JP5911810B2 - Method for producing regulatory T cells - Google Patents

Description

  The present invention relates to a method for producing a cell population containing