JP6273653B2 - Colitis or colorectal cancer model non-human animal and use thereof - Google Patents

Description

  The present invention relates to a colitis or colon cancer model non-human animal and use thereof.

  Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are intractable diseases whose causes are unknown. In particular, ulcerative colitis is likely to cause cancer, and the mechanism leading to carcinogenesis remains unclear.

  In order to develop a treatment method for colitis or colon cancer, it is useful to use a model animal that develops colitis and colon cancer. As a model animal that develops colitis, a mouse model is known in which ulcerative colitis is induced by allowing a mouse to drink water containing 2% dextran sulfate for 1 week (for example, Non-Patent Document 1). See).

Okayasu I., et al., A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice., Gastroenterology, 98 (3), 694-702, 1990.

  However, in the mouse model described in Non-Patent Document 1, colorectal cancer hardly occurs. Accordingly, an object of the present invention is to provide a model non-human animal that spontaneously develops colitis and colon cancer.

The present invention includes the following aspects.
(1) A colitis or colon cancer model non-human animal deficient in RAS-related protein-1a (Rap1a) and RAS-related protein-1b (Rap1b) in a T cell-specific manner.
(2) Any of the following non-human animals:
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal.
(3) T cells lacking Rap1a and Rap1b.
(4) The T cell according to (3) collected from the non-human animal of colitis or colon cancer model described in (1).
(5) A colitis or colon cancer model non-human animal which is an immunodeficient non-human animal transplanted with the T cell according to (3) or (4).
(6) The non-human animal according to (2) or the non-human animal according to (2) and the Rap1a ^{f / f} Rap1b ^{f / f} non-human animal are crossed, and Rap1a and Rap1b are specifically T cells. A non-human animal lacking Rap1 and Rap1b specifically lacking Rap1a and Rap1b, wherein the non-human animal is a colitis or colon cancer model non-human animal. Production method.
(7) A method for producing a non-human animal of colitis or colon cancer model, comprising a step of transplanting the T cell according to (3) or (4) into an immunodeficient non-human animal.
(8) On the L-selectin ligand, Mucosal Addressin Cell Adhesion Molecule-1 (MadCAM-1) or P-selectin of lymphocytes deficient in Rap1a and Rap1b in the presence or absence of the test substance A step of measuring a rolling activity, and when the rolling activity in the presence of the test substance is lower than the rolling activity in the absence of the test substance, the test substance is colitis or A method for screening a therapeutic agent for colitis or colon cancer, comprising the step of determining that the therapeutic agent is colon cancer.
(9) Breeding the colitis or colon cancer model non-human animal according to (1) or (5) under administration or non-administration of a test substance, and colitis or colon cancer of the non-human animal Measuring the degree of onset of the disease, and the degree of onset of colitis or colon cancer under administration of the test substance compared with the degree of onset of colitis or colon cancer under non-administration of the test substance A method for screening a therapeutic agent for colitis or colon cancer, comprising the step of determining that the test substance is a therapeutic agent for colitis or colon cancer.
(10) A step of culturing lymphocytes lacking Rap1a and Rap1b in the presence or absence of a test substance, and a step of measuring phosphorylation level of Ezrin / Radixin / Moesin (ERM) protein in the lymphocytes And the phosphorylation level in the presence of the test substance is higher than the phosphorylation level in the absence of the test substance, the test substance is colitis or colon cancer And a method of screening for a therapeutic agent for colitis or colon cancer, comprising the step of determining that the therapeutic agent is.
(11) a step of culturing lymphocytes deficient in Rap1a and Rap1b in the presence or absence of a test substance, and a step of measuring the kinase activity of Lymphocyte-oriented kinase (LOK) protein in the lymphocytes; When the kinase activity in the presence of the test substance is increased compared to the kinase activity in the absence of the test substance, the test substance is a therapeutic agent for colitis or colon cancer. A method for screening for a therapeutic agent for colitis or colorectal cancer.

  According to the present invention, a model non-human animal that spontaneously develops colitis and colon cancer can be provided. The model non-human animal is useful for elucidating the pathogenesis of colitis or colon cancer and for developing a treatment method.

2 is a photograph showing the result of immunoblotting in Experimental Example 1. 6 is a graph showing the results of a flow adhesion assay of Experimental Example 2. 6 is a photograph showing the result of immunoblotting in Experimental Example 3. 6 is a graph showing the results of a flow adhesion assay of Experimental Example 4. 10 is a graph showing the results of flow cytometry analysis in Experimental Example 5. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 6. FIG. 10 is a graph showing the results of a detachment assay of Experimental Example 7. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 8. FIG. 14 is a fluorescence micrograph showing the results of Experimental Example 9. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 10. FIG. (A) And (b) is the fluorescence micrograph which shows the result of Experimental example 11. FIG. (C) is a graph showing the proportion of cells exhibiting tether-like membrane elongation in Experimental Example 11. In Experimental example 12, it is a fluorescence micrograph which shows the localization of L-selectin. (A) is the microscope picture of the control cell and Rap1KD cell which introduce | transduced LifeAct-mCherry which is a marker of actin polymerization in Experimental example 13. FIG. (B) And (c) is a graph which shows the result of Experimental example 13. FIG. 14 is a photomicrograph showing the results of Experimental Example 14. 16 is a confocal microscope image showing the results of Experimental Example 15. 18 is a graph showing the results of Experimental Example 16. In Experimental example 16, it is a fluorescence micrograph which shows a mode that T cell adhered to the high endothelial venule in a mesenteric lymph node. (A)-(c) is a graph which shows the result of the experiment example 17. FIG. (A) And (b) is a graph which shows the result of Experimental example 18. FIG. (A) And (b) is a graph which shows the result of the flow cytometry analysis of Experimental example 19. FIG. (A)-(d) is a graph which shows the result of the experiment example 20. FIG. (A)-(c) is a graph which shows the result of the flow cytometry analysis of Experimental example 21. FIG. (A)-(c) is a fluorescence micrograph which shows the result of Experimental example 22. FIG. (A) And (b) is a graph which shows the result of the flow cytometry analysis of Experimental example 23. FIG. In Experimental example 24, it is the graph which measured the change of the body weight of the Rap1 ^{− / −} mouse and the Rap1a ^{f / f} Rap1b ^{f / f} mouse as a control. In Experimental Example 24, it is a photograph showing representative colon morphology of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. In Experimental Example 24, it is a graph which shows the result of having evaluated the damage by the colitis of Rap1 ^{− / −} mouse based on observation with an optical microscope. (A) to (h) are photographs showing the results of hematoxylin and eosin staining of paraffin-embedded tissue sections of the colons of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice in Experimental Example 24 and observed with an optical microscope. is there. (A) is a photomicrograph showing a crypt abscess at the site of inflammation of the large intestine of Rap1 ^{− / −} mice in Experimental Example 24 (magnification 40 times). (B) is a microphotograph showing epithelioid cell granulomas in the large intestine of Rap1 ^{− / −} mice in Experimental Example 24 (magnification 40 times). (A) is a graph showing the results of flow cytometry analysis of Experimental Example 25. (B) is a graph showing the ratio of IL-17, IFN-γ or IL-4 producing cells in CD4 positive T cells in the lamina propria of colon of Rap1 ^{− / −} mice in Experimental Example 25. In Experimental Example 26, Rap1 ^{f / f} mice and Rap1 ^{- / - [3} H] CD4 + naive T cells from mice - is a graph showing thymidine incorporation. In Experimental example 27, it is a graph which shows the result of having measured the cytokine production amount of the CD4 positive naive T cell derived from a Rap1 ^{f / f} mouse and a Rap1 ^{− / −} mouse. It is a graph which shows the result of Experimental example 28. 42 is a graph showing the results of flow cytometry analysis in Experimental Example 29. 6 is a graph showing the results of T _reg suppression assay of Experimental Example 30. 14 is a graph showing the results of flow cytometry analysis of Experimental Example 31. 14 is a graph showing the results of flow cytometry analysis of Experimental Example 31. 14 is a photograph showing the result of immunoblotting in Experimental Example 32. It is a graph which shows the result of the detachment assay of Experimental example 33. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 34. FIG. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 35. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 36. (A) And (b) is a photograph which shows the result of Experimental example 37. FIG. (A) And (b) is a graph which shows the result of the flow cytometry analysis of Experimental example 38. FIG. 42 is a photograph showing a result of immunoblotting in Experimental Example 39. It is a graph which shows the result of Experimental example 40. (A) And (b) is a microscope picture which shows the result of Experimental example 41. FIG. 4 is a photograph showing the result of immunoblotting in Experimental Example 42. 4 is a photograph showing the result of immunoblotting in Experimental Example 43. 14 is a photograph showing the result of immunoblotting in Experimental Example 44. 6 is a photograph showing the result of immunoblotting in Experimental Example 45. 14 is a photograph showing a result of immunoblotting in Experimental Example 46. 6 is a photograph showing the result of immunoblotting in Experimental Example 47. 6 is a photograph showing the result of immunoblotting in Experimental Example 48. 6 is a photograph showing the result of immunoblotting in Experimental Example 49. 6 is a photograph showing the result of immunoblotting in Experimental Example 50. 6 is a photograph showing the result of immunoblotting in Experimental Example 51. 6 is a photograph showing the result of immunoblotting in Experimental Example 52. In Experimental example 52, it is a graph which shows the result of having measured rolling activity. (A) And (b) is a graph which shows the result of having performed the flow adhesion assay in Experimental example 52. FIG. (A) And (b) is a graph which shows the result of the flow cytometry analysis in Experimental example 53. FIG. (C) is a graph showing the percentage of IL-17, IFN-γ and IL-4 producing cells as a percentage. It is a photograph which shows the result of the immunoblotting of Experimental example 54. (A) is a confocal micrograph in Experimental Example 55. FIG. (B) is the graph which showed the ratio of the cell which formed the bleb once or more in 1 minute in Experimental example 55 in percentage. 14 is a graph showing the results of flow cytometry analysis of Experimental Example 56. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 57. FIG. (A) And (b) is a graph which shows the result of the flow cytometry analysis of Experimental example 58. FIG. In Experiment 59, mice without transplantation of cells, CD4-positive was introduced only vector ^{Rap1 - / -} _{T EM} cells transplanted mice (control), and Ezrin mutant CD4 + was introduced (T567E) Rap1 ^{- / -} the T _EM cell is a graph of change in body weight of the transplanted mice. In Experimental Example 60, it is a graph which shows the result of having evaluated the damage by the colitis of a mouse | mouth based on optical microscope observation. In Experimental Example 60, it is a microscope picture of the large intestine of the mouse | mouth which transplanted the cell. 6 is a fluorescence micrograph showing the results of Experimental Example 61. FIG. 6 is a confocal micrograph showing the result of Experimental Example 62. FIG. 6 is a confocal micrograph showing the result of Experimental Example 62. FIG. It is a confocal microscope image which shows the result of Experimental example 62. In Experimental example 63, it is an image which shows the result of having introduce | transduced the sensor of Rap1 activity based on fluorescence resonance energy transfer (FRET) into the control cell, and stimulating with CXCL12. It is a figure showing a result of example 64 of an experiment. It is a photograph which shows the result of the immunoblotting of Experimental example 65. 7 is a photograph showing the result of immunoblotting in Experimental Example 66. 7 is a photograph showing the result of immunoblotting in Experimental Example 67. 6 is a photograph showing the result of immunoblotting in Experimental Example 68. 7 is a photograph showing the result of immunoblotting in Experimental Example 69. 6 is a photograph showing the result of immunoblotting in Experimental Example 70. 7 is a photograph showing the result of immunoblotting in Experimental Example 71. 14 is a graph showing the results of Experimental Example 72. 14 is a graph showing the results of Experimental Example 72. It is a graph which shows the result of Experimental example 73. (A) And (b) is a graph which shows the result of Experimental example 74. FIG. 7 is a photograph showing the results of Experimental Example 75. 7 is a graph showing the results of a flow adhesion assay of Experimental Example 76. In Experimental example 77, it is a graph which shows the result of having measured the speed | rate of rolling on LS12 cell of a control cell or FLNa / bKD cell. 7 is a graph showing the results of a flow adhesion assay of Experimental Example 78. In Experimental example 79, it is a graph which shows the result of having pre-treated the control cell or FLNa / bKD cell with the anti-LFA-1 antibody, and measuring the rolling speed | rate on LS12 cell. 6 is a graph showing the results of a flow adhesion assay of Experimental Example 80. 6 is a photograph showing the result of immunoblotting in Experimental Example 81. It is a graph which shows the result of the flow adhesion assay of Experimental example 82. In Experimental example 82, the graph which shows the result of having examined the influence of the anti-LFA-1 antibody with respect to the speed | rate of rolling on LS12 endothelial cell in absence of CCL21 of FLNa ^{f / f} T cell and FLNa <^-/-> T cell. It is. (A) And (b) is a graph which shows the result of the flow adhesion assay of Experimental example 83. (A)-(d) is a graph which shows the result of Experimental example 84. FIG. (A) And (b) is a graph which shows the result of the transwell migration assay of Experimental example 85. FIG. (A)-(c) is a graph which shows the result of the flow cytometry analysis of Experimental example 86. FIG. 2 is a photograph showing the result of immunoblotting in Experimental Example 87. FIG. It is a graph which shows the result of the flow adhesion assay of Experimental example 88. 10 is a graph showing the results of Experimental Example 89.

[Colonitis or colorectal cancer model non-human animal]
(First embodiment)
In one embodiment, the present invention provides a colitis or colon cancer model non-human animal. The colitis or colon cancer model non-human animal of this embodiment is deficient in Rap1a and Rap1b in a T cell-specific manner.

  As described later in the Examples, mice lacking Rap1a and Rap1b specifically for T cells spontaneously develop colitis and colon cancer. Therefore, the model non-human animal of this embodiment is useful as a model non-human animal that spontaneously develops colitis and colon cancer.

  The non-human animal is not particularly limited, and examples thereof include mice, rats, rabbits, pigs, cows, monkeys, chickens and the like. The model non-human animal of this embodiment is deficient in Rap1a and Rap1b in a T cell specific manner. For example, the Genbank accession number of the mouse Rap1a gene is NM_145541, and the Genbank accession number of the mouse Rap1b gene is NM_024457. When the non-human animal is an animal other than a mouse, it is sufficient that Rap1a and Rap1b in each animal species are deficient.

  In the model non-human animal of the present embodiment, the lack of Rap1a and Rap1b means that the expression of functional Rap1a protein and Rap1b protein is suppressed or deficient. For example, part or all of the Rap1a allele on the genome and part or all of the Rap1b allele may be deleted (knocked out). For example, the expression of Rap1a and Rap1b is suppressed (knocked out) by introduction of siRNA, shRNA, etc. May be down).

Examples of the method of deleting Rap1a and Rap1b specifically for T cells include a method using a Cre recombinase-LoxP system. For example, a non-human animal (flox non-human animal) in which each of the Rap1a allele and the Rap1b allele is sandwiched with a LoxP sequence that is a target sequence of Cre recombinase (hereinafter referred to as “Rap1a ^{f / f} Rap1b ^{f / f} non-human animal”) And a non-human animal introduced with an expression unit in which a gene encoding a Cre recombinase protein is linked downstream of a promoter of a gene that is specifically expressed in T cells.

  Examples of genes that are specifically expressed in T cells include lymphocyte-specific protein tyrosine kinase (Lck), CD4, and the like. Hereinafter, a non-human animal having an expression unit in which a gene encoding a Cre recombinase protein is linked downstream of a promoter of the Lck gene may be referred to as “Lck-Cre non-human animal”. A non-human animal having an expression unit in which a gene encoding a Cre recombinase protein is linked downstream of the promoter of the CD4 gene may be referred to as a “CD4-Cre non-human animal”.

When Rap1a ^{f / f} Rap1b ^{f / f} non-human animal and Lck-Cre non-human animal or CD4-Cre non-human animal are crossed, non-human deficient in Rap1a and Rap1b specifically in T cells An animal appears.

This process will be described in more detail. As an example, a case where a Rap1a ^{f / f} Rap1b ^{f / f} non-human animal and a CD4-Cre non-human animal are crossed will be described. Rap1a ^{f / f} Rap1b ^{f / f The} primary hybrid (F ₁ ) genotype obtained by crossing a non-human animal with a CD4-Cre non-human animal is CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} And the phenotype is normal.

