely local CD62L.sup. character in intestinal tissues and are acutely diminished in relative abundance in the inflamed tissues of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease and were immediately stained with the indicated antibodies. All plots display CD62L vs CD103 contour plots of CD8.sup.+ gated cells.

[0157] FIG. 12. CD8.sup.+CD62L.sup.CD103.sup.+ T-cells are highly enriched for CD38 and CCR9 expression in intestinal tissues of CD patient where CCR9 expression is acutely diminished in inflamed tissues. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease and were immediately stained with the indicated antibodies. All plots display CD38 vs CCR9 of CD8.sup.+CD62L.sup.CD103.sup.+ cells (left panels) CD8.sup.+CD62L.sup.+CD103.sup. (right panels).

[0158] FIG. 13. CD8.sup.+GranzymeB.sup.+ T-cells generally do not express CD103 in MLN of patients but may express CD103.sup.+ in LP. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease and were immediately stained with the indicated antibodies. All plots display CD103 vs GranzymeB contour plots of CD8.sup.+ gated cells.

[0159] FIG. 14. Relative abundance of CD8.sup.+CD103.sup.+CCR9.sup.+ T-cells is acutely diminished in the inflamed tissues of CD patients. Single cell suspensions prepared from indicated resected tissues of a representative CD patient with ileocaecal disease and were immediately stained with the indicated antibodies. All plots display CD103 vs CCR9 contour plots of CD8.sup.+ gated cells.

[0160] FIG. 15. 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 and were immediately stained with the indicated antibodies. All plots display HLA-DR vs CD103 of CD45.sup.+CD11c.sup.hiCD80.sup.+ cells.

[0161] FIG. 16. T-cell Migratory-type surface markers correlated with CD8.sup.+CD103.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 CD8 lymphocytes expressed as CD8 vs CD103 dotplot. B) CD8.sup.+CD103.sup. cells expressed as SSC vs CD54 contour plot. C) CD8.sup.+CD103.sup.+ cells expressed as SSC vs CD54 contour plot. D) Summary of ranked preferable migratory markers of X.sup.+/X.sup. condition for identification of regulatory T-cells.

[0162] FIG. 17. Example of T-cell Functional-type surface markers correlated with CD8.sup.+CD103.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 CD8 lymphocytes expressed as CD8 vs CD103 dotplot. B) CD8.sup.+CD103.sup. cells expressed as SSC vs CD58 contour plot. C) CD8.sup.+CD103.sup.+ cells expressed as SSC vs CD58 contour plot.

[0163] FIG. 18. Summary of T-cell Functional-type surface markers correlated with CD8.sup.+CD103.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 as in FIG. 17. Table summarises ranked preferable functional markers of Y.sup.+/Y.sup. condition for identification of regulatory 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 may also exist.

[0164] FIG. 19. Purification of CD8.sup.+7.sup.highCD103.sup.+ cells from peripheral blood. 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 subgates for single cells in B) and CD8+ cells in C). Plot D) redisplays total CD8+ lymphocytes as Beta7-integrin vs CD103 and defines a gate around the rare sub-population Beta7-integrin.sup.Hi CD103k. Plot E) shows the enrichment of the rare Beta7-integrin.sup.Hi CD103.sup.+ sub-population of CD8 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.

[0165] FIG. 20. In vitro Expansion of CD8.sup.+7.sup.highCD103.sup.+ T-cells. The subset of CD8.sup.+7.sup.highCD103.sup.+ enriched for Tregs was highly purified by FACS (FIG. 19), and was cultured over several days. The proliferation curve displays the expansion of a starting pool of 80,000 sorted cells reaching almost 40 millions cells over 13 days of culture.

[0166] FIG. 21. Peripheral CD8.sup.+CD103.sup.+ T-cells have skewed V usage compared to total CD8 cells. PBMC recovered from blood of three healthy donors over ficoll were immediately labelled with CD45, CD8 and CD103 antibodies in addition panels of V-specific antibodies and analysed by flow cytometry. Coverage of donor C11 (top panel) was CD8.sup.+48.56% and CD8.sup.+CD103.sup.+ 73.48%. Coverage of donor C26 (middle panel) was CD8.sup.+69.46% and CD8.sup.+CD103.sup.+ 66.57%. Coverage of donor C34 (bottom panel) was CD8.sup.+ 49.43% and CD8.sup.+CD103.sup.+ 52.42%.

[0167] Based on experiments, the inventors have made the following observations: CD8 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 putative CD8 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. These cells may optionally be repatterned to express correct homing receptors with addition of specific recombinant protein and chemical stimuli 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 CD8+ populations observed in peripheral circulation, when compared to non-mucosal populations.

[0168] The general fundamental difference between CD4 and CD8 cells is that CD4 cells primarily engage with MHCII-antigen complexes, while CD8 engage MHCI-antigen complexes. In this sense, CD4 cells can be considered to engage antigens derived extrinsically to the cell, while CD8 engage antigens derived intrinsically.

[0169] CD8 T-cells are primarily considered to be cytotoxic effectors that eliminate virally infected and tumour host cells, for instance. While CD8 cells have been recognised to contain regulatory immunosuppressive subsets, there are few if any surface markers that reliably identify CD8 Treg cells. There have been several markers proposed to partly define CD8 Tregs, or at least subsets of CD8 Tregs. These include FOXP3, similar to that of CD4 Tregs, CD28 and CD103. What was of significant interest to was the proposition that CD8 Tregs were positive for CD103. We considered it likely that this is simply coincidental to the fact a that a large proportion of CD8 Tregs have a propensity to dominantly recirculate to the small intestine as part of oral tolerance mechanisms. Indeed, it stands to reason that a large proportion of CD8 Tregs are involved with the small intestinal mucosa, being by far the largest site of direct interaction between potential pathogens and the immune system. The possibility that small-intestinal homing and retention phenotype is indicative of CD8 Treg characteristics in the peripheral blood of humans was thus investigated.

[0170] FIG. 1 presents an analysis of blood from a healthy donor where CD8 cells are gated and displayed as 7 vs 4 dotplots. 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 FIG. 1a are displayed as CD103 vs CCR9 contour plots in FIG. 1b to FIG. 1e. As anticipated, the 4.sup.+7.sup.hi population is highly enriched for CD103, with some 97% of all cells expressing CD103. This population is also highly enriched for CCR9 expression (FIG. 1b).

[0171] 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. 2a simply shows gated CD8 cells as a CD8 vs CD103 dotplot. From here, total gated CD103 and CD103+ cells are displayed a 7 vs 4 dotplots in FIGS. 2b and 2c respectively. The negative population appears as a standard pool of CD8 T-cells with regard to 7 and 4 expression, although strikingly lack the expression of a 4.sup.+7.sup.hi population (FIG. 2b). In contrast the CD103+ population is highly enriched for the 4.sup.+7.sup.hi (FIG. 2bc, and compare FIG. 1a). 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.

[0172] This data can be used to visualise the quite clear expression of CD103 on the 7.sup.hi population (FIG. 3a). This CD8.sup.+7.sup.hiCD103.sup.+ population is highly enriched for 4.sup.+CCR9.sup.+ cells (FIGS. 3b and 3c).

[0173] To investigate whether the identified cells of CD8.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+ are of a Treg nature, CD8 cytotoxic markers granzyme B and perforin were analysed within the various populations presented above. This was conducted on the background of higher E7 resolution where two distinct subsets CD103+ cells can be distinguished based on their 7 expression (FIG. 4a). Of all CD8 T-cells in circulation, some 30% are positive for either GranzymeB or Perforin and GranzymeB cytotoxic markers (FIG. 4b) However, almost no cytotoxic marker expression is observed in the CD8.sup.+7.sup.highCD103.sup.+ or CD8.sup.+7.sup.+CD103.sup.+ (FIGS. 4c and d). These data are consistent with a Treg character of CD8.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+ cells.

[0174] To further expand the observations from FIGS. 1 to 4, the CCR9-Granzyme relationships of CD8 cells were investigated (FIG. 5). As expected, CD8.sup.+7.sup.highCD103.sup.+ are highly enriched for CCR9 expression and do not express GranzymeB (FIG. 5c). Further to this, inside this CD8.sup.+7.sup.highCD103.sup.+ population, CCR9.sup. cells have around triple the relative abundance of GranzymeB.sup.+ cells. Interestingly, the bulk of remaining CCR9+ cells in the peripheral CD8 pool are of CD8.sup.+7.sup.+CD103.sup. character (FIG. 5e), while almost no CCR9 expression is observed on CD8.sup.+7.sup.+CD103.sup.+ (FIG. 5d). This latter CD8.sup.+7.sup.+CD103.sup.+ population do not ubiquitously express 4 integrin, suggesting a distinct tissue origin of these cells (not shown). In general, CCR9+ CD8 cells do not express significant levels of cytotoxic Tcell markers.