Subsequently, when CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal and Rap1a ^{f / f} Rap1b ^{f / f} non-human animal are crossed, CD4-Cre ^+/− Rap1a ^{f / F} Rap1b ^{f / f} non-human animals appear. CD4-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animals lack Rap1a and Rap1b in a T cell-specific manner.

  Here, when an Lck-Cre non-human animal is used instead of a CD4-Cre non-human animal, “CD4-Cre” in the above description may be read as “Lck-Cre”. In addition, non-human animals deficient in Rap1a and Rap1b can also be obtained in a T cell-specific manner by mating non-human animals with genotypes other than those described above. Details will be described later.

(Second Embodiment)
In one embodiment, the present invention provides a non-human animal with colitis or colon cancer, which is an immunodeficient non-human animal transplanted with T cells deficient in Rap1a and Rap1b.

  T cells lacking Rap1a and Rap1b will be described later. As described later in the Examples, when T cells lacking Rap1a and Rap1b were transplanted into mice whose immune system was destroyed by irradiation, spontaneous colitis and colon cancer were observed.

  Therefore, the model non-human animal of this embodiment is useful as a model non-human animal that spontaneously develops colitis and colon cancer.

  Examples of immunodeficient non-human animals include irradiated mice, SCID mice, NOD-scid mice, NOD / Shi-scid-IL2Rγnull mice (NOG mice), and the like.

  In the model non-human animal of this embodiment, the species of T cells lacking Rap1a and Rap1b and the species of the immunodeficient non-human animal may be the same or different. For example, T cells lacking Rap1a and Rap1b may be derived from humans, and immunodeficient non-human animals may be mice.

[Non-human animals]
In one embodiment, the present invention provides any of the following non-human animals.
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal.

Here, “wt” means that the locus is wild type. These non-human animals can be obtained from their offspring by mating Rap1a ^{f / f} Rap1b ^{f / f} non-human animals with CD4-Cre non-human animals or Lck-Cre non-human animals, for example. it can.

Among the above non-human animals, CD4-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal lacks Rap1a and Rap1b in a T cell-specific manner, and does not have colitis or colon cancer model Become a human animal.

Among the above non-human animals, CD4-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animals, Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} Non-human animals other than non-human animals have normal phenotypes. Non-human animals with normal phenotypes are easy to maintain because they do not develop colitis or colon cancer.

If necessary, any of the above non-human animals, or any of the above non-human animals and a Rap1a ^{f / f} Rap1b ^{f / f} non-human animal can be crossed to provide colitis or a colon cancer model non-human animal. Can be easily obtained.

More specifically, when any of the above non-human animals or any of the above non-human animals is mated with a Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, CD4-Cre ^{+ /} -Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animals appear. These non-human animals are deficient in Rap1a and Rap1b in a T cell-specific manner and become colitis or colon cancer model non-human animals.

Therefore, it can also be said that the above-mentioned non-human animals and Rap1a ^{f / f} Rap1b ^{f / f} non-human animals are non-human animals for producing colitis or colon cancer model non-human animals.

  Here, “mating any of these non-human animals” refers to mating one species selected from these non-human animals and one species selected from these non-human animals. And mating with a non-human animal other than the one selected from these non-human animals.

More specifically, when any one selected from the following non-human animals is crossed, CD4-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal Appear. In addition, the following non-human animals are those that do not have the Rap1a ^{wt / wt} or Rap1b ^{wt / wt} genotype among the above-mentioned non-human animals.
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal.

CD4-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal, and Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
One non-human animal selected from the group consisting of:
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal, and Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
When one kind of non-human animal selected from the group consisting of the above is crossed, non-human animals deficient in Rap1a and Rap1b appear in the progeny.

[T cells deficient in Rap1a and Rap1b]
In one embodiment, the present invention provides T cells deficient in Rap1a and Rap1b. As will be described later in the Examples, colitis or a colon cancer model non-human animal can also be produced by transplanting the T cells of this embodiment into an immunodeficient non-human animal.

  The T cells of this embodiment may be, for example, T cells collected from the above-described colitis or colon cancer model non-human animal of the first embodiment. Collection of T cells from non-human animals can be performed by a conventional method. For example, lymphocytes collected from blood or the like of human or non-human animals may be stained with an antibody that specifically recognizes T cells, and T cells may be sorted with a cell sorter, or magnetic beads, magnet stands, etc. You may use and sort.

  The T cell of this embodiment may be derived from a human or a non-human animal. In addition, the T cell of this embodiment may be a primary cell or an established cell line.

  The T cell of this embodiment may be, for example, a T cell in which a part or all of the Rap1a allele on the genome and a part or all of the Rap1b allele are deleted (knocked out). Alternatively, for example, it may be a T cell in which expression of Rap1a and Rap1b is suppressed (knocked down) by introduction of siRNA, shRNA and the like.

  For example, the Genbank accession number of the human Rap1a gene is NM_001010935, and the Genbank accession number of the human Rap1b gene is NM_001010942. When the T cell species of the present embodiment is an animal other than a human, it is sufficient that Rap1a and Rap1b in each animal species are deficient.

[Method for producing non-human animal model of colitis or colorectal cancer]
(First embodiment)
In one embodiment, the present invention relates to any one of the following non-human animals, or any one of the following non-human animals and a Rap1a ^{f / f} Rap1b ^{f / f} non-human animal, A step of obtaining a non-human animal deficient in Rap1a and Rap1b, wherein the non-human animal deficient in Rap1a and Rap1b specifically for T cells is a colitis or colon cancer model non-human animal. A method for producing a non-human animal is provided.
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal.

Colitis or colon cancer model non-human animals should be maintained as the above-described non-human animals, Rap1a ^{f / f} Rap1b ^{f / f} non-human animals, Lck-Cre non-human animals, CD4-Cre non-human animals, etc. Can do. In particular, non-human animals having a normal phenotype are easy to maintain because they do not develop colitis or colon cancer. Then, when non-human animals are mated when necessary, colitis or colon cancer model non-human animals can be produced.

(Second Embodiment)
In one embodiment, the present invention provides a method for producing a non-human animal with colitis or a colon cancer model, comprising the step of transplanting the above-described T cells lacking Rap1a and Rap1b into an immunodeficient non-human animal.

  A non-human animal model of colitis or colorectal cancer can also be produced by the production method of the present embodiment.

[Method of screening for therapeutic agent for colitis or colon cancer]
(First embodiment)
In one embodiment, the present invention relates to the rolling activity of lymphocytes lacking Rap1a and Rap1b on L-selectin ligand, MadCAM-1 or P-selectin in the presence or absence of a test substance. And when the rolling activity in the presence of the test substance is lower than the rolling activity in the absence of the test substance, the test substance is And a method for screening a therapeutic agent for colitis or colon cancer, comprising the step of determining that the therapeutic agent is a therapeutic agent.

  As the test substance, for example, a compound library can be used, and the same applies to the screening methods of other embodiments described later.

  The lymphocyte deficient in Rap1a and Rap1b may be the T cell deficient in Rap1a and Rap1b described above. Alternatively, it may be a cell obtained by knocking out or knocking down Rap1a and Rap1b in a lymphocyte cell which is a primary cell or a cell line. Here, the lymphocyte cells may be T cells, for example, lymphocytes other than T cells such as B cells.

  In the screening method of the present embodiment, “on L-selectin ligand” means, for example, the surface of a vascular endothelial cell expressing a ligand of L-selectin protein, that is, a peripheral lymph node addressin (PNad) molecule. Alternatively, it may be a support surface coated with peripheral lymph node addressin molecules.

  Examples of peripheral lymph node addressin molecules include Glycosylation-dependent cell adhesion molecule (GlyCAM), Nepmucin (CD300LG), hexasulfated sialyl Lewis X (sLeX), and the like.

  Further, “on MadCAM-1” may be, for example, the surface of a vascular endothelial cell expressing MadCAM-1 protein or the surface of a support coated with MadCAM-1 protein. For example, the Genbank accession number of the mouse MadCAM-1 protein is NP_038619, and the Genbank accession number of the human MadCAM-1 protein is NP_570116.

  Further, “on P-selectin” may be, for example, the surface of a vascular endothelial cell expressing P-selectin protein, or the surface of a support coated with P-selectin protein. For example, the Genbank accession number of the mouse P-selectin protein is NP_035477, and the Genbank accession number of the human P-selectin protein is NP_002996.

  Rolling is a phenomenon in which lymphocytes roll on the surface of L-selectin ligand, MadCAM-1 or P-selectin. An increase in rolling activity means that the frequency of occurrence of rolling is increased, and a decrease in rolling activity means that the frequency of occurrence of rolling is reduced. The frequency of occurrence of rolling can be measured, for example, by measuring the number of times that lymphocytes have been rolled per unit time.

More specifically, for example, a lymphocyte is placed in a chamber having a surface having the above-mentioned L-selectin ligand, MadCAM-1 or P-selectin, the medium is perfused, and the lymphocyte rolling is measured. Rolling activity can be measured by an adhesion assay. The medium can be perfused by, for example, generating a laminar flow of about ² dyn / cm ² using a syringe pump.

  The process by which lymphocytes adhere to endothelial cells can be divided, for example, into rolling, temporary adhesion (0.5-10 seconds residence time) and stable adhesion (more than 10 seconds residence time). In the screening method of the present embodiment, when measuring the occurrence frequency of rolling, the frequency of occurrence of temporary adhesion and the frequency of occurrence of stable adhesion may be included, or the frequency of occurrence of only rolling by excluding these. May be measured.

  As described below in the Examples, the inventors have found that lymphocytes lacking Rap1a and Rap1b are compared to lymphocytes lacking Rap1a and Rap1b on L-selectin ligand, on MadCAM-1 or P -Clarified that the rolling activity on selectin is significantly increased. Therefore, the test substance that suppresses the increase in the rolling activity can be a therapeutic agent for colitis or colon cancer.

(Second Embodiment)
In one embodiment, the present invention provides a step of breeding the above-described colitis or colon cancer model non-human animal under administration or non-administration of a test substance, and development of colitis or colon cancer in the non-human animal. And the degree of onset of colitis or colon cancer under administration of the test substance is lower than the degree of onset of colitis or colon cancer under non-administration of the test substance A screening method for a therapeutic agent for colitis or colon cancer, comprising the step of determining that the test substance is a therapeutic agent for colitis or colon cancer.

  The non-human animal of colitis or colon cancer model may be the non-human animal of the first embodiment described above or the non-human animal of the second embodiment described above.

  The test substance may be administered orally by a method such as addition to drinking water, addition to food, direct drinking, or parenteral administration by, for example, intravenous injection.

  A method for measuring the degree of onset of colitis or colorectal cancer is not particularly limited. For example, a method for measuring change in body weight, a method for evaluating by microscopic observation of a tissue section of the large intestine, a method for measuring the amount of a cancer marker Etc.

  A test substance that reduces the degree of onset of colitis or colon cancer when administered to a non-human animal of colitis or colon cancer model can be a therapeutic agent for colitis or colon cancer.

(Third embodiment)
In one embodiment, the present invention comprises culturing lymphocytes deficient in Rap1a and Rap1b in the presence or absence of a test substance, and measuring the phosphorylation level of ERM protein in the lymphocytes. When the phosphorylation level in the presence of the test substance is increased compared to the phosphorylation level in the absence of the test substance, the test substance may be colitis or colon cancer. And a method for screening a therapeutic agent for colitis or colon cancer, comprising the step of determining that the therapeutic agent is a therapeutic agent.

  As lymphocytes deficient in Rap1a and Rap1b, the same lymphocytes as those in the screening method of the first embodiment described above can be used.

  Examples of the ERM protein include Ezrin protein, Radixin protein, and Moesin protein. For example, the Genbank accession number of the mouse Ezrin protein is NP — 033536, and the Genbank accession number of the human Ezrin protein is NP — 00104547. For example, the Genbank accession number of the mouse Radixin protein is NP_001098086, and the Genbank accession number of the human Radixin protein is NP_001247421. Further, for example, the Genbank accession number of the mouse Moesin protein is NP — 034963, and the Genbank accession number of the human Moesin protein is NP — 002435.

  As will be described later in Examples, the inventors have revealed that phosphorylation level of ERM protein is decreased in lymphocytes lacking Rap1a and Rap1b. Therefore, when lymphocytes lacking Rap1a and Rap1b are cultured in the presence of the test substance, the test substance that suppresses the decrease in the phosphorylation level of the ERM protein is a therapeutic agent for colitis or colon cancer. obtain.

(Fourth embodiment)
In one embodiment, the present invention comprises culturing lymphocytes lacking Rap1a and Rap1b in the presence or absence of a test substance, and measuring the kinase activity of LOK protein in the lymphocytes. When the kinase activity in the presence of the test substance is increased compared to the kinase activity in the absence of the test substance, the test substance is a therapeutic agent for colitis or colon cancer. A method for screening for a therapeutic agent for colitis or colon cancer.

  As lymphocytes deficient in Rap1a and Rap1b, the same lymphocytes as those in the screening method of the first embodiment described above can be used.

  For example, the Genbank accession number of the mouse LOK protein is NP_033314, and the Genbank accession number of the human LOK protein is NP_005981.

  As will be described later in Examples, the inventors have found that the kinase activity of LOK protein is decreased in lymphocytes deficient in Rap1a and Rap1b. Therefore, when lymphocytes lacking Rap1a and Rap1b are cultured in the presence of the test substance, the test substance that suppresses the decrease in the kinase activity can be a therapeutic agent for colitis or colon cancer.

The kinase activity of LOK protein is, for example, obtained by reacting lymphocyte debris with myelin basic protein (MBP), which is a substrate of LOK protein, in a buffer containing [γ- ³² P] ATP and incorporated into MBP. It can be measured by measuring the radioactivity of ³² P.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Cells and mice]
CD3-positive T cells or CD4-positive T cells are obtained from the lymph nodes or spleens of Rap1-deficient mice or filamin (FLN) -deficient mice using MidiMACS (Miltenyi Biotech) or cell sorter (model “moFloXDP”, Beckman Coulter). separated. FLNa exon 2-3 RAPlA is RAPlA ^{f / f} mice sandwiched by LoxP sequences, Rap1b ^{f / f} mice exon 1 is sandwiched by LoxP sequences raplB, exons 2-7 FLNa is sandwiched by LoxP sequences ^{f / f} mice were maintained in a specific pathogen removal environment. These mice were crossed with Lck-Cre mice or CD4-Cre mice to obtain mice lacking Rap1a, Rap1b or FLNa specifically for T cells. All animal experiments were conducted according to the protocol approved by Kitasato University Animal Experiment Committee.

[Antibodies and reagents]
Fluorescent-labeled anti-mouse CD3, CD4, CD8, CD62L, CD44, CXCR3, CCR7, α 4 β 7, Foxp3 antibodies (eBioscience, Inc.), anti-mouse LFA-1 antibody (Santa Cruz Biotechnology), anti-human LFA- 1 antibody (TS2 / 4, TS1 / 18; American Type Culture Collection), anti-T7 antibody (Novagen), anti-RFP antibody (MBL), anti-Spa-1, FLNa, FLNb antibody (Bethyl), anti-Rap1 antibody (BD Bioscience), anti-ERM, anti-phosphorylated ERM antibody (Cell Signaling), antibody against LFA-1 activation epitope (MEM148, Abcam), anti-FLAG antibody (Invitrogen), anti-β-actin antibody (Sigma), anti Halo antibody and HaloLink ™ resin (Promega), peroxidase-labeled goat anti-rat IgG antibody, peroxidase-labeled goat anti-rat IgG antibody (Cell Signaling) were used for flow cytometry, immunoprecipitation and immunoblotting. An antibody against an activation epitope of LFA-1 (KIM127) is described in T.W. Provided by Dr. Springer (Harvard Medical School, Boston). Mouse CXCL12, CCL21, and CXCL10 were purchased from R & D Systems. Cytokine production was measured using BD ™ Cytometric Bead Array (CBA) solutions (BD Biosciences).

[DNA construct]
Rap1 dominant negative mutant Rap1N17 and Rap1 dominant active mutant Rap1V12 are pcDNA3.1 vector (Life Technologies) or lentiviral vector (CSII-EF-MCS, RIKEN, Dr. Hiroyuki Miyoshi) Provided)) and expressed.