[0175] It is hypothesised that CD8.sup.+7.sup.+CD103.sup. cells with CCR9 expression could represent recent thymic emigrants. To address this, CCR9 and CD62L relationships were investigated in the CD8.sup.+7.sup./+/highCD103.sup./+ populations (FIG. 6). First we may observe the fact that in total CD8 T-cells CD62L negativity correlates with a CCR9.sup.high phenotype, while CD62L positivity correlates with a CCR9.sup.+ phenotype (FIG. 6b). Consistent with CD8.sup.+7.sup.+CD103.sup.CCR9.sup.+ cells representing recent thymic emigrants, these cells are enriched for CD62L positivity (FIG. 6e). Conversely, CD8.sup.+7.sup.highCD103.sup.+ do not express significant levels of CD62L, suggesting that they could be enriched for a mucoscal emigrant population considering their high expression of 4, 7, CD103 and CCR9 (FIG. 6c).

[0176] To further confirm both thymic emigrant nature of CD8.sup.+7.sup.+CD103.sup. cells, and indeed the expected antigen-experienced nature of CD8.sup.+7.sup.hiCD103.sup.+ cells, the expression of CCR7 and CD45RA was analysed on these subpopulations. Firstly, nearly all CCR9.sup.+CD45RA.sup. cells in peripheral circulation were contained within the CD8.sup.+7.sup.hiCD103.sup.+ population (FIG. 7 b and c), confirming their activated nature and supporting that this is a population of activated/proliferating mucosal emigrants. Conversely, the CD8.sup.+7.sup.+CD103.sup. cells that express also CCR9 are almost exclusively express CD45RA (FIG. 7e), supporting the expectation that these cells represent nave recent thymic emigrants. In addition it is observed that CD8.sup.+7.sup.+CD103.sup.+ cells, which do not generally express CCR9 are of an activated CD45RA nature (FIG. 7d). This suggests that these cells could be activated emigrants from distinct mucosal, or nonmucosal tissues. The recent thymic emigrant nature of CD8.sup.+7.sup.+CD103.sup. cells was further confirmed by the high enrichment of CCR7 expression within this population (FIG. 8e).

[0177] Considering the observations above, it was considered that CD patients with active disease might show a distortion of mucosal emigrant CD8 populations in peripheral circulation. These various populations of CD8.sup.+ were investigated in the peripheral blood of a cohort of 10 CD patients with active disease and 10 healthy controls by flow cytometry (FIG. 9). No difference was observed in the percentage of 4.sup.+7.sup.high cells among all CD8.sup.+ lymphocytes (FIG. 9a), nor a difference in total CD103.sup.+ cells among all CD8+ lymphocytes (FIG. 9b) between CD patients and healthy controls. However, the percentage of cells expressing CCR9 was significantly diminished in CD patients in both CD8.sup.+4.sup.+7.sup.hiCD103.sup.+ and CD8.sup.+4.sup.+7.sup.+CD103.sup.+ peripheral cell populations (FIG. 9c). This is underscored by reduced percentage of CCR9+ cells among all CD8.sup.+CD103.sup.+ and CD8.sup.+CD103.sup. cells alike (FIG. 9d). Among total CD8+ cells, a reduced percentage of cells carried CD62L.sup.CCR9.sup.+ marking, while the percentage of CD62L.sup.+CCR9.sup.+ cells was unchanged (FIG. 9e). Taken together, these results suggest sufficient export CD8.sup.+4.sup.+7.sup.hi(CD103.sup.+) cells from mucosa in CD patients, but an acute defect in the CCR9 expression in these cells. Conversely, the total percentage of CD62L.sup.+CCR9.sup.+ recent thymic emigrants remains unchanged.

[0178] To further investigate the possibility that CD8 cells derived from the intestinal mucosa with migratory behaviours, CD8 cells within normal and inflamed tissues of the small bowel from CD patients were analysed. First it was confirmed that GranzymeB.sup.+ CD8 Tcells are largely of a local character in intestinal tissues, since GranzymeB and CD62L expression are mutually exclusive in MLN (FIG. 10). Importantly, one does not observe CD62L expression in LP tissues shw. Interestingly, the CD8.sup.+GranzymeB.sup.+ population is of the same relative abundance in inflamed and normal tissues of CD patients (FIG. 10). This suggests that the inflammation in these tissues is not directly driven by an expansion of CD8 cytotoxic T-cells.

[0179] Similar to the cytotoxic CD8 T-cells present in the MLN and LP, CD103.sup.+CD8 are largely of a local character in these intestinal tissues, since CD103 and CD62L expression are mutually exclusive (FIG. 11). In contrast, while GranzymeB.sup.+ cells are present with the same relative abundance in normal and inflamed tissues, the CD103.sup.+ CD8 T-cell population is significantly diminished in both SLN and inflamed LP (FIG. 11).

[0180] To further characterise the nature of CD103.sup.+ CD8 cells in MLN and LP, expression of CD38 and CCR9 was investigated (FIG. 12). CD8.sup.+CD62L.sup.CD103.sup.+ T-cells are highly enriched in normal MLN for CD38 and CCR9 expression. This is in contrast to the low expression of both CD38 and CCR9 in CD8.sup.+CD62L.sup.+CD103.sup. T-cells, which supports the locally-experienced and migratory nature of the CD8.sup.+CD62L.sup.CD103.sup.+ subset. Strikingly, the expression of both CCR9 is strongly diminished on CD8.sup.+CD62L.sup.CD103.sup.+ cells of the SLN when compared to normal MLN (FIG. 12). CD8.sup.+CD62L.sup.CD103.sup.+ cells in the normal LP are almost exclusively CD38.sup.+CCR9+ in nature, in further support of the local education and migratory nature of CD8.sup.+CD62L.sup.CD103.sup.+ cells. Similar SLN, inflamed LP shows are reduction in CCR9 expression, but in this tissue maintain CD38 expression.

[0181] In analogy to the blood analyses presented in FIGS. 4 to 6, the CD103 expressing CD8 cells in the MLN and SLN largely do not express GranzymeB (FIG. 13). In contrast, however, GranzymeB cells in both inflamed and normal LP strongly express CD103, however there is notable reduction in the CD103 expression on both GranzymeB positive and negative cells in the inflamed LP. Collectively, these observations suggest that resident cells of the LP, regardless of functional phenotype, are capable of expressing CD103. However, cells migrating extrinsically, or raised environments other than the LP but endowed with LP homing properties, are largely of noncytotoxic nature. Additional cues not investigated could be responsible for cytotoxic T-cell retention in the LP, or migrating cytotoxic cells are culled in non-mucosal environments. Overall these results points towards a defect in patterning of non-cytotoxic T-cell migratory cues.

[0182] To directly visualise a hypothesised defect in migratory behaviour of CD8 cells, CD103 and CCR9 double positivity was investigated in the small bowel tissues of CD patients (FIG. 14). In normal MLN CD8.sup.+CD103.sup.+CCR9.sup.+ cells account for roughly 22% of all CD8 cells, while this acutely diminished to 3% in SLN. Similarly, a vast majority of cells in normal LP are double positive for CD103 and CCR9, while both expression of CCR9 or CCR9/CD103 is reduced in inflamed LP.

[0183] Overall these results are suggestive of a defect in patterning of non-cytotoxic T-cell migratory cues. The DC subset that is responsible for patterning this receptor expression on T-cells are known to be a CD103.sup.+ DC subset. The possibility of a numerical deficiency in this CD103.sup.+ DC population was tested as a possible cause of CCR9 and CD103 deficiency. FIG. 15 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.sup.+ 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).

[0184] To further identify migratory markers on peripheral mucosal emigrant CD8.sup.+ cells high throughput screening was conducted on the basis of CD103.sup.+ expression. FIG. 16 a to c shows an example of an identified marker, CD54, that is highly enriched in CD8.sup.+ CD103.sup.+. FIG. 16d summarises all key markers identified in this screen, and classifies them from high relevance (1) to lower relevance (3).