  Subclone cDNA encoding filamin a (FLNa) repeats 1-3, 4-7, 8-11 and 21 or cDNA encoding the cytoplasmic region of β2 into pGEX vector (GE Healthcare Biosciences) It was expressed as a glutathione-S-transferase (GST) fusion protein in the BL21 strain. FLNa cDNA linked with a Halo tag was purchased from Kazusa DNA Research Institute. Moreover, the repeat 3 of FLNa was deleted using KOD-Plus-mutageness kit (Toyobo) and subcloned into pcDNA3.1 vector (Life Technologies).

  Human Ezrin that mimics the phosphorylation state is an Ezrin mutant in which threonine at position 567 of cDNA (pFN21AE1417, Kazusa DNA Laboratory) is single-point substituted with glutamic acid using a mutageness kit and has a FLAG tag at the N-terminus. As a subcloning, it was subcloned into the NotI site of pcDNA3.1 vector (Life Technologies).

  The expression vector of LOK to which the FLAG tag was linked was prepared by subcloning human LOK cDNA and FLAG cDNA into EcoRI / XhoI and XhoI / XbaI sites of pcDNA3.1 vector (Life Technologies), respectively.

  The expression vector of Ral-GDS-RBD to which the EGFP tag is bound is the cDNA encoding EGFP and the RBD of human Ral-GDS (amino acids 772 to 868), NheI / HindIII and BamHI of pcDNA3.1 vector (Life Technologies). / EcoRI site to introduce each.

  Rap1 activity sensor based on Fluorescence Resonance Energy Transfer (FRET) is a Rap1 bound to yellow fluorescent protein (YFP) tag, and Rap1 bound to RAPL-RBD bound to monomeric fluorescent protein (mTurquoise) tag And made on pcDNA3.1 vector. All constructs were confirmed by sequencing of the base sequence.

[RNA interference]
BAF / LFA-1 cells using lentiviral vectors (provided by RIKEN, Dr. Hiroyuki Miyoshi) with or without GFP targeting RAP1a, Rap1b, FLNa, FLNb or control scrambled shRNA Introduced. shRNA was introduced downstream of the H1 promoter cassette of the lentiviral vector. Preparation and concentration of lentiviral particles were performed by conventional methods. The gene transfer efficiency was 90% or more.

  The base sequence of the sense strand of Rap1a siRNA is shown in SEQ ID NO: 1. The base sequence of the sense strand of Rap1b siRNA is shown in SEQ ID NO: 2. The base sequence of the sense strand of FLNa siRNA is shown in SEQ ID NO: 3. The base sequence of the sense strand of FLNb siRNA is shown in SEQ ID NO: 4.

[Flow adhesion assay]
Flow adhesion assays were performed on human endothelial cell line LS12 or on plates coated with Nepmucin, GlyCAM-1 or MadCAM-1-IgG (5 μg / mL in phosphate buffer (PBS)). Here, MadCAM-1-IgG means a fusion protein of MadCAM-1 protein and a constant region of IgG protein.

Human endothelial cell line LS12, which expressed or did not express mouse ICAM-1, was cultured on fibronectin-coated discs one day before the experiment. The monolayer of LS12 was treated with TNFα for the last 6 hours. In addition, the monolayer was optionally treated with a chemokine (CXCL12, CCL21 or CXCL10, 0.5 μM) before being placed in the flow chamber (model “FCS2”, Bioptechs). Laminar flow was generated using an automatic syringe pump (Harvard Paretas). Lymphocytes (1 × 10 ⁶ cells / mL) or BAF / L-selectin / LFA-1 cells (1 × 10 ⁵ cells / mL) are suspended in RPMI 1640 medium containing pre-warmed 10% bovine serum and 1 mM HEPES. At 37 ° C. and ² dyn / cm ² . In some experiments, cells were treated with the antibodies described. A phase difference image under a microscope field of 0.32 mm ² was recorded at 3.3 msec intervals using an Olympus Plan Fluor DL10 × / 0.3 NA objective lens. Lymphocyte frame-by-frame movement and velocity were calculated by automatically tracking individual cells using MetaMorph software (Molecular Devices). Interactions with LS12 cells, PNAd or MadCAM-1 were classified based on rolling, temporary adhesion (0.5-10 seconds residence time) and stable adhesion (more than 10 seconds residence time). The frequency of cells showing rolling, temporary adhesion and stable adhesion per minute was shown.

[T _reg inhibition assay]
Naive T cells (CD4 ⁺ CD45RB ^high CD25 ⁻ ) and regulatory T cells (T _reg ; CD4 ⁺ CD45RB ^low CD25 ⁺ ) were sorted from spleen cells and lymph node-derived single cell suspensions by FACS sorting. After sorting, naive T cells were labeled with 5,6-carboxyfluorescein diacetate (CFSE, Invitrogen) and prepared at 5 × 10 ⁵ cells / mL in complete RPMI medium. _Unlabeled T _reg cells were prepared at 2.5 × 10 ⁵ cells / mL. Subsequently, these cells were co-cultured on round bottom 96-well plates such that the ratios of T _reg cells: naive T cells were 1: 2 and 1: 4. Subsequently, cells were stimulated with 1 μg / mL soluble anti-CD3 antibody and 2 μg / mL anti-CD28 antibody. After 48 hours, cells were harvested and the proliferation of naive T cells was determined by CFSE dilution and flow cytometric analysis.

[Intracellular cytokine staining]
CD4 positive T cells were collected from the medium and phorbol-12-myristate-13-acetate (PMA, 50 ng / mL) and ionomycin (1 μg / mL) in the presence of brefeldin A (10 μg / mL) at 37 ° C., 4 ° C. Stimulated for hours. After staining with PECy7-labeled anti-CD4 antibody (RM4-5), the cells were fixed with 4% paraformaldehyde-phosphate buffer (PBS) for 10 minutes and permeated with HBSS buffer containing 0.5% saponin and 1% bovine serum. Treated with FITC-labeled anti-interferon (IFN) -γ antibody (XMG1.1), PE-labeled anti-interleukin (IL) -17A antibody (TC11-18H10), and APC-labeled anti-IL-4 antibody (11B11) for 30 minutes Stained. Subsequently, the cells were washed twice with HBSS buffer containing 0.5% saponin and 1% bovine serum, suspended in HBSS buffer containing 1% bovine serum, and analyzed by flow cytometry.

[Statistical analysis]
When the experimental groups were subjected to multiple comparisons, analysis of variance (ANOVA) followed by Bonferroni-corrected two-sided multiple t-test was performed, and when comparisons were made between two groups, Student's two-sided t-test was performed. In any case, a P value of less than 5% was considered significant.

<I. Rap1 deficiency promoted L-selectin-dependent rolling>
[Experimental Example 1]
The inventors previously used a cell line (BAF / LFA-1 / L-selectin) in which human L-selectin and LFA-1 were expressed in BAF, which is a pro-B cell line, and LS12, which is an endothelial cell. Thus, an experimental system was established that reconstituted the adhesion cascade of lymphocytes mediated by L-selectin and LFA-1.

  In order to examine the role of Rap1 in the above experimental system, the inventors knocked down Rap1 in BAF / LFA-1 / L-selectin cells by introducing a lentivirus expressing shRNA targeting Rap1a and Rap1b. did.

  BAF / LFA-1 / L-selectin cells (hereinafter sometimes referred to as “Rap1KD” cells) into which lentiviruses encoding shRNA against Rap1a and Rap1b were introduced were prepared. As a control, BAF / LFA-1 / L-selectin cells into which a lentivirus encoding scrambled shRNA was introduced were used. Cell disruptions of these cells were subjected to immunoblotting, and Rap1 protein was detected using an anti-Rap1 antibody.

  FIG. 1 is a photograph showing the results of immunoblotting. Representative results from three experiments are shown. Actin was detected as a loading control. As a result, it was confirmed that the expression level of Rap1 protein in Rap1a and Rap1b knockdown cells (Rap1KD cells) was reduced to about 20% of the control cells BAF / LFA-1 / L-selectin cells.

[Experiment 2]
Control cells, Rap1KD cells, Spa-1 expressing cells, Rap1KD cells expressing Rap1N17 and Rap1KD cells expressing Rap1V12 on LS12 cells in the presence or absence of 100 nM CXCL12, 2 dyn / cm Perfused under ² laminar flows. Here, Rap1N17 is a dominant negative mutant of Rap1, and Rap1V12 is a dominant active mutant of Rap1. Spa-1 is a RTP1-specific GTPase activating protein.

  Subsequently, a digital image showing the interaction between each cell and the endothelial cell LS12 was taken at 30 frames / second. More than 100 cell adhesion events were measured and classified as rolling, temporary adhesion, and stable adhesion as shown in FIG. In FIG. 2, the data represent the mean value ± standard error in three independent experiments. In FIG. 2, “* 1” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the control cells. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 1% compared to Rap1KD cells.

  It is known that LFA-1 mediated cell adhesion induced by CXCL12 is inhibited by Rap1 deficiency. In contrast, surprisingly, the frequency of Rap1KD cell rolling on endothelial cells was significantly increased in the absence of CXCL12.

  In addition, Spa-1 expression inhibited cell adhesion induced by CXCL12, but did not increase the frequency of rolling events. This result indicates that a decrease in Rap1-GDP promotes rolling.

  Consistent with this result, rolling frequency did not decrease when Rap1V12, a dominant active mutant, was expressed in Rap1KD cells, but rolling frequency decreased when Rap1N17, a dominant negative mutant, was expressed in Rap1KD cells. . This result indicates that the conversion from GDP to GTP facilitates capture and subsequent rolling.

[Experiment 3]
Subsequently, the inventors analyzed the function of Rap1 in the lymphocyte adhesion cascade using primary mouse T cells.

In order to produce a Rap1a and Rap1b conditional double knockout mouse (Rap1CKO), a mouse in which the Rap1a allele and the Rap1b allele are respectively sandwiched by loxP sequences (hereinafter sometimes referred to as “Rap1 ^{f / f} ” mouse). Lck-Cre mice or CD4-Cre mice expressing Cre recombinase specifically for T cells were mated. Among the obtained offspring, mice lacking Rap1 specifically for T cells (hereinafter sometimes referred to as “Rap1 ^{− / −} ” mice) were obtained.

Rap1 ^{f / f} mouse-derived T cells and Rap1 ^{− / −} mouse-derived cell debris were subjected to immunoblotting using an anti-Rap1 antibody. FIG. 3 is a photograph showing the results of immunoblotting. Representative results from three experiments are shown. Actin protein was detected as a loading control.

As a result, it was confirmed that Tap cells derived from Rap1 ^{− / −} mice are deficient in Rap1 protein.

[Experimental Example 4]
Subsequently, Rap1 ^{f / f} mouse-derived T cells (hereinafter sometimes referred to as “Rap1 ^{f / f} T cells”) and Rap1 ^{− / −} mouse-derived T cells (hereinafter referred to as “Rap1 ^{− / −} T cells”). Was perfused under laminar flow of ² dyn / cm ^{2 in} the presence or absence of 100 nM CCL21 on LS12 cells expressing mouse ICAM-1.

A digital image showing the interaction between T cells and endothelial cells was taken at 30 frames / second under laminar flow. Adhesion events of 100 or more cells were measured and classified as rolling, temporary adhesion, and stable adhesion as shown in FIG. In FIG. 4, the data represent the mean value ± standard error in three independent experiments. In FIG. 4, “*” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the total frequency in the control Rap1 ^{f / f} T cells.

As a result, Rap1 ^{− / −} T cells lacked chemokine-induced stable adhesion events on endothelial cells. However, it showed a significantly increased rolling frequency in the absence of CCL21 compared to Rap1 ^{f / f} T cells.

[Experimental Example 5]
FIG. 5 is a graph showing representative results of flow cytometry showing the expression characteristics of CCR7 in naive T cells derived from mesenteric lymph nodes of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. FIG. 24 (a) shows LFA-1 expression characteristics of naive T cells and effector memory (E / M) T cells derived from mesenteric lymph nodes of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. It is a graph which shows the typical result of the flow cytometry analysis shown. As shown in FIGS. 5 and 24 (a), the expression levels of LFA-1 and CCR7 in Rap1 ^{f / f} T cells and Rap1 ^{− / −} T cells were similar.

[Experimental Example 6]
Subsequently, the inventors determined the effect of anti-LFA-1 antibody or anti-L-selectin antibody on the interaction of Rap1 ^{f / f} T cells and Rap1 ^{− / −} T cells with LS12 cells in the absence of CCL21. It was investigated.

Rap1 ^{f / f} T cells and Rap1 ^{− / −} T cells were pretreated in the presence or absence of anti-LFA-1 antibody or anti-L-selectin antibody, and adhesion events of each cell were examined in the same manner as described above. . FIG. 6A is a graph showing the frequency of rolling, temporary adhesion, and stable adhesion of each cell.

As a result, anti-LFA-1 antibody treatment did not affect the occurrence of rolling of Rap1 ^{− / −} T cells, but anti-L-selectin antibody treatment completely inhibited the occurrence of rolling in any cell. . This result indicates that Rap1 deficiency promotes L-selectin-dependent rolling on endothelial cells.

FIG. 6 (b) is a graph showing the rate of rolling of Rap1 ^{f / f} T cells or Rap1 ^{− / −} T cells on LS12 endothelial cells in the absence of CCL21. Each cell was pretreated in the presence or absence of anti-LFA-1 antibody. In FIG. 6 (b), the data represents the mean value ± standard error in three experiments (n = 100 for each group). In FIG. 6 (b), “*” indicates that there is a significant difference at a risk rate of less than 0.3% compared to the control Rap1 ^{f / f} T cells.

The rate of rolling was reduced in Rap1 ^{− / −} T cells compared to Rap1 ^{f / f} T cells. Also, this result was not affected by pretreatment of cells with anti-LFA-1 antibody.

[Experimental Example 7]
Subsequently, adhesion events on endothelial cells were examined using Rap1KD cells. First, Rap1V12, a dominant active mutant of Rap1, was expressed in BAF / LFA-1 / L-selectin cells or Rap1KD cells as control cells. Subsequently, the LFA-1 / ICAM-1-dependent binding of each cell was measured by placing it in a quiescent state by a detachment assay.

Each cell was incubated on ICAM-1 for 10 minutes and then placed under laminar flow ( ² dyn / cm ² ) to count strongly adherent cells that were resistant to laminar flow. The results are shown in FIG. Data represent mean ± standard error in triplicate experiments (n = 50 per group). As a result, it was revealed that cells in which Rap1V12 was expressed exhibited binding activity to ICAM-1.

[Experimental Example 8]
Subsequently, the effect of anti-LFA-1 antibody or anti-L-selectin antibody on the interaction of control cells and Rap1KD cells (BAF / LFA-1 / L-selectin cells) with LS12 cells in the absence of CXCL12 investigated. As control cells, cells into which scrambled shRNA was introduced were used.

Control cells and Rap1KD cells were pretreated in the presence or absence of anti-LFA-1 antibody or anti-L-selectin antibody and perfused on LS12 cells under laminar flow of ² dyn / cm ² . Subsequently, the adhesion event of each cell was examined in the same manner as described above.

  FIG. 8A is a graph showing the frequency of rolling, temporary adhesion, and stable adhesion of each cell. In FIG. 8 (a), the data represents the mean value ± standard error in three experiments (n = 150 for each group).

  As a result, anti-LFA-1 antibody treatment did not affect the occurrence of rolling of control cells, but anti-L-selectin antibody treatment completely inhibited the occurrence of rolling in any cell.

  This result further supports that Rap1 deficiency promotes L-selectin-dependent rolling on endothelial cells.

  FIG. 8 (b) is a graph showing the rate of rolling of control cells and Rap1KD cells on LS12 endothelial cells in the absence of CXCL12. Each cell was pretreated in the presence or absence of anti-LFA-1 antibody. In FIG. 8 (b), the data represents the mean value ± standard error in three experiments (n = 150 for each group). In FIG. 8 (b), “*” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the control cells.

[Experimental Example 9]
GFP was expressed in control cells and Rap1KD cells (green fluorescent protein), and the rolling state of each cell was observed under a laminar flow using an objective lens with a magnification of 63 times. FIG. 9 is a fluorescence micrograph showing the observation results. Shown are sequential images of control cells that do not interact with endothelial cells, control cells that roll fast (> 400 μm / sec) on endothelial cells, and Rap1KD cells that roll slowly (<100 μm / sec). The scale bar indicates 5 μm.

[Experimental Example 10]
To further verify the dependence on L-selectin, we examined the adhesion events of control cells and Rap1KD cells on plates with purified GlyCAM-1 or Nepmucin (CD300LG) immobilized. .