[0185] Similarly, a high throughput screen was conducted to identify surface markers associated with proinflammatory or regulatory function on peripheral mucosal emigrant CD8.sup.+ Tcells on the basis of CD103.sup.+ expression. FIG. 17 shows an example of one identifled marker, CD58 that is highly enriched in CD8.sup.+ CD103.sup.+. FIG. 18 summarises all key markers identified in this screen, and classifies them from high relevance (1) to lower relevance (3). In general, positively correlated markers are associated with regulatory functions, while negatively correlated markers are associated with proinflammatory functions.

[0186] In order to assess the feasibility of recovering CD8.sup.+7.sup.hiCD103.sup.+ T-cells from peripheral blood at high purity, PBMCs from healthy donors were labelled and sorted on the basis of these defined markers (FIG. 19). FIG. 19 a to d show the basic gating strategy of FACS-based purification of these cells, and FIG. 19e displays achieved purity of greater than 99%.

[0187] As proof of concept that CD8.sup.+7.sup.hiCD103.sup.+ purified from peripheral blood of could be expanded as a therapeutic population, cells purified by FACS as described in FIG. 19 were expanded with recombinant stimuli in vitro. FIG. 20 displays a representative growth curve of such an expansion.

[0188] Finally, to test the hypothesis that mucosal emigrant CD8+ cells in peripheral circulation are in some way clonally restricted due to their activated, emigrant and recirculating nature, a assessment of V usage among CD8.sup.+CD103.sup.+ in peripheral circulation was conducted (FIG. 21). Across three healthy donors, the usage of V segments was markedly different between CD8.sup.+CD103.sup.+ and the total pool of CD8.sup.+ lymphocytes. This indirectly supports the proposal that CD8.sup.+CD103.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.

Experimental Material and Methods

Material

[0189] 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 (F1B27) 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), CD11 b BV510 (ICRF44), CD11 b PE-Cy7 (ICRF44), CD11c BV421 (B-Iy6), CD11c BV605 (B-Iy6), CD11c PE (B-Iy6), 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 (T066), CD39 FITC (T066), 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 (BN13), CD152 BV786 (BN13), 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 (BUCCR9), CD199 APC (112509), CD303 BV421 (201A), CD357 APC (62), Annexin V APC, 7 integrin BV421 (F1B504), 7 integrin BV605 (F1B504), 7 integrin PE (F1B504), 7 integrin APC (F1B504), 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 (T039), HLA-DR, DP, DQ Alexa 647 (T39), HLA-DQ FITC (Tu169), IFN-g Alexa 647 (4S.63), 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 (6032F6), Ig light chain PE (G20-193), Light chain A PE (JDC-12), IgM BV605 (G20-127), IgM FITC (G20-127), IgM FITC IgM PE-Cy5 (G20-127), Lineage cocktail FITC, Perforin BV421 (5G9), 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 (T10139.1A-31), TCR FITC (61), TCR -1 FITC (11F2), TCR PE-CF594 (61), 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 (H129), CD1d (CD1d42), CD30 (BerH8), CD54 (LB-2), CD2 (RPA-2.10), CD31 (WM59), CD55 (IA10), CD3 (HIT 3a), CD32 (FL18.26), CD56 (6159), 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 (C638, NL07), CD61 (VI-PL2), CD7 (M-T701), CD37 (M-6371), 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 (H110a), CD41a (HIP8), CD64 (10.1), CD11a (G43-25B), CD41b (HIP2), CD66 (a,c,d,e) (61.1/CD66), CD11 b (D12), CD42a (ALMA.16), CD66b (G10F5), CD11c (B-Iy 6), CD42b (HIP1), CD66f (IID10), CD13 (WM15), CD43 (1G10), CD69 (FN50), CD14 (M5E2), CD44 (G44-26), CD70 (Ki-24), CD15 (H198), CD45 (H130), CD71 (M-A712), CD15s (CSLEX1), CD45RA (HI100), CD72 (J4-117), CD16 (3G8), CD45R6 (MT4), CD73 (AD2), CD18 (6.7), CD45RO (UCHL1), CD74 (M-B741), CD19 (H1619), CD46 (E4.3), CD75 (LN1), CD20 (2H7), CD47 (66H12), CD77 (5B5), CD21 (B-Iy 4), CD48 (T U145), CD79b (C63-1), CD22 (H1622 CD49a SR84 CD80 L307.4 CD23 EBVCS-5 CD49b AK-7 CD81 JS-81), CD24 (ML5), CD49c (C3 II.1), CD83 (H615e), 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 (134136), 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 (4134-1), CD195 (2D7/CCR5), CD97 (VIM3b), CD137 (Ligand C65-485), CD196 (11A9), CD98 (UM7F8), CD138 (Mil 5), 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 (C6R-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 (H464), CD151 (14A2.H1), CD229 (HLy9.1.25), CD108 (KS-2), CD152 (BN13), 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 (1101), CD118 (12D3), CD162 (KPL-1), CD271 (C40-1457), CD119 (GIR-208), CD163 (GHI/61), CD273 (MIH18), CD120a (MABTNFR1-A1), CD164 (N666), 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 (61), Ms IgG2b IC (27-35), CD305 (DX26), HPC (13139), Ms IgG3 IC (J606), CD309 (89106), HLA-A,B,C (G46-2.6), CD49f (GoH3), CD314 (1D11), HLA-A2 (13137.2), 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 (61311), CD210 (3F9), CD335 (9E2/NKp46), Disialoganglioside GD2 (14.G2a), CD212 (266/12beta 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 (T10139.1A-31), TRA-1-60 (TRA-1-60), Integrin 7 (F16504), 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; 10Test Beta Mark TCR V Kit from Beckman Coulter.

Patients and Tissue Preparation

[0190] 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/mIgentamycin 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. 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.

Patients and Blood Preparation

[0191] 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.

Direct Cell Purification by FACS

[0192] 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 Software (BD Biosciences) to acquire data. Final analyses utilized FlowJo software (Tree Star Inc.).

Expansion of Sorted Cell Populations

[0193] 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).

Row Cytometry

[0194] 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.).

Statistics

[0195] All data was expressed as meanSEM. Pair wise comparisons were two-tailed MannWhitney 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.

ItemsSpecific Issues

[0196] 1. Treg cells for use in the treatment of an inflammatory disease of the gastrointestinal tract, the Treg cells have signatures for
i) identifying that the T-cells are regulatory Tcells,
ii) identifying that the Treg cells are tissue type tropic, i.e they can migrate to the diseased tissue,
iii) identifying that the Treg cells are tropic with respect to the diseased tissue of the gastrointestinal tract, i.e. they are homing cells,
iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the target tissue of gastrointestinal tract, and/or
v) identifying that the Treg cells are retained in the target tissue of the gastrointestinal tract,
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).
2. Tregs for use according to item 1, wherein the inflammatory disease is Crohn's disease or ulcerative colitis.
3. Tregs for use according to item 2, wherein the disease is Crohn's disease which is located in the small bowel.
4. Treg cells for use according to any of the preceding items for the treatment of an inflammatory disease of the small bowel, the Treg cells have signatures for
i) identifying that the T-cells are regulatory Tcells,
ii) identifying that the Treg cells are mucosal tropic,
iii) identifying that the Treg cells are small bowel tropic, and optionally the Treg cells have signatures for
iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the small bowel, and/or
v) identifying that the Treg cells are retained in the small bowel.
5. Treg cells for use according to item 4 having signatures for
iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the small bowel.
6. Treg cells for use according to any of items 3-5 having signatures for
v) identifying that the Treg cells are retained in the small bowel.
7. 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 CD8.sup.+, or CD8.sup.+CD28.sup.+.
8. 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.+.
9. 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 is 47.sup.highE.sup.+ or 4.sup.+.sup.highE.sup.+.
10. Treg cells for use according to any of the items 3-9, wherein the signatures for identifying that the Treg cells are small bowel tropic is CCR9.sup.+.
11. 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.+.
12. Treg cells for use according to any of the preceding items, wherein the Treg cells comprise a signature selected from the following signatures:
CD8.sup.+4.sup.+7.sup.+CCR9.sup.+
CD8.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+
CD8.sup.+CD28.sup.+4.sup.+7.sup.+CCR9.sup.+,
CD8.sup.+CD28.sup.+4.sup.+7.sup.highE.sup.+CCR9.sup.+
CD8.sup.+4.sup.+7.sup.+X.sup.+
CD8.sup.+4.sup.+7.sup.highE.sup.+X.sup.+
CD8.sup.+CD28.sup.+4.sup.+7.sup.+X.sup.+
CD8.sup.+CD28.sup.+4.sup.+7.sup.highE.sup.+X.sup.+
CD8.sup.+4.sup.+7.sup.+
CD8.sup.+4.sup.+7.sup.highE.sup.+
CD8.sup.+CD28.sup.+4.sup.+7.sup.+
CD8.sup.+CD28.sup.+4.sup.+7.sup.highE.sup.+
wherein X is the signature relating to tropism of the diseased part of the gastrointestinal part and may be X.sup.+ or X.sup., wherein 4.sup.+ may be substituted with 4.