Control cells and Rap1KD cells were perfused under a laminar flow of ² dyn / cm ² on plates fixed with purified GlyCAM-1 or Nepmucin (CD300LG). Subsequently, cell adhesion events were measured as described above.

  FIG. 10A is a graph showing the results. Data represent mean ± standard error in triplicate experiments (n = 50 per group). In FIG. 10 (a), “* 1” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the control cells.

  FIG. 10 (b) is a graph showing the rolling speed of control cells and Rap1KD cells on a plate on which GlyCAM-1 or Nepmucin (CD300LG) is fixed. In FIG. 10 (b), “* 2” indicates that there is a significant difference at a risk rate of less than 5% compared to the control cells.

  As a result, in the Rap1KD cells, the rolling frequency increased 10 times or more compared to the control cells. In addition, the rolling speed on the plate on which GlyCAM-1 or Nepmucin (CD300LG) was fixed was significantly reduced.

[Experimental Example 11]
In order to investigate the rolling morphology of control cells and Rap1KD cells, GFP was expressed in control cells and Rap1KD cells, introduced into the flow chamber, and observed for GFP fluorescence using a 63x magnification objective lens under laminar flow did. FIG. 11A is a fluorescence micrograph showing the observation result of the fluorescence of GFP. The scale bar indicates 5 μm.

  As a result, the slowly rolling (<100 μm / sec) Rap1KD cells showed some tether-like membrane elongation. In addition, when the tether adhered to the endothelial cells and extended toward the upstream side of the flow, the instantaneous velocity decreased to 50 μm / second or less. In contrast, control cells rolling fast (> 400 μm / sec) did not show a clear overhang on the endothelial cells.

  FIG. 11 (b) shows successive images of Rap1KD cells rolling slowly. The scale bar indicates 5 μm. FIG. 11 (c) is a graph showing the percentage of cells exhibiting tether-like membrane elongation. Data represent the mean ± standard error of the results of three independent experiments (n = 50 for each group). In FIG. 11 (c), “*” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the control cells.

[Experimental example 12]
FIG. 12 is a fluorescence micrograph showing the localization of L-selectin in control cells and BAF / LFA-1 cells (Rap1KD) in which Rap1 was knocked down. A photograph of two representative cells is shown. “DIC” indicates a differential interference microscope image. The scale bar indicates 5 μm. The arrow in the figure indicates a bleb-like protrusion.

  As a result, no difference was observed in the localization of L-selectin between control cells and Rap1KD cells. However, Rap1KD cells have been shown to form multiple blebs containing L-selectin.

[Experimental Example 13]
Control cells and Rap1KD cells were observed with a confocal microscope. FIG. 13A is a photomicrograph of control cells and Rap1KD cells into which LifeAct-mCherry, which is a marker for actin polymerization, was introduced. Shown are photographs of two representative cells in at least 3 independent experiments. The scale bar indicates 5 μm.

  FIG. 13 (b) is a graph showing the number of blebs formed by control cells and Rap1KD cells per minute, and FIG. 13 (c) is a graph showing bleb maintenance time in Rap1KD cells (each group). N = 50). As a result, the number of blebs and the maintenance time were significantly higher in Rap1KD cells compared to control cells.

[Experimental Example 14]
Subsequently, the inventors incubated control cells and Rap1KD cells at 37 ° C. in the absence of chemokines. FIG. 14 is a photograph showing microscopic images of these cells. As a result, it became clear that Rap1KD cells show active bleb formation in the absence of chemokine stimulation. In addition, as described later, the control cells temporarily showed bleb formation after stimulation with chemokines.

[Experimental Example 15]
FIG. 15 is a representative confocal microscope image showing bleb elongation and retraction of Rap1KD cells into which LifeAct-mCherry, an actin biosensor, was introduced. Digital images were taken every 5 seconds. The arrow in the figure indicates a bleb. The scale bar is 5 μm. Unlike other known cell protrusions, it is known that bleb elongation does not involve actin polymerization, so that bleb formation can be confirmed using this as an index.

  The above results indicate that Rap1-GDP suppresses L-selectin dependent capture and rolling on endothelial cells by limiting tether formation.

<II. Rap1 deficiency impairs T cell homeostasis>
[Experimental Example 16]
Rap1 ^{f / f} T cells or Rap1 ^{− / −} T cells were passed through high endothelial venules in the mesenteric lymph nodes of mice, and the frequency of rolling or adhesion was measured. FIG. 16 is a graph showing the results. Data represent the mean ± standard error of the results of 4 independent experiments. In FIG. 16, “*” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the corresponding Rap1 ^{f / f} T cells.

FIG. 17 is a fluorescence micrograph showing a state in which T cells adhere to high endothelial venules in mesenteric lymph nodes. In vivo images showing adhesion of lymphocytes were taken 30 minutes after intravenous administration of Rap1 ^{f / f} T cells or Rap1 ^{− / −} T cells labeled with different labels. Representative results in 3 independent experiments are shown.

As a result, the frequency of rolling of Rap1 ^{− / −} T cells on high endothelial venules was significantly higher than that of Rap1 ^{f / f} T cells. However, Rap1 ^{− / −} T cells are mixed with Rap1 ^{f / f} T cells of the same number of cells and transplanted into mice, and 30 minutes later, they accumulate on high endothelial venules in mesenteric lymph nodes. I didn't.

[Experimental Example 17]
Rap1 ^{f / f} T cells and Rap1 ^{− / −} T cells were transformed into 5,6-carboxyfluorescein diacetate (CFSE, Invitrogen) and 5- (and-6)-(((4-chloromethyl) benzoyl) amino. ) Each was labeled with tetramethylrhodamine (CMTMR, Invitrogen), mixed with the same number of cells, and intravenously administered to normal mice. One hour later, lymphocytes in the blood and mesenteric lymph nodes of recipient mice were analyzed by flow cytometry. 18 (a) and 18 (b) are graphs showing experimental results. Representative results from 4 experiments are shown. The numbers above the squares indicate the ratio of Rap1 ^{− / −} T cells to Rap1 ^{f / f} T cells. In FIG. 18, “Blood” represents blood and “LN” represents a lymph node (the same applies hereinafter).

FIG. 18 (c) is a graph showing the ratio of Rap1 ^{− / −} T cells to Rap1 ^{f / f} T cells in mesenteric lymph nodes (n = 4 experiments). “*” In FIG. 18C indicates that there is a significant difference at a risk rate of less than 0.1% compared to Rap1 ^{f / f} T cells. As a result, it was revealed that Rap1 ^{− / −} T cells home to the mesenteric lymph nodes with an efficiency of less than 10% of Rap1 ^{f / f} T cells.

[Experiment 18]
The number of T cells in blood and mesenteric lymph nodes of 4-5 week old Rap1 ^{f / f} mice and Rap1 ^{− / −} mice was counted. FIGS. 19A and 19B are graphs showing the results (n = 10). Data represent mean ± standard error.

FIG. 19 (a) shows the result of blood, and FIG. 19 (b) shows the result of mesenteric lymph node. In FIG. 19 (b), “*” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the Rap1 ^{f / f} mouse. In FIG. 19, “mLN” represents a mesenteric lymph node (the same applies hereinafter).

As a result, at 4-5 weeks of age, the number of T cells in the mesenteric lymph nodes of Rap1 ^{− / −} mice decreased to about 10% of the number of T cells in the mesenteric lymph nodes of Rap1 ^{f / f} mice. It became clear to do. At the same time, it was revealed that the number of T cells in the blood of Rap1 ^{− / −} mice increased as compared with the number of T cells in the blood of Rap1 ^{f / f} mice.

[Experimental Example 19]
20 (a) and 20 (b) are graphs showing representative flow cytometry characteristics of lymphocytes derived from mesenteric lymph nodes of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. FIG. 20 (a) shows the results of Rap1 ^{f / f} mice, and FIG. 20 (b) shows the results of Rap1 ^{− / −} mice. The horizontal axis shows the expression intensity of CD62L in the cell population gated on CD3 positive cells, and the vertical axis shows the expression intensity of CD44 in the cell population gated on CD3 positive cells.

As a result, it was revealed that 90% or more of mesenteric lymph node-derived T cells of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice have a naive T cell phenotype.

[Experiment 20]
21 (a)-(d) show blood, mesenteric lymph nodes, Peyer's patch (PP), colonic mucosa lamina propria (Colon) of 9-week-old Rap1 ^{f / f} mice and Rap1 ^{− / −} mice, respectively. LP) is a graph showing the number of naive T cells and CD4 positive effector memory T cells (hereinafter sometimes referred to as “T _EM ” cells). Data represent mean ± standard error.

21 (b) to (c), “* 1” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the Rap1 ^{f / f} mouse. Further, “* 2” represents that there is a significant difference at a risk rate of less than 0.1% compared to the Rap1 ^{f / f} mouse. In addition, “* 3” indicates that there is a significant difference at a risk rate of less than 0.5% compared to the Rap1 ^{f / f} mouse. In addition, “* 4” indicates that there is a significant difference at a risk rate of less than 0.1% compared to Rap1 ^{f / f} mice.

[Experiment 21]
22 (a)-(c) show representative flow cytometric properties of lymphocytes in blood, mesenteric lymph nodes, lamina propria of the colon of Rap1 ^{f / f} and Rap1 ^{− / −} mice. It is a graph.

  FIG. 22 (a) shows the blood result, FIG. 22 (b) shows the result of the mesenteric lymph node, and FIG. 22 (c) shows the result of the colonic mucosa lamina propria. The horizontal axis shows the expression intensity of CD62L in the cell population gated on CD4 positive cells, and the vertical axis shows the expression intensity of CD44 in the cell population gated on CD4 positive cells.

In 8 weeks of age or older ages, Rap1 ^{- / -} CD62L in mesenteric lymph nodes and Peyer's patches of mice ^- the percentage of CD44 ⁺ T _EM cells, 62.5 ± 4.3% and 84.2% ± 6 Increased to 4%. In contrast, in Rap1 ^{f / f} mice, they were 5.8 ± 1.8% and 39.3 ± 3.2%, respectively.

The number of _{T EM} cells in blood and mesenteric lymph nodes, Rap1 ^{f / f} mice and Rap1 ^{- / -} did not differ significantly between the mouse. The number of _{T EM} cells in Peyer's patches, Rap1 compared to Rap1 ^{f / f} mice ^{- / -} was lower in mice. Nevertheless, the number of _{T EM} cells in colonic lamina propria, Rap1 compared to Rap1 ^{f / f} mice ^{- / -} was increased to more than four times in mice. This result is in contrast to the result that Rap1 ^{− / −} naive T cells failed to home to the mesenteric lymph nodes.

[Experimental example 22]
Frozen sections of the colons of Rap1 ^{f / f} and Rap1 ^{− / −} mice were stained with anti-CD4 antibody and anti-CD8 antibody and observed with a fluorescence microscope.

FIGS. 23A to 23C are fluorescence micrographs showing the results of staining with anti-CD4 antibody and anti-CD8 antibody. FIG. 23 (a) shows the results of Rap1 ^{f / f} mice (40 × magnification). FIG. 23 (b) shows the results of Rap1 ^{− / −} mice (40 × magnification). FIG.23 (c) is the fluorescence-microscope photograph which expanded the area | region c of FIG.23 (b) (magnification 200 times).

As a result, it was revealed that CD4-positive T cells, not CD8-positive T cells, infiltrated the colonic mucosa lamina propria of Rap1 ^{− / −} mice.

[Experimental example 23]
FIGS. 24 (a) and (b) show LFA-1 of naive T cells and effector memory (E / M) T cells derived from mesenteric lymph nodes of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. 24 (a)) and α ₄ β ₇ (FIG. 24 (b)) are representative results of flow cytometry analysis showing expression characteristics. As a result, high expression of alpha ₄ beta _7, the number of intestinal directivity of _{T EM} cells, Rap1 ^{f / f} mice and Rap1 ^{- / -} were exactly equal in mesenteric lymph nodes of mice.

These results are loss of Rap1 indicates to promote homing to the colon of T _EM cells.

<III. Rap1 conditional knockout mice spontaneously developed colitis with colon cancer at 12 weeks of age>
[Experimental example 24]
The inventors crossed a Rap1a ^{f / f} Rap1b ^{f / f} mouse (hereinafter sometimes referred to as “Rap1 ^{f / f} mouse”) with an Lck-Cre mouse or a CD4-Cre mouse to thereby obtain a T cell. Mice specifically lacking Rap1a and Rap1b (hereinafter sometimes referred to as “Rap1 ^{− / −} mouse”) were prepared.

As a result of analysis of Rap1 ^{− / −} mice, it became clear that at 9 weeks of age, a debilitating disease developed, and weight loss and diarrhea were observed.

FIG. 25 shows a Rap1 ^{− / −} mouse (hereinafter sometimes referred to as “− / −”) and a Rap1a ^{f / f} Rap1b ^{f / f} mouse (hereinafter sometimes referred to as “f / f”) as a control. .) Is a graph obtained by measuring a change in body weight (n = 10). Body weight was measured every day and expressed as a percentage based on the weight at the start of measurement. Data represent mean ± standard error.

In FIG. 25, “* 1” indicates that there is a significant difference at a risk rate of less than 2% compared to the corresponding Rap1 ^{f / f} mouse, and “* 2” indicates a significant difference at a risk rate of less than 1% “* 3” indicates that there is a significant difference at a risk rate of less than 0.1%.

Rap1 ^{− / −} mice were observed to develop intussusception and prolapse at 12 weeks of age. Histological analysis revealed that all Rap1 ^{− / −} mice developed severe colitis.

FIG. 26 is a photograph showing representative colon morphology of 8-12 week old Rap1 ^{f / f} and Rap1 ^{− / −} mice. “# 1” indicates intussusception in the colon of Rap1 ^{− / −} mice. “# 2” indicates an adenoma (cancer) that developed between the cecum and colon of Rap1 ^{− / −} mice. The bar in FIG. 26 represents 1 cm.

FIG. 27 is a graph showing the results of evaluating damage caused by colitis in Rap1 ^{− / −} mice based on observation with an optical microscope. The vertical axis represents the histological score. The histological score was evaluated based on the following criteria. Data represent mean ± standard error. In FIG. 27, “*” represents that there is a significant difference at a risk rate of less than 0.1% compared to the corresponding Rap1 ^{f / f} mouse.
Grade 0: No change is observed.
Grade 1: Minimal isolated scattered infiltration of inflammatory cells into the mucosa with or without minimal thickening of the epithelium.
Grade 2: Mild isolated to diffuse inflammatory cell infiltration, often extending to the submucosa, accompanied by erosion, mild to moderate epithelial thickening and mild to moderate mucin from goblet cells With a deletion of.
Grade 3: Often transmural, with moderate inflammatory cell infiltration, moderate to severe epithelial thickening and mucin loss.
Grade 4: often transmural, with significant inflammatory cell infiltration with crypt abscesses and sometimes ulcers, marked epithelial thickening and mucin loss.
Great 5: Prominent transmural inflammation with severe ulceration and loss of intestinal glands.

Subsequently, paraffin-embedded tissue sections of colons of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice were stained with hematoxylin and eosin, and the tissue structure was observed with an optical microscope. FIG. 28 (a) is a photomicrograph showing the cecal tissue structure of Rap1 ^{f / f} mice (magnification 40 times). FIG. 28 (b) is a photomicrograph showing the tissue structure of the colon of Rap1 ^{f / f} mice (magnification 40 times). FIG. 28 (c) is a photomicrograph showing the tissue structure of the inflammatory site of the large intestine of Rap1 ^{− / −} mice (magnification 40 times). FIG. 28D is a photomicrograph in which the region (d) in FIG. 28B is enlarged (magnification 200 times). FIG. 28 (e) is a photomicrograph in which the region (e) in FIG. 28 (b) is enlarged (magnification 200 times). The bar represents 200 μm. FIGS. 28 (d) and 28 (e) show that lymphocytes are infiltrating and intestinal glands disappear in the inflammatory site of the colon of Rap1 ^{− / −} mice. FIG. 28 (f) is a photomicrograph showing the tissue structure of adenoma that developed in Rap1 ^{− / −} mice. FIG. 28G is an enlarged micrograph of the region (g) in FIG. 28F (magnification 200 times). FIG. 28 (h) is an enlarged micrograph of the region (h) in FIG. 28 (f) (magnification 200 times).

FIG. 29 (a) is a photomicrograph showing a crypt abscess in the inflamed site of the large intestine of Rap1 ^{− / −} mice (magnification 40 times). The lower right region of FIG. 29A is an enlarged micrograph of the region surrounded by the square in FIG. 29A (magnification 200 times).

FIG. 29 (b) is a photomicrograph showing epithelioid granulomas in the large intestine of Rap1 ^{− / −} mice (40 × magnification). The lower right region of FIG. 29B is a micrograph obtained by enlarging the region surrounded by the square in FIG. 29B (magnification 200 times).

FIG. 29 (c) is a photomicrograph showing the tissue structure of a region where the intestinal glands of Rap1 ^{− / −} mice have disappeared (magnification 40 times). FIG. 29 (d) is an enlarged micrograph of the region (d) in FIG. 29 (c) (magnification 200 times). FIG. 29 (e) is a fluorescence micrograph showing the result of staining the same region as FIG. 29 (d) with anti-CD3 antibody and anti-B220 antibody (magnification 200 times). CD3 is a T cell marker and B220 is a B cell marker.

As shown in FIGS. 28-29, Rap1 ^{− / −} mice often showed crypt abscesses and epithelioid granulomas. These are characteristic of Crohn's disease. However, in contrast to large intestine damage-dependent inflammation observed in T cell-independent colitis induced by sodium dextran sulfate, Rap1 ^{− / −} mice have both severe ulcers and neutrophil infiltration. I was not able to admit. Furthermore, these symptoms in Rap1 ^{− / −} mice were always accompanied by the development of tubular adenomas associated with colitis with mild atypia.

[Experiment 25]
Subsequently, the cytokine secretion characteristics of CD4 ⁺ effector memory T (T _EM ) cells in the lamina propria of Rap1 ^{− / −} mice were examined (n = 5). Specifically, T cells in the lamina propria of Rap1 ^{− / −} mice were stimulated with phorbol-12-myristate-13-acetate (PMA) and ionomycin. Subsequently, using flow cytometry, based on the production of interferon (IFN) -γ, interleukin (IL) -17 and IL-4, T _H 1 cells and T _H 17 cells in CD4 positive cells, or The proportion of T _H 1 cells and T _H 2 cells was measured.

FIG. 30A is a graph showing the results of flow cytometry analysis. FIG. 30 (b) is a graph showing the ratio of IL-17, IFN-γ or IL-4 producing cells in CD4-positive T cells in the lamina propria of colon of Rap1 ^{− / −} mice. Data represent mean ± standard error.

As a result, in Rap1 ^{− / −} mice, pathogenic T _H 17 effector cells and T _H 1 effector cells formed in the mesenteric lymph nodes and Peyer's patches are homing to the colon and causing colitis. It became clear.

[Experiment 26]
Subsequently, the inventors used CD4 positive naive T cells derived from Rap1 ^{f / f} mice and Rap1 ^{− / −} mice (hereinafter sometimes referred to as “ _TN cells”) as anti-CD3 antibodies and anti-CD28 antibodies. Stimulated and examined the effect of Rap1 deficiency on proliferation and cytokine production.

FIG. 31 is a graph showing the amount of [ ³ H] -thymidine incorporation of CD4 positive naive T cells derived from Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. Primary CD4-positive naive T cells derived from lymph nodes and spleens of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice were unstimulated (indicated as “None” in FIG. 31), only with anti-CD3 antibody (in FIG. 31). , Indicated as “CD3”) or an anti-CD3 antibody and an anti-CD28 antibody (indicated as “CD3 / CD28” in FIG. 31), and the amount of ³ [H] -thymidine incorporation after 48 hours from the start of the stimulation. Measured (n = 3). Data represent the mean ± standard error of the results of three independent experiments. In FIG. 31, “* 1” indicates that there is a significant difference at a risk rate of less than 1% compared to the corresponding Rap1 ^{f / f} mouse-derived T cells, and “* 2” indicates a risk rate of less than 2%. Represents a significant difference.

  As a result, T cells deficient in Rap1 showed suppressed proliferation against cross-linking of the T cell receptor complex in the presence and absence of anti-CD28 antibody compared to control T cells.

[Experiment 27]
FIG. 32 is a graph showing the results of measuring cytokine production levels of CD4-positive naive T cells derived from Rap1 ^{f / f} mice and Rap1 ^{− / −} mice. Primary CD4-positive naive T cells derived from lymph nodes and spleens of Rap1 ^{f / f} mice and Rap1 ^{− / −} mice were either unstimulated (indicated as “−” in FIG. 32) or anti-CD3 antibody and anti-CD28 antibody ( In FIG. 32, it was stimulated with “+”. Three days after the start of stimulation, the amount of cytokine in the culture supernatant of each cell was measured (n = 3). Data represent the mean ± standard error of the results of three independent experiments. In FIG. 32, “* 1” indicates that there is a significant difference at a risk rate of less than 1% compared to the corresponding Rap1 ^{f / f} mouse-derived T cells, and “* 2” is a risk rate of less than 5%. Represents a significant difference.

In Rap1 ^{f / f} T cells and Rap1 ^{− / −} T cells, no difference was found in the production amounts of IFN-γ, IL-10, TNF and IL-6. On the other hand, the production of IL-2, IL-4, IL-17 by Rap1 ^{− / −} T cells was decreased or slightly increased compared to Rap1 ^{f / f} T cells.

[Experiment 28]
FIG. 33 shows the percentage of Foxp3 positive cells based on total CD4 positive T cells in mesenteric lymph nodes, Peyer's patch and colonic lamina propria of 9 week old Rap1 ^{f / f} and Rap1 ^{− / −} mice. (N = 10). Data represent mean ± standard error.

[Experimental example 29]
FIG. 34 is a graph showing representative results of flow cytometric analysis of cells in the lamina propria of colon of Rap1 ^{f / f} and Rap1 ^{− / −} mice. The results in FIG. 34 show that lymphocytes gated on CD4 positive cells express Foxp3.

[Experiment 30]
FIG. 35 is a graph showing the results of a T _reg inhibition assay. The specific experimental procedure is as described above. Control CD4-positive naive T cells (indicated as “T _con ” in FIG. 35) were mixed with Rap1 ^{f / f} T _reg cells or Rap1 ^{− / −} T _reg cells at the ratios shown in FIG. Stimulation was carried out with anti-CD28 antibody for 48 hours, and the growth inhibitory effect of naive T cells by T _reg cells was examined.

From the above results, the ratio of Foxp3 positive cells and the suppressive action in mesenteric lymph nodes, Peyer's patch or colonic mucosa lamina propria of Rap1 ^{− / −} mice decreased compared to similar cells derived from Rap1 ^{f / f} mice. It became clear that they did not.

The above results indicate that the increase in T _H 17 effector cells or T _H 1 effector cells in the colon of Rap1 ^{− / −} mice is not dependent on the increased proliferation or cytokine production of Rap1 deficient T cells.

<IV. Loss of Rap1 was to promote the rolling of _{T EM} cell>
[Experimental example 31]
In order to study the Rap1-deficient _{T EM} cell homing activity, Rap1 ^{f / f} mice and Rap1 ^{- / -} CD4 + naive T cells from mice _were cultured under inducing conditions _T H 17. FIG. 36 and FIG. 37 shows CD44, CD62L, α ₄ β ₇ , CXCR3, PSGL when CD4 positive naive T cells derived from Rap1 ^{f / f} mice and Rap1 ^{− / −} mice were cultured under T _H 17 induction conditions. It is a graph which shows the typical result which shows the expression characteristic of -1 and LFA-1.

As a result, lack of Rap1 did not significantly affect differentiation into T _H 17 cells and T _H 1 cells. In addition, the lack of Rap1 did not significantly affect the expression of α ₄ β ₇ , PSGL-1, LFA-1, and CXCR3.

[Experiment 32]
^{Subsequently,} the ^Rap1 _f / f _{T EM} cells were incubated 37 ° C. 15 seconds crushed in the presence or absence of 100 nM CXCL10. Cell debris was subjected to pull-down assay using RalGDS-RBD fusion protein and analyzed by immunoblotting using anti-Rap1 antibody.

FIG. 38 is a photograph showing representative results in three independent experiments. As a ^{result, Rap1} _f / f _{T EM} cells revealed that activated by stimulation with CXCL10.

[Experimental Example 33]
FIG. 39 shows Rap1 ^{f / f} CD4 positive naive T cells, Rap1 ^{− / −} CD4 positive naive T cells, Rap1 ^{f / f} CD4 positive effector T cells measured by a detachment assay in the presence or absence of chemokines. It is a graph which shows the result of having measured LFA-1-ICAM-1 binding or (alpha) _{4 (} beta) _7- MadCAM-1 coupling | bonding of a Rap1 ^-/- CD4 positive effector cell after putting it into a resting state.

Each cell was incubated on ICAM-1 or MadCAM-1 for 10 minutes and then placed under laminar flow ( ² dyn / cm ² ) to count strongly adherent cells that were resistant to laminar flow. Data represent mean ± standard error in triplicate experiments (n = 50 per group). In FIG. 39, “* 1” indicates that there is a significant difference at a risk rate of less than 1% compared to the naive T cells of the control. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 2% compared to the control _TEM cells.

As a result, Rap1 ^{- / -} naive T cells or ^{Rap1 - /} - of _{T EM} cells of LFA-1 or alpha ₄ beta _7, binding activity to ICAM-1 or MadCAM-1 is significantly lower compared to control cells It was. On the other _hand, basic _{coupling T EM} cells bind to ICAM-1 or MadCAM-1 was increased. This was considered to be due to increased expression of LFA-1 or alpha ₄ beta _7.

[Experimental example 34]
We, under laminar flow, deficiency of Rap1 was studied the effect on the adhesion cascade of T _EM cells to endothelial cells expressing P- selectin and LFA-1.

T _H 17 induced ^Rap1 _f / f _{T EM} cells or differentiated under ^{Rap1 - /} - and _{T EM} cells, on LS12 cells expressing P- selectin and mouse ICAM-1, the presence of 100 nM CXCL10 or In the absence, it was perfused under laminar flow of ² dyn / cm ² .

A digital image showing the interaction between _TEM cells and endothelial cells was taken at 30 frames / second under laminar flow. Adhesion events of 100 or more cells were measured and classified into rolling, temporary adhesion, and stable adhesion as shown in FIG. 40 (a). In FIG. 40 (a), the data represents the mean value ± standard error in three independent experiments. In FIG. 40 (a), "* 1", as compared to the control of ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference hazard rate of less than 2%. Further, "* 2", compared to control ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference risk rate of less than 5%.

Figure 40 (b) is, in the absence of ^{CXCL10, Rap1} _f / f _{T EM} cells or ^{Rap1 - /} - is a graph showing the speed of rolling on endothelial cells of _{T EM} cells. In Figure 40 (b), "*", as compared to the control of ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference risk rate of less than 5%.

As a result, ^{Rap1 - / -} _{T EM} ^cells, compared to ^Rap1 _f / f _{T EM} cells in the presence and absence of CXCL10, significantly showed rolling and adhesion more frequent. Moreover, ^{Rap1 - / -} _{T EM} cells speed rolling ^was significantly slower compared to the rate of ^Rap1 _f / f _{T EM} cell rolling.

These results, ^{Rap1 - /} - in _{T EM} cells, indicating that PSGL-1 / P- selectin-dependent rolling is increased.

[Experimental Example 35]
Subsequently, the inventors under laminar flow, deficiency of Rap1 was studied the effect on the adhesion cascade of _{T EM} cells to endothelial cells expressing MadCAM-1.

T _H 17 induced ^Rap1 _f / f _{T EM} cells or differentiated under ^{Rap1 - /} - and _{T EM} cells, on LS12 cells expressing MadCAM-1, the presence or absence of 100nM CXCL10, 2dyn Perfusion under a laminar flow of / cm ² .

A digital image showing the interaction between _TEM cells and endothelial cells was taken at 30 frames / second under laminar flow. Adhesion events of 100 or more cells were measured and classified into rolling, temporary adhesion, and stable adhesion as shown in FIG. In FIG. 41 (a), the data represents the mean value ± standard error in three independent experiments. In FIG. 41 (a), "* 1", as compared to the control of ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference hazard rate of less than 0.1%. Further, "* 2", compared to control ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference hazard rate of less than 2%.

Figure 41 (b) is, in the absence of ^{CXCL10, Rap1} _f / f _{T EM} cells or ^{Rap1 - /} - is a graph showing the speed of rolling on endothelial cells of _{T EM} cells. In Figure 41 (b), "*", as compared to the control of ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference hazard rate of less than 0.3%.

As a result, ^{Rap1 - / -} _{T EM} ^cells, compared to ^Rap1 _f / f _{T EM} cells, significantly showed rolling and adhesion more frequent. Moreover, ^{Rap1 - / -} _{T EM} cells speed rolling ^was significantly slower compared to the rate of ^Rap1 _f / f _{T EM} cell rolling.

[Experimental Example 36]
We also in laminar flow, ^{Rap1 - /} - of _{T EM} cells were examined rolling and adhesion to purified MadCAM-1 above.

^{_{T H 17 Rap1 f / f T}} EM cells were differentiated under inducing conditions or ^{Rap1 - /} - _T the _EM cells, MadCAM-1 which was purified by immobilized plates, the presence or absence of 100 nM CXCL10, Perfusion was performed under a laminar flow of ² dyn / cm ² .

Rap1 ^{- /} - The _{T EM} cells, previously, were introduced with lentiviral vectors expressing allow holding the Rap1N17 a dominant negative mutant of Rap1. As a control, cells into which an empty lentiviral vector was introduced were used.

In laminar _flow, and take digital images showing the interaction of _{T EM} cells and MadCAM-1 at 30 frames / sec. Adhesion events of 100 or more cells were measured and classified into rolling, temporary adhesion, and stable adhesion as shown in FIG. In FIG. 42 (a), the data represents the mean value ± standard error in three independent experiments. 42 in (a), "* 1", as compared to ^Rap1 _f / f _{T EM} cells in control unstimulated, indicating that there is a significant difference hazard rate of less than 2%. Further, "* 2", as compared to a corresponding ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference risk rate of less than 1%.

Figure 42 (b) is, in the absence of ^{CXCL10, Rap1} _f / f _{T EM} cells or ^{Rap1 - /} - is a graph showing the speed of rolling on endothelial cells of _{T EM} cells. Figure 42 in (b), "*", as compared to the control of ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference risk rate of less than 0.5%.

As a result, ^{Rap1 - / -} _{T EM} cells in the presence or absence of a ^CXCL10, as compared to ^Rap1 _f / f _{T EM} cells, significantly showed rolling and adhesion more frequent. Further, ^Rap1 on MadCAM ^{- / -} _{T EM} cells speed rolling ^was significantly slower compared to the rate of ^Rap1 _f / f _{T EM} cell rolling. Moreover, ^{Rap1 - /} - in _{T EM} cells, expression of Rap1N17 a dominant negative mutant of Rap1 significantly inhibited the frequency of adhesion events onto MadCAM.

[Experiment 37]
Figure 43 (a) and (b), 1 [mu] M 5,6-carboxyfluorescein diacetate (CFSE, Invitrogen) ^Rap1 _f / f _{T EM} cells labeled with (FIG. 43 (a)) and ^{Rap1 - / -} _{T EM} It is a figure which shows the result of having observed the mode of rolling on MadCAM of a cell (FIG.43 (b)) using the objective lens of 63 times magnification under laminar flow. Figure 43 (a) and (b) ^Rap1 in time shown in f / _{f T EM} cells and ^{Rap1 - /} - shows a representative image of _{T EM} cells.

^/ - ^{- Rap1} slow rolling is less than 100 [mu] m / sec _{T EM} cells showed elongation of some of the tether-like film. This is similar to L-selectin dependent rolling. ^{However, Rap1} _f / f _{T EM} cells did not show a clear projection on MadCAM.

[Experiment 38]
Subsequently, ^{Rap1 - /} - in order to compare _{T EM} cells and ^Rap1 _f / f _{T EM} ability of homing of cells to the colon were injected into normal mice and labeling the cells separately. Specifically, ^{Rap1 - / -} _{T EM} cells were labeled with 1 [mu] M 5,6-carboxyfluorescein diacetate (CFSE, Invitrogen). ^{Moreover, Rap1} _f / f _{T EM} cells 10 [mu] M 5-(and -6) - were labeled with (((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (CMTMR, Invitrogen). ⁶ × ⁶ cells were mixed and injected intravenously into C57BL / 6 mice. Ten hours later, cells derived from the lamina propria of the colon were analyzed by flow cytometry.

44 (a) and 44 (b) are graphs showing the results of flow cytometry analysis. Figure 44 (a) is, ^{Rap1 - /} - shows the percentage of _{T EM} cells and ^Rap1 _f / f _{T EM} cells are representative results of three experiments. The numbers on the square in FIG. 44 (b) in, ^{Rap1 - /} - shows the percentage of _{T EM} cells and ^Rap1 _f / f _{T EM} cells.

Consistent with enhanced rolling and adhesion to MadCAM-1 above was homed to colon ^{Rap1 - / -} _{T EM} number of cells ^was about 4-fold higher number of ^Rap1 _f / f _{T EM} cells.

These results indicate that loss of Rap1 promotes the homing of the large intestine of pathogenic T _EM cells. As a result, spontaneous colitis is considered to be induced.

<V. Rap1 deficiency reduced phosphorylation of Ezrin and Moesin proteins>
[Experimental Example 39]
The inventors examined whether Ezrin / Radixin / Moesin (ERM) protein is controlled by Rap-1 GDP. As ERM protein, Ezrin protein and Moesin protein were examined.

  Cells in which Rap1 of BAF cells, which is a pro-B cell line, was knocked down (Rap1KD) were stimulated with 100 nM CXCL12 for the time shown in FIG. As a control, normal BAF cells were used. The cell lysate was immunoreacted using an antibody against Ezrin protein phosphorylated at the 567th threonine residue, an antibody against Moesin protein phosphorylated at the 558th threonine residue, an anti-Ezrin antibody and an anti-Moesin antibody. Detected by blotting. FIG. 45 is a photograph showing the results of immunoblotting. Representative results from three independent experiments are shown.

  As shown in FIG. 45, lymphocytes expressed Ezrin protein and Moesin protein. In control BAF cells, Ezrin and Moesin proteins were highly phosphorylated. However, when stimulated with CXCL12, phosphorylation levels decreased to less than half.

  In contrast, the amount of basal phosphorylation of ERM protein in unstimulated Rap1KD cells was 1/3 that of control cells. This result indicates that Rap1-GDP is essential for maximal phosphorylation of ERM protein in unstimulated cells.

[Experimental Example 40]
Subsequently, the inventors introduced sense iRNA specific for Rap1a and Rap1b into BAF cells using a GFP-expressing lentiviral vector to obtain Rap1KD cells. As control cells, cells in which scrambled control iRNA was introduced into BAF cells using a lentiviral vector expressing GFP were used.

  Rap1KD cells and control cells were incubated at 37 ° C. for 10 minutes in the presence and absence of 100 nM CXCL12. Subsequently, each cell was stained with an antibody against phosphorylated Ezrin protein and an antibody against phosphorylated Moesin protein.

  Subsequently, each cell was observed with a fluorescence microscope, and the proportion of cells having 5 times or more phosphorylated Ezrin protein or phosphorylated Moesin protein in the uropod was measured in comparison with the side portion and rear portion of the cell. FIG. 46 is a graph showing the measurement results.

  Data represent the mean ± standard error of the results of three independent experiments (n = 40 for each group). In FIG. 46, “*” represents that there is a significant difference at a risk rate of less than 0.1% compared to the control cells.

  It is known that uropod formation induced by CXCL12 depends on phosphorylation of ERM protein. As shown in FIG. 46, it was revealed that uropod formation induced by CXCL12 is also reduced in Rap1KD cells.

[Experimental example 41]
47 (a) and (b) are fluorescence micrographs (“pERM”) of control cells (FIG. 47 (a)) and Rap1KD cells (FIG. 47 (b)), antibodies against phosphorylated Ezrin protein and phosphorylated Moesin. The result of dyeing an antibody against a protein is shown, and “GFP” shows the detection result of fluorescence of GFP.) And differential interference micrograph (DIC). Representative results are shown. The scale bar is 5 μm.

[Experimental Example 42]
Subsequently, the inventors forcibly expressed Spa-1, which is a GTPase activator of Rap1, and examined its influence. First, an expression vector for Spa-1 was introduced into BAF cells. As a control, cells in which only the vector was introduced into BAF cells were used. Subsequently, these cell disruptions were subjected to immunoblotting and detected with an anti-Spa-1 antibody.

  FIG. 48 is a photograph showing the results of immunoblotting. As a result, it was confirmed that Spa-1 was forcibly expressed.

[Experimental Example 43]
Subsequently, control cells and cells in which Spa-1 was forcibly expressed were incubated and disrupted at 37 ° C. in the presence of 100 nM CXCL12 for the time shown in FIG. Subsequently, the cell disruption was subjected to a pull-down assay using RalGDS-RBD fusion protein and analyzed by immunoblotting using an anti-Rap1 antibody.

  As a result, it was shown that the expression of Spa-1 inhibits the activation of Rap1 by CXCL2 stimulation. This inhibition reached a maximum 15 seconds after CXCL2 stimulation.

[Experimental Example 44]
Subsequently, control cells and cells in which Spa-1 was forcedly expressed were incubated and disrupted in the presence of 100 nM CXCL12 at 37 ° C. for the time shown in FIG. Subsequently, the cell debris was subjected to immunoblotting using an antibody against phosphorylated Ezrin protein, an antibody against phosphorylated Moesin protein, an anti-Ezrin antibody and an anti-Moesin antibody.

  FIG. 50 is a photograph showing the results of immunoblotting. Representative results from three independent experiments are shown. As a result, phosphorylation of ERM protein was about 1.6 times higher than that in control cells at all time points after stimulation with CXCL2.

[Experimental Example 45]
In naive T cells, ERM protein exists in a phosphorylated activated state, and it is known that phosphorylation of ERM protein disappears in 15 seconds when stimulated with CCL21.

Therefore, the inventors examined phosphorylation of Ezrin protein and Moesin protein in Rap-1 ^{f / f} naive T cells and Rap-1 ^{− / −} naive T cells. First, naive T cells were stimulated with 100 nM CCL21 for the time shown in FIG. Subsequently, each cell disruption was subjected to immunoblotting using an antibody against phosphorylated Ezrin protein, an antibody against phosphorylated Moesin protein, an anti-Ezrin antibody and an anti-Moesin antibody.

  FIG. 51 is a photograph showing the results of immunoblotting. Representative results from three independent experiments are shown. Actin protein was detected as a loading control.

As a result, it is clear that the basal level of ERM protein phosphorylation in Rap-1 ^{− / −} naive T cells is reduced to about half (52%) that of Rap-1 ^{f / f} naive T cells. became.

[Experimental example 46]
T _H 17 induced ^Rap1 _f / f _{T EM} cells or cultured under conditions ^{Rap1 - /} - and _{T EM} cells were stimulated for 15 seconds at 100 nM CXCL10. The disrupted cells were subjected to immunoblotting using an antibody against phosphorylated Ezrin protein, an antibody against phosphorylated Moesin protein, an anti-Ezrin antibody and an anti-Moesin antibody.
FIG. 52 is a photograph showing the results of immunoblotting.

As a result, ERM proteins in T _EM cells is highly phosphorylated, it revealed that phosphorylation level rapidly decreased by CXCL10 stimulation. In the absence of CXCL10 stimulation, ^{Rap1 - /} - phosphorylation level of ERM proteins _{T EM} cells ^was less than 1/3 that of ^Rap1 _f / f _{T EM} cells.

[Experimental example 47]
It is known that Rho-related kinase (Rock) phosphorylates ERM protein. Therefore, the inventors examined whether the activity of Rho protein is affected by the lack of Rap1.

  Specifically, Rho protein bound to GTP was recovered by pull-down assay using a fusion protein (GST-Rhotekin RBD) of Rho binding region (RBD) of Rhotekin protein and GST protein.

  Control cells and Rap1KD cells were stimulated with 100 nM CXCL12 for the time shown in FIG. 53 and then disrupted, and a pull-down assay using GST-Rhotekin RBD protein was performed. Subsequently, Rho protein and total Rho protein bound to GTP were detected by immunoblotting using an anti-Rho antibody. FIG. 53 is a photograph showing the results of immunoblotting. Representative results of 4 independent experiments.

  As shown in FIG. 53, in control cells, Rho protein was activated 3 minutes after CXCL12 stimulation. In Rap1KD cells, no reduction in Rho protein activation was observed.

  This result indicates that Rho protein activity is not involved in the reduction of ERM protein phosphorylation in Rap1KD cells.

[Experimental Example 48]
It is known that LOK protein phosphorylates lymphocyte ERM protein. Therefore, the inventors examined whether the activity of the LOK protein is affected by the lack of Rap1.

  LOS protein (FLAG-LOK) conjugated with a FLAG tag was expressed in COS cells, an African green monkey kidney cell line. At the same time, Rap1V12 or Rap1N17 bound with a T7 tag was also expressed. Rap1N17 is a dominant negative mutant of Rap1.

  Subsequently, cell debris was immunoprecipitated with Halo-linked resin and immunoblotted with anti-T7 antibody.

  FIG. 54 is a photograph showing the results of immunoblotting. Representative results from four independent experiments are shown. As a result, it was revealed that Rap1-GDP, not Rap1-GTP, binds to the LOK protein.

[Example 49]
Subsequently, the kinase activity of the LOK protein in Rap1N17-expressing COS cells or Rap1V12-expressing COS cells was measured. FIG. 55 is a photograph showing the results of measuring kinase activity. Representative results from three experiments are shown.

  As a result, it became clear that the binding between LOK protein and Rap1-GDP increased the kinase activity of LOK protein.

[Experimental Example 50]
Subsequently, FLAG-LOK was expressed in control cells and Rap1KD cells, and stimulated with 100 nM CXCL12 for the time shown in FIG. Subsequently, the kinase activity of the LOK protein was measured. FIG. 56 is a photograph showing the results of measuring kinase activity. Representative results from three experiments are shown.

  As a result, it was revealed that the kinase activity of the LOK protein in Rap1KD cells was reduced to 18 ± 2.6% of the control cells. CXCL12 stimulation rapidly reduced the kinase activity of the LOK protein by 1/3. This result was consistent with the kinetics of decreased ERM protein phosphorylation described above.

[Example 51]
^{Subsequently, Rap1} _f / f _{T EM} cells or ^{Rap1 - /} - and _{T EM} cells were time stimulus shown in Figure 57 at 100 nM CXCL10. Subsequently, the kinase activity of the LOK protein in each cell was measured. FIG. 57 is a photograph showing the results of measuring kinase activity. Representative results from three experiments are shown.

As a result, ^{Rap1 - / -} _{T EM} kinase activity of LOK proteins in cells, revealed that was reduced to 32% of control cells in the absence of CXCL10 stimulation.

These results show that Rap1-GDP is required for activation of LOK protein, it plays an important role in the phosphorylation of ERM proteins of naive T cells and _{T EM} cells.

<VI. Expression of Ezrin protein mimicking phosphorylation state reduced rolling in Rap1-deficient T cells>
[Experimental Example 52]
It was examined whether reduced phosphorylation of Ezrin protein and Moesin protein in Rap1KD cells promotes L-selectin-dependent rolling. Specifically, a mutant (T567E) that mimics the phosphorylation state of Ezrin protein was introduced into control cells and Rap1KD cells.

  First, a sense iRNA specific to Rap1a and Rap1b was introduced into a BAF cell using a lentiviral vector expressing GFP to obtain a Rap1KD cell. As control cells, cells in which scrambled control iRNA was introduced into BAF cells using a lentiviral vector expressing GFP were used.

  A lentiviral vector expressing an Ezrin mutant (T567E) linked with a FLAG tag was introduced into these cells. As a control, cells into which only the vector was introduced were used. Subsequently, these cell disruptions were subjected to immunoblotting and detected with an anti-FLAG antibody. Actin protein was detected as a loading control.

  FIG. 58 is a photograph showing the results of immunoblotting. Representative results of 4 independent experiments are shown. As a result, it was confirmed that the Ezrin mutant (T567E) could be forcibly expressed.

  Subsequently, rolling of these cells on Nepmucin (CD300LG) was measured in the same manner as described above. FIG. 59 is a graph showing the results of measuring rolling activity. Data represent mean ± standard error in 3 independent experiments. In FIG. 59, “*” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the control Rap1KD cells.

  Subsequently, the rolling of the above cells on LS12 cells was measured in the same manner as described above. FIG. 60 (a) is a graph showing the results of measuring the rolling activity. Data represent mean ± standard error in 3 independent experiments. In FIG. 60 (a), “* 1” indicates that there is a significant difference at a risk rate of less than 0.5% compared to control cells into which scrambled control iRNA was introduced. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 0.1% compared to control cells into which scrambled control iRNA was introduced.

  In addition, “* 3” indicates that there is a significant difference at a risk rate of less than 0.2% compared to the control Rap1KD cells. In addition, “* 4” indicates that there is a significant difference at a risk rate of less than 0.5% compared to the control Rap1KD cells.

  FIG. 60 (b) is a graph showing the rolling speed of each cell. Data represent mean ± standard error in triplicate experiments (n = 150 for each group). In FIG. 60 (b), “*” represents that there is a significant difference at a risk rate of less than 1% compared to cells in which only the vector was introduced into Rap1KD cells.

  As a result, as shown in FIG. 60 (a), by expressing the Ezrin mutant (T567E), the frequency of rolling of Rap1KD cells on Nepmucin or on endothelial cells in the absence of chemokine was increased. Compared with significantly decreased.

  Moreover, the expression of the Ezrin mutant (T567E) in the control cells decreased the frequency of rolling on the endothelial cells in the presence and absence of chemokines, similar to the expression of the Ezrin mutant (T567E) in Rap1KD cells. .

  In addition, as shown in FIG. 60 (b), the rolling speed of Rap1KD cells was significantly increased by expressing the Ezrin mutant (T567E).

[Experimental Example 53]
By flow cytometric analysis, cytokine secretion characteristics of T cells derived from mesenteric lymph nodes of Rap1 ^{− / −} mice that spontaneously developed colitis were measured (n = 5). CD4-positive _TEM cells derived from lymph nodes of mice that developed colitis were cultured in the presence of anti-CD3 antibody and anti-CD28 antibody for 3 days. Subsequently, stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin was performed to analyze cytokine secretion characteristics. 61 (a) and 61 (b) are graphs showing the results of flow cytometry. FIG. 61 (c) is a graph showing the percentage of IL-17, IFN-γ and IL-4 producing cells as a percentage. Data represent mean ± standard error.

[Experimental Example 54]
Subsequently, ^{Rap1 - / -} _{T EM} cells introduced with lentiviral vectors expressing Ezrin mutant _(T567E), were cultured under inducing conditions _T H 17. As a control, cells into which only the vector was introduced were used. Subsequently, these cell disruptions were subjected to immunoblotting and detected with an anti-FLAG antibody.

  FIG. 62 is a photograph showing a result of immunoblotting. Representative results from three independent experiments are shown. Actin protein was detected as a loading control. As a result, it was confirmed that the Ezrin mutant (T567E) could be forcibly expressed.

[Experimental Example 55]
Subsequently, Ezrin mutant (T567E) was forcibly expressed ^Rap1 _f / f _{T EM} cells and ^{Rap1 - /} - in the form of _{T EM} cells were examined by confocal microscopy. FIG. 63 (a) shows a typical confocal micrograph.

FIG. 63 (b) is a graph showing the percentage of cells that formed blebs at least once per minute (n = 50). In Figure 63, "* 1", as compared to the control of ^Rap1 _f / f _{T EM} cells, indicating that there is a significant difference hazard rate of less than 0.1%. Further, "* 2", the control of Rap1 ^- / - T _EM cells as compared to only was introduced cell vector, indicating that there is a significant difference hazard rate of less than 0.2%.

[Experimental Example 56]
Figure 64 is a control cell and Ezrin mutant (T567E) CD4-positive expressed ^{Rap1 - /} - in _{T EM} cells is a graph showing the expression profile of CXCR3 and α ₄ β _7.

As a result, ^Rap1 expressed control cells and Ezrin mutant (T567E) ^{- / -} _{T EM} cells showed expression of comparable CXCR3 and α ₄ β _7. On the other hand, ^Rap1 was expressed Ezrin mutant (T567E) ^{- / -} _{T EM} cells showed significantly reduced blebbing compared to control cells.