and any of the signatures may also comprise CD62L.sup.CD38.sup.+
13. Treg cells for use according to any of items 3-12, wherein the small bowel disease is Crohn's disease.
14. A method for treating a patient suffering from an inflammatory disease of the gastrointestinal tract, the method comprises
a) isolating Treg cells defined in any one of items 1-13 from a tissue sample obtained from a patient suffering from the inflammatory disease of the gastrointestinal tract,
b) expanding the Treg cells in vitro,
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
ii) identifying that the Treg cells are tissue type tropic,
iii) identifying that the Treg cells are diseased tissue tropic,
iv) identifying that the Treg cells are emigrant cells, i.e. they originate from the target tissue, and/or
v) identifying that the Treg cells are retained in the target tissue,
d) administering the Treg cells obtained from b) or c) to the patient.
15. A method according to item 14, wherein the expanded Treg cells from step b) or c) have features as defined in any one of items 1-13.
16. A method according to item 14 or 15, wherein the tissue sample is from peripheral blood of the patient.
17. A method according to any of items 12-15, wherein the inflammatory disease of the gastrointestinal tract is Crohn's disease.
18. A method for obtaining Treg cells as defined in any one of items 1-13, the method comprises
a) isolating Treg cells defined in any one of items 1-13 from a tissue sample obtained from a patient suffering from an inflammatory disease of the gastrointestinal tract,
b) expanding the Treg cells in vitro,
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
ii) identifying that the Treg cells are tissue type tropic,
iii) identifying that the Treg cells are diseased tissue tropic relating to the diseased part of the gastrointestinal tract,
iv) identifying that the Treg cells are emigrant cells, i.e. the originates from the target tissue of the gastrointestinal tract, and/or
v) identifying that the Treg cells are retained in the target tissue of the gastrointestinal tract.
19. A method according to item 18, wherein the inflammatory disease of the gastrointestinal tract is Crohn's disease such as Crohn's disease located in the small bowel.
20. A method according to item 18 or 19, 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.
21. A method according to any of items 18-20 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.
22. A method according to any of items 18-21, 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.
23. A method for obtaining Treg cells as defined in any one of items 1-13, the method comprising
a) providing Treg cells comprising a signature selected from CD8.sup.+,
CD8.sup.+7.sup.highE.sup.+,
CD8.sup.+CD28.sup.+,
CD8.sup.+CD28.sup.+.sup.highE.sup.+,
and the above-mentioned signatures may further comprise the signature CD62L.sup. CD38.sup.+,
and
b) re-patterning the Treg cells to further comprise the signature 47.sup.+, 4.sup.+7.sup.+, 4.sup.+7.sup.+X or 4.sup.+7.sup.+CCR9.sup.+, wherein X is as defined herein before.
24. A method according to item 23, 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
25. A method for obtaining Treg cells as defined in any one of items 1-13, the method comprising
a) providing Treg cells comprising a signature selected from
CD8.sup.+CCR9.sup.+,
CD8.sup.+7.sup.highE.sup.+CCR9.sup.+,
CD8.sup.+CD28.sup.+CCR9.sup.+,
CD8.sup.+CD28.sup.+7.sup.highE.sup.+CCR9.sup.+,
and the above-mentioned signatures may further comprise the signature CD62L.sup. CD38.sup.+,
and
b) re-patterning the Treg cells to further comprise the signature 47.sup.+ or 4.sup.+7.sup.+.
26. A method according to item 24, 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.
27. A pharmaceutical composition comprising Treg cells as defined in any of items 1-13 dispersed in an aqueous medium.
28. Treg cells as defined in any of items 1-13.