[Experimental Example 57]
Vector only or Ezrin mutant (T567E) CD4-positive expressed ^{Rap1 - / -} _{T EM cells, T} H 17 inducing conditions, the MadCAM-1 which was purified by immobilized plates, with or without the 100 nM CXCL10 In the presence, it was perfused under laminar flow of ² dyn / cm ² .

In laminar _flow, and take digital images showing the interaction of _{T EM} cells and MadCAM-1 at 30 frames / sec. Adhesion events of 100 or more cells were measured and classified into rolling, temporary adhesion, and stable adhesion as shown in FIG. 65 (a).

In FIG. 65 (a), the data represents the mean value ± standard error in three independent experiments. In FIG. 65 (a), “* 1” indicates that there is a significant difference at a risk rate of less than 2% compared to the control _TEM cells. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 0.2% compared to the corresponding _TEM cells.

Figure 65 (b) is, in the absence of CXCL10, CD4-positive ^{Rap1 - /} - is a graph showing the speed of rolling on MadCAM-1 of _{T EM} cells. In FIG. 65 (b), “*” indicates that there is a significant difference at a risk rate of less than 0.5% compared to the control _TEM cells.

As shown in FIG. 65 (a) and (b), the expression of Ezrin mutant (T567E), in the presence or absence of CXCL10, ^{Rap1 - /} - rolling and adhesion of _{T EM} cells significantly suppressed Increased rolling speed.

[Example 58]
Subsequently, we, Ezrin mutant (T567E) CD4-positive expressed ^{Rap1 - /} - was investigated homing to _{T EM} cells of the colon. As control cells, CD4-positive was introduced only vector ^{Rap1 - /} - were used _{T EM} cells.

5 × 10 ⁶ cells each stained with CFSE or CMTMR were mixed and injected intravenously into C57BL / 6 mice. Ten hours later, cells derived from the lamina propria of the colon were analyzed by flow cytometry.

FIG. 66 (a) is a graph showing the results of flow cytometry. The numbers on the square in FIG. 66 (a) in control cells and Ezrin mutant (T567E) expressed ^{Rap1 - /} - shows the percentage of _{T EM} cells. Figure 66 (b) is in the colon lamina propria, ^Rap1 was expressed Ezrin mutant (T567E) ^{- / -} is a graph showing the ratio of control cells of _{T EM} cells (n = 3). In FIG. 66 (b), “*” represents that there is a significant difference at a risk rate of less than 0.1% compared to the control cells.

As a result, ^Rap1 was expressed Ezrin mutant (T567E) ^{- / -} of _{T EM} cells, the ability to home to the colon was reduced to 36.5 ± 5.3% of control cells. ^/ - ^{- Rap1} expressed Ezrin mutant (T567E) _{T EM} cells and control cells, in the blood were the same number exists.

[Experiment 59]
Subsequently, the inventors transplanted CD4-positive Rap1 ^{− / −} pathogenic T cells into normal irradiated mice, and whether or not the expression of Ezrin mutant (T567E) suppresses the development of colitis. investigated.

Figure 67 is a mouse that is not transplanted cells, CD4-positive was introduced only vector ^{Rap1 - / -} _{T EM} cells transplanted mice (control), and Ezrin mutant CD4 + was introduced (T567E) Rap1 ^{- / -} It is the graph which measured the body weight of the mouse _| mouth which transplanted the _TEM cell every day, and showed it with the percentage on the basis of the body weight at the time of a measurement start (n = 3 about each group). Data represent mean ± standard error. In FIG. 67, “*” represents that there is a significant difference at a risk rate of less than 2% compared to control cells.

[Experimental Example 60]
FIG. 68 is a graph showing the results of evaluating the damage caused by colitis in each mouse based on observation with an optical microscope. The vertical axis represents the histological score. The histological score was evaluated based on the criteria described above. Data represent mean ± standard error. In Figure 68, "*" is, CD4-positive was introduced only vector ^{Rap1 - / -} _{T EM} cells compared to mice transplanted with the, indicating that there is a significant difference hazard rate of less than 0.1%.

  FIG. 69 is a photomicrograph showing a typical tissue structure of the large intestine in a mouse transplanted with cells. A paraffin-embedded tissue section of the colon of each mouse was stained with hematoxylin and eosin, and a representative histological structure observed with a light microscope is shown (magnification 40 times). The scale bar indicates 200 μm.

As a result, CD4-positive ^{Rap1 - /} - mice transplanted with _{T EM} cells in 3 weeks, developed debilitating disease characterized weight loss, diarrhea. However, CD4-positive ^Rap1 was introduced Ezrin mutant (T567E) ^{- / -} mice transplanted with _{T EM} cells, at least 3 weeks after implantation, showed no symptoms as described above.

[Experimental Example 61]
Figure 70, CD4-positive ^{Rap1 - / -} _{T EM} cells mice were implanted and Ezrin mutant CD4 + was introduced (T567E) ^{Rap1 - / -} and _{T EM} cells frozen sections of colon mice transplanted with anti-CD4 antibody and It is a typical fluorescence micrograph showing the result of staining with an anti-CD8 antibody.

As a result, CD4-positive was introduced Ezrin mutant (T567E) ^{Rap1 - / -} in the colon of mice transplanted with _{T EM} cells, CD4-positive ^{Rap1 - / -} _{T EM} cells compared to colon transplanted mice, CD4 It was revealed that infiltration of positive _TEM cells was reduced.

The above results indicate that Rap1-GDP suppresses L-selectin, P-selectin or α ₄ β _7- dependent rolling via phosphorylation of ERM protein and prevents migration of CD4-positive pathogenic T cells to the colon. By suppressing, it shows that the onset of colitis is inhibited.

<VII. Filamine inhibited LFA-1 activation>
[Experimental Example 62]
The intracellular localization of Rap1-GTP after chemokine stimulation was examined. In the following experiments, Ral guanine nucleotide dissociation inhibitor (GDS) -RBD (Ras binding domain) -mCherry (RalGDS-RBD) was used as a reporter.

  Control cells expressing a fusion protein of RalGDS-RBD and mCherry were stimulated with 100 nM CXCL12 for the time shown in FIG. FIG. 71 is a photograph showing the results of observing the above cells with a confocal microscope. The scale bar represents 5 μm.

  FIG. 72 is a confocal micrograph showing how RalGDS-RBD-mCherry is localized in the cell membrane 3 seconds after stimulation with CXCL12. A fusion protein of RalGDS-RBD and mCherry was expressed in wild-type cells, and changes in its localization by CXCL12 stimulation were examined. FIG. 72 shows representative results of 5 experiments. The scale bar indicates 5 μm. The graph below each photograph in FIG. 72 shows a line profile of the fluorescence intensity of RalGDS-RBD-mCherry along the XY line in each photograph.

  As a result, before CXCL12 stimulation, RalGDS-RBD-mCherry protein was diffused throughout the cells. And it accumulated on the cell membrane 5 seconds after CXCL12 stimulation.

  As shown in FIG. 71, blebs appeared several seconds after Rap1 activation, followed by membrane folds. FIG. 73 is a confocal microscope image showing a pseudo color image of the fluorescence intensity of RalGDS-RBD-mCherry. 70 seconds after CXCL12 stimulation, RalGDS-RBD-mCherry was localized in the perinuclear region. The scale bar represents 5 μm. Finally, RalGDS-RBD-mCherry was localized at the leading edge of polarized cells.

[Experimental Example 63]
FIG. 74 is an image showing the results of introduction of a Rap1 activity sensor based on fluorescence resonance energy transfer (FRET) into control cells and stimulation with 100 nM CXCL12 for the time shown in FIG. The fluorescence intensity ratio image of mTurquoise / CFP corresponding to FRET efficiency is shown. Representative results of five experiments are shown. The scale bar indicates 5 μm.

  As shown in FIG. 74, Rap1 activation occurred at the cell membrane within 5 seconds of CXCL12 stimulation, followed by the perinuclear region. Finally, Rap1 activation was restricted to the leading edge of the cell.

[Experimental Example 64]
The inventors further investigated the relationship between Rap1 activation and adhesion of lymphocytes to endothelial cells under laminar flow using RalGDS-RBD-mCherry. Control cells expressing RalGDS-RBD-mCherry were perfused under laminar flow of ² dyn / cm ² on LS12 cells in the presence of 100 nM CXCL12. A digital image of the cell adhesion area was taken at 1 frame per second using a 63x objective lens under laminar flow. FIG. 75 is a diagram showing representative results of three experiments.

  As a result, it was revealed that when RalGDS-RBD-mCherry was clearly accumulated in a part of the contact area between the control cells and the endothelial cells, the cells stopped through the Rap1 activation site.

  These results indicate that Rap1 activation occurs within the cell membrane within a few seconds of chemokine stimulation and induces LFA-1-dependent cell adhesion on endothelial cells.

[Example 65]
Subsequently, the inventors examined whether filamin (FLN), an actin-binding protein, was bound to the cytoplasmic domain of LFA-1. Specifically, binding between Rap1V12 or Rap1N17 and FLNa was examined by pull-down assay.

  Rap1V12, Rap1N17, αL, and β2 bound with a T7 tag were introduced into COS cells. 48 hours after gene transfer, the cells are disrupted, and a pull-down assay using a fusion protein of FLNa repeats 1-3, 4-7, 8-11 and 21 and GST is performed, and anti-T7 antibody or anti-β2 antibody is used. Detection was performed by immunoblotting. FIG. 76 shows representative results of three experiments.

  As a result, Rap1V12, not Rap1N17, bound to the fusion protein of FLNa repeats 1 to 3 and GST. However, the other areas examined were either very weakly bound or not bound at all. In addition, β2 of LFA-1 bound to the repeat 21 of FLNa.

[Experimental Example 66]
Rap1V12, Rap1N17, αL, and β2 bound with a T7 tag were introduced into COS cells. Cells were disrupted 48 hours after gene transfer, the cell disruption was subjected to immunoprecipitation using anti-FLNa antibody, and the precipitate was detected by immunoblotting using anti-T7 antibody. FIG. 77 is a photograph showing the results of immunoprecipitation. Representative results from 4 experiments are shown. As shown in FIG. 77, Rap1-GTP but not Rap1-GDP bound to FLNa in the cells.

[Example 67]
Subsequently, the inventors examined the binding between FLNa lacking repeats 1 to 3 and Rap1V12.

  Specifically, Halo-wild type FLNa, Halo-FLNa (Δ1), Halo-FLNa (Δ2), and Halo-FLNa (Δ3) were introduced into COS cells. Here, FLNa (Δ1) represents a repeat 1 deletion mutant of FLNa. FLNa (Δ2) represents a repeat 2 deletion mutant of FLNa. FLNa (Δ3) represents a repeat 3 deletion mutant of FLNa.

  Subsequently, 48 hours after gene introduction, the cells were disrupted, the cell disruption was subjected to immunoprecipitation using Halolink ™ resin, and the precipitate was detected by immunoblotting using anti-T7 antibody or anti-FLNa antibody. FIG. 78 is a photograph showing the results of immunoprecipitation. Representative results from three independent experiments are shown.

  As a result, Rap1V12 bound to FLNa (Δ1) and FLNa (Δ2), but not to FLNa (Δ3). This result indicates that repeat 3 of FLNa is the most important region for binding to Rap1-GTP.

[Experimental Example 68]
Subsequently, the inventors examined whether the binding between FLNa and Rap1-GTP affects the binding between FLNa and the cytoplasmic region of β2. Specifically, Halo-FLNa, Rap1V12 and Rap1N17 bound to T7 tag, αL, and β2 bound to mRFP tag were introduced into COS cells. Cells were disrupted 48 hours after gene transfer, the cell disruption was subjected to immunoprecipitation using Halolink ™ resin, and the precipitate was immunoprecipitated using anti-mRFP antibody, anti-T7 antibody, anti-Halo antibody and anti-αL antibody. Detected by blotting. FIG. 79 is a photograph showing the results of immunoprecipitation. Representative results from three independent experiments are shown.

  As a result, it was revealed that FLNa binds to Rap1V12. As a result, the binding between FLNa and β2 was decreased as compared to the binding between FLNa and β2 in cells in which Rap1N17 was expressed.

[Example 69]
The membrane fraction of BAF / LFA-1 cells expressing Rap1V12 (T7-Rap1V12) bound only with the vector or T7 tag was pulled down using a fusion protein of β2 cytoplasmic region and GST (β2cyt-GST).

  FLNa, total FLNa and T7-Rap1V12 bound to β2cyt-GST were analyzed by immunoblotting using anti-FLNa antibody and anti-T7 antibody. FIG. 80 is a photograph showing a result of immunoblotting. Representative results from three independent experiments are shown.

  As a result, the amount of FLNa pulled down by β2cyt-GST from the membrane fraction of Rap1V12-expressing cells was 32% of the control cells.

[Example 70]
Since Rap1 is activated by chemokine stimulation, the inventors investigated whether CXCL12 stimulation reduced the binding of FLN and β2 depending on Rap1-GTP. Cells expressing vector alone or Spa-1 were stimulated with 100 nM CXCL12 for 1 minute and disrupted. Cell debris was immunoprecipitated using anti-β2 antibody, and the precipitate was detected by immunoblotting using anti-FLNa antibody.

  FIG. 81 is a photograph showing a result of immunoblotting. Representative results from three independent experiments are shown. As a result, the amount of FLNa immunoprecipitated with anti-β2 antibody in the control cells after CXCL12 stimulation was 28% of the amount of FLNa immunoprecipitated with anti-β2 antibody in the control cells without CXCL12 stimulation.

  On the other hand, the amount of FLNa immunoprecipitated with anti-β2 antibody in Spa-1 expressing cells after CXCL12 stimulation was 68% of the amount of FLNa immunoprecipitated with anti-β2 antibody in Spa-1 expressing cells without CXCL12 stimulation. there were.

  This result shows that CXCL12 stimulation decreases the binding of β2 and LFNa in control cells, but does not decrease the binding of β2 and LFNa in Spa-1 expressing cells. That is, inhibition of Rap1 activation by the expression of Spa-1 suppressed dissociation between LFA-1 and β2 cytoplasmic region after CXCL12 stimulation.

[Experimental Example 71]
Subsequently, the inventors examined Rap-dependent dissociation between FLN and β2 in the adhesion cascade. First, FLNa and FLNb were knocked down (KD) with shRNA. Lentivirus encoding scrambled shRNA (control), shRNA against FLNa or shRNA against FLNb was introduced into BAF / LFA-1 / L-selectin cells, and cell debris was analyzed by immunoblotting. FIG. 82 is a photograph showing the results of immunoblotting. Representative results from three independent experiments are shown. Actin protein was detected as a loading control. As a result, it was confirmed that 85% or more of FLNa and FLNb were knocked down.

[Experimental Example 72]
LFA-1 activation in control cells, FLNaKD cells, FLNbKD cells, and FLNa / bKD cells was examined by flow cytometric analysis using an antibody against an activation epitope of LFA-1 (KIM127).

  FIG. 83 is a graph showing the results. The data was normalized based on the expression level of LFA-1 using an anti-LFA-1 antibody (TS1 / 18). Data represent the mean value of three experimental results ± standard error. In FIG. 83, “*” indicates that there is a significant difference with a risk rate of less than 1%, compared to the control cells.

  Subsequently, the same experiment was performed by flow cytometry analysis using another antibody (MEM148) against an activated epitope of LFA-1, and the activity of LFA-1 in control cells, FLNaKD cells, FLNbKD cells, and FLNa / bKD cells. We studied

  FIG. 84 is a graph showing the results. The data was normalized based on the expression level of LFA-1 using an anti-LFA-1 antibody (TS1 / 18). Data represent the mean value of three experimental results ± standard error. In FIG. 84, “*” represents that there is a significant difference with a risk rate of less than 1%, compared with the control cells.

  As a result, the expression of the epitope showing the activation of LFA-1 recognized by the antibodies KIM127 and MEM148 was significantly increased by the knockdown of FLN. This result indicates that FLN limits the activation of LFA-1.

[Experimental Example 73]
Subsequently, the inventors examined whether knockdown of FLN increases the mobility of LFA-1 by FRAP (fluorescence recovery after photofading). FIG. 85 is a graph showing the percentage recovery after 1 minute from photobleaching in control cells and FLNa / bKD cells expressing LFA-1 labeled with red fluorescent protein (mRFP). Data represent the mean of three experimental results ± standard error (n = 20 for each group). In FIG. 85, “*” represents that there is a significant difference at a risk rate of less than 0.5% compared to the control cells.

  As a result, the mobility of LFA-1 in FLNa / bKD cells was significantly higher than that in control cells.

[Experimental example 74]
FIGS. 86 (a) and (b) show that control cells (FIG. 86 (a)) and FLNa / bKD cells (FIG. 86 (b)) co-expressing αL-mRFP and β2 were used with a laser having a wavelength of 567 nm. 3 is a graph showing typical results of photobleaching and quantitative FRAP analysis.

[Experimental Example 75]
FIG. 87 is a photograph showing a typical result of FRAP analysis using BAF cells in which αL-mRFP and β2 were co-expressed. Squares indicate areas where photo bleaching has been performed. The scale bar represents 5 μm.

[Experimental Example 76]
Control cells, FLNaKD cells, FLNbKD cells and FLNa / bKD cells were perfused on LS12 cells under a laminar flow of ² dyn / cm ² . Digital images showing the interaction of each cell were taken at 30 frames / second and classified into rolling, temporary adhesion, and stable adhesion (n = 150 for each group).

  FIG. 88 is a graph showing the results. In FIG. 88, “* 1” indicates that there is a significant difference at a risk rate of less than 2% compared to the total frequency in the control cells. “* 2” indicates that there is a significant difference at a risk rate of less than 1% compared to the total frequency in control cells.

  Consequently, FLNa or FLNb deficiency in BAF / LFA-1 / L-selectin cells increases the frequency of rolling and adhesion, and double knockdown of FLNa and b can increase these frequencies additively. It became clear.

[Experimental Example 77]
FIG. 89 is a graph showing the rolling speed of control cells or FLNa / bKD cells on LS12 cells. Data represent the mean of three experimental results ± standard error (n = 150 for each group). In FIG. 89, “*” indicates that there is a significant difference with a risk rate of less than 1%, as compared to the control cells.

  As a result, the rolling speed of FLNa / bKD cells was significantly slower than that of control cells.

[Experimental Example 78]
Control cells and FLNa / bKD cells were pretreated in the presence or absence of anti-L-selectin antibody or anti-LFA-1 antibody and perfused on LS12 cells under laminar flow of ² dyn / cm ² . Digital images showing the interaction of each cell were taken at 30 frames / second and classified into rolling, temporary adhesion, and stable adhesion.

  FIG. 90 is a graph showing the results. Data represent the mean of three experimental results ± standard error (n = 150 for each group). In FIG. 90, “*” indicates that there is a significant difference at a risk rate of less than 1% compared to the total frequency in the control cells.

  As a result, it became clear that the frequency of interaction events of cells was significantly reduced by pretreatment of cells with anti-LFA-1 antibody.

[Experimental Example 79]
Control cells or FLNa / bKD cells were pretreated with anti-LFA-1 antibody and the rolling rate on LS12 cells was examined. FIG. 91 is a graph showing the effect of anti-LFA-1 antibody on the rolling rate of control cells or FLNa / bKD cells.

  In FIG. 91, “* 1” indicates that there is a significant difference at a risk rate of less than 2% compared to control cells. “* 2” indicates that there is a significant difference at a risk rate of less than 5% compared to FLNa / bKD cells not pretreated with anti-LFA-1 antibody.

  As a result, it became clear that the rolling speed of FLNa / bKD cells was significantly increased by the anti-LFA-1 antibody treatment.

[Experimental Example 80]
Control and FLNa / bKD cells were laminar flowed ² dyn / cm ² on LS12 cells in the presence or absence of 100 nM CXCL12, in the presence or absence of anti-L-selectin antibody or anti-LFA-1 antibody. Perfused.

  FIG. 92 is a graph showing experimental results. Data represent the mean of three experimental results ± standard error (n = 150 for each group). In FIG. 92, “* 1” indicates that there is a significant difference at a risk rate of less than 2% compared to the total frequency in control cells stimulated with CXCL12 and not treated with anti-LFA-1 antibody. In addition, “* 2” indicates that there is a significant difference with a risk rate of less than 1% compared to the total frequency in control cells stimulated with CXCL12 and not treated with anti-LFA-1 antibody.

  From the above results, it was revealed that FLN deficiency mainly induces LFA-1-dependent rolling and adhesion.

<VIII. FLNa-deficient T cells showed enhanced LFA-1 mediated function>
[Experimental Example 81]
Subsequently, the inventors examined the function of FLNa in the adhesion cascade of primary T cells. In order to produce a conditional knockout mouse of FLNa, a mouse in which the FLNa allele is sandwiched between loxP sequences (hereinafter sometimes referred to as “FLNa ^{f / f} ” mouse) and T cell-specific expression of Cre recombinase. Lck-Cre mice were mated. Among the obtained offspring, mice lacking FLNa specifically for T cells (hereinafter sometimes referred to as “FLNa ^{− / −} ” mice) were obtained.

T cells derived from FLNa ^{f / f} mice and FLNa ^{− / −} mice were analyzed by immunoblotting using anti-FLNa antibody or anti-FLNb antibody. Actin protein was detected as a loading control. FIG. 93 is a photograph showing representative results of three independent experiments.

As a result, it was confirmed that FLNa ^{− / −} mouse-derived T cells (hereinafter sometimes referred to as “FLNa ^{− / −} T cells”) do not express FLNa protein. However, FLNb expression increased slightly in a compensatory manner.

[Experiment 82]
Adhesion events of FLNa ^{f / f} T cells and FLNa ^{− / −} T cells on LS12 cells were measured under laminar flow as described above. FIG. 94 is a graph showing the results. Data represent the mean ± standard error of the results of three independent experiments. In FIG. 94, “* 1” indicates that there is a significant difference at a risk rate of less than 0.1%, compared with the total frequency in the corresponding FLNa ^{f / f} T cells. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 0.1% compared to the total frequency in FLNa ^{− / −} T cells that were not treated with anti-LFA-1 antibody. In addition, “* 3” indicates that there is a significant difference at a risk rate of less than 3% compared to the total frequency in FLNa ^{− / −} T cells not treated with anti-LFA-1 antibody.

FIG. 95 is a graph showing the effect of anti-LFA-1 antibody on the rate of rolling of FLNa ^{f / f} T cells and FLNa ^{− / −} T cells on LS12 endothelial cells in the absence of CCL21. Data represent the mean ± standard error of the results of three independent experiments. In FIG. 95, “* 1” indicates that there is a significant difference at a risk rate of less than 1% compared to FLNa ^{f / f} T cells. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 1% compared to FLNa ^{− / −} T cells that were not treated with anti-LFA-1 antibody.

As a result, it was clarified that FLNa ^{− / −} T cells showed a significant increase in the frequency of LFA-1-dependent rolling and a significant decrease in the rolling speed compared to FLNa ^{f / f} T cells. . In addition, the pretreatment with the anti-LFA-1 antibody significantly decreased the frequency of FLNa ^{− / −} T cell interaction events and significantly increased the rolling speed.

[Experimental example 83]
FLNa ^{f / f} T cells and FLNa ^{− / −} T cells were perfused under laminar flow of ² dyn / cm ² on MadCAM-1 in the absence of CCL21. FIG. 96 (a) is a graph showing the results of measuring cell adhesion events in the same manner as described above. Data represent mean ± standard error in triplicate experiments (n = 100 for each group).

FIG. 96 (b) is a graph showing the rolling speed of FLNa ^{f / f} T cells and FLNa ^{− / −} T cells. Data represent mean ± standard error in triplicate experiments (n = 100 for each group).

As a result, FLNa ^{- / -} between the T cell and FLNA ^{f / f} T cells, alpha ₄ beta ₇ significant difference to the speed of the frequency and the rolling of the rolling of the MadCAM-1 is dependent of rolling I was not able to admit.

[Experimental Example 84]
97 (a) and (b) show the displacement of FLNa ^{f / f} T cells and FLNa ^{− / −} T cells on ICAM-1 in the presence or absence of CCL21 (FIG. 97 (a)). ) And speed (FIG. 97 (b)) (n = 50 for each group). In FIG. 97, “* 1” indicates that there is a significant difference at a risk rate of less than 2% compared to Rap1 ^{f / f} T cells. In addition, “* 2” indicates that there is a significant difference at a risk rate of less than 5% compared to Rap1 ^{f / f} T cells.

97 (c) and (d) are representative diagrams showing the trajectories of FLNa ^{f / f} T cells and FLNa ^{− / −} T cells on ICAM-1 in the presence of CCL21. Each line shows the trajectory of one cell.

As a result, migration and speed on ICAM-1 was significantly increased in FLNa ^{− / −} T cells compared to FLNa ^{f / f} T cells.

[Experimental Example 85]
The inventors examined the chemotaxis of FLNa ^{− / −} T cells to CCL21 by a transwell migration assay. First, purified FLNa ^{f / f} T cells and FLNa ^{− / −} T cells were placed in 500 μL of 1% fetal calf serum-RPMI medium on uncoated transwell inserts with 3 μm diameter pores. Seeding at a concentration of 10 ⁶ cells / mL.

  Subsequently, CCL21 having a concentration shown in FIG. 98 (a) was added to the medium (500 μL) in the lower chamber. Subsequently, cell migration was performed at 37 ° C. for 1 hour. Subsequently, the transwell insert was removed, and the number of cells moved to the lower chamber was counted.

FIG. 98 (a) is a graph showing the results. The number of cells that have moved to the lower chamber relative to the total number of cells is shown as a percentage. Data represent mean ± standard error in 3 independent experiments. FIG. 98 (b) is a graph showing the results of a similar experiment performed using a transwell insert coated with ICAM-1. Data represent mean ± standard error in 3 independent experiments. “*” Indicates that there is a significant difference at a risk rate of less than 0.2% compared to the corresponding FLNa ^{f / f} T cells.

As a result, in experiments with uncoated transwell inserts, the number of cells attracted by CCL21 and migrated to the lower chamber was found to be different between FLNa ^{f / f} T cells and FLNa ^{− / −} T cells. There wasn't. On the other hand, the number of FLNa ^{− / −} T cells that passed through the ICAM-1 coated transwell insert was significantly increased at low concentrations of CCL21 compared to FLNa ^{f / f} T cells.

[Experimental example 86]
FLNa ^{f / f} T cells and FLNa ^{− / −} T cells were labeled with CFSE or CMTMR, mixed in the same number of cells, and injected intravenously into normal mice. One hour later, lymphocytes in blood and peripheral lymph nodes were analyzed by flow cytometry. FIGS. 99 (a) and 99 (b) are graphs showing the results of flow cytometry analysis in blood (FIG. 99 (a)) and lymph nodes (FIG. 99 (b)). The numbers above the squares in the graph indicate the ratio of FLNa ^{− / −} T cells to FLNa ^{f / f} T cells.

FIG. 99 (c) is a graph showing the ratio of FLNa ^{− / −} T cells to FLNa ^{f / f} T cells in lymph nodes (n = 4). “*” Indicates that there is a significant difference at a risk rate of less than 2% compared to FLNa ^{f / f} T cells. As a result, it was revealed that the number of FLNa ^{− / −} T cells homing to the lymph node was significantly larger than that of FLNa ^{f / f} T cells.

  These results support that FLNa is an inhibitor of LFA-1-dependent primary T cell adhesion and migration.

[Experiment 87]
In order to investigate whether Rap1-dependent dissociation of FLN from β2 is associated with chemokine-induced adhesion, the following experiment was performed. First, wild-type (WT) FLNa or FLNa (Δ3) bound with a Halo tag was introduced into BAF / LFA-1 / L-selectin cells. Subsequently, these cell disruptions were subjected to immunoblotting using an anti-Halo antibody.

  FIG. 100 is a photograph showing the results of immunoblotting. Representative results from three experiments are shown. As a result, it was confirmed that wild type FLNa and FLNa (Δ3) were expressed.

[Experimental Example 88]
Subsequently, adhesion events on LS12 cells of cells expressing wild type FLNa or FLNa (Δ3) were measured in the same manner as described above under laminar flow. FIG. 101 is a graph showing the results. Data represent mean ± standard error in 3 independent experiments. “* 1” indicates that there is a significant difference at a risk rate of less than 3% compared to the total frequency in control cells that were not treated with anti-LFA-1 antibody. “* 2” indicates that there is a significant difference at a risk rate of less than 1% compared to the sum of the frequencies in the corresponding control cells.

[Experimental Example 89]
FIG. 102 is a graph showing the results of examining the expression of KIM127 epitope in the presence or absence of CXCL12 in cells expressing only vector, wild type FLNa or FLNa (Δ3) (n = 5 for each group). ). “*” Indicates that there is a significant difference at a risk rate of less than 5% compared to cells expressing wild-type FLNa.

  As a result, the expression of FLNa (Δ3) in BAF / LFA-1 / L-selectin cells significantly decreased the frequency of cell adhesion by CXCL12 stimulation compared to cells expressing wild-type FLNa, and the KIM127 antibody Significantly inhibited the formation of the recognized LFA-1 expanded conformation.

  The above results indicate that the binding of Rap1-GTP to FLNs is important for LFA-1 activation, and LFA-1 activation is essential for chemokine-stimulated cell adhesion.

  According to the present invention, a model non-human animal that spontaneously develops colitis and colon cancer can be provided. The model non-human animal is useful for elucidating the pathogenesis of colitis or colon cancer and for developing a treatment method.

Claims (8)

  A method for spontaneously developing colitis or colorectal cancer in a non-human animal, wherein RAS-related protein-1a (Rap1a) and RAS-related protein-1b (Rap1b) are deleted in a T cell-specific manner in the non-human animal. A method comprising the steps. The above-mentioned step of deleting Rap1a and Rap1b can be performed by crossing any of the following non-human animals or any of the following non-human animals with Rap1 ^{f / f} Rap1b ^{f / f} non-human animals. The method according to claim 1, which is a step of obtaining a non-human animal deficient in Rap1a and Rap1b.
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^+/− Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
CD4-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^+/- Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{wt / wt} Rap1b ^{f / f} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / wt} Rap1b ^{wt / wt} non-human animal,
Lck-Cre ^{+ / +} Rap1a ^{f / f} Rap1b ^{wt / wt} non-human animal.   A colitis or colon cancer model non-human animal, which is an immunodeficient non-human animal transplanted with T cells lacking Rap1a and Rap1b.   A method for producing a non-human animal of colitis or colon cancer model, comprising a step of transplanting T cells lacking Rap1a and Rap1b into an immunodeficient non-human animal. Rolling activity of lymphocytes deficient in Rap1a and Rap1b on L-selectin ligand, Mucosal Addressin Cell Adhesion Molecule-1 (MadCAM-1) or P-selectin in the presence or absence of the test substance Measuring process;
When the rolling activity in the presence of the test substance is lower than the rolling activity in the absence of the test substance, the test substance is a therapeutic agent for colitis or colon cancer A determining step;
A screening method for a therapeutic agent for colitis or colon cancer. Rearing a non-human animal deficient in Rap1a and Rap1b, or an immunodeficient non-human animal transplanted with a T cell deficient in Rap1a and Rap1b under administration or non-administration of a test substance;
Measuring the degree of onset of colitis or colon cancer in the non-human animal;
When the degree of onset of colitis or colon cancer under administration of the test substance is lower than the degree of onset of colitis or colon cancer under non-administration of the test substance, the test substance Determining that it is a treatment for colitis or colon cancer;
A screening method for a therapeutic agent for colitis or colon cancer. Culturing lymphocytes lacking Rap1a and Rap1b in the presence or absence of a test substance;
Measuring the phosphorylation level of Ezrin / Radixin / Moesin (ERM) protein in the lymphocytes;
When the phosphorylation level in the presence of the test substance is increased compared to the phosphorylation level in the absence of the test substance, the test substance is used to treat colitis or colon cancer. Determining that it is an agent;
A screening method for a therapeutic agent for colitis or colon cancer. Culturing lymphocytes lacking Rap1a and Rap1b in the presence or absence of a test substance;
Measuring the kinase activity of Lymphocyte-oriented kinase (LOK) protein in the lymphocytes;
When the kinase activity in the presence of the test substance is increased compared to the kinase activity in the absence of the test substance, the test substance is a therapeutic agent for colitis or colon cancer. Determining that there is,
A screening method for a therapeutic agent for colitis or colon cancer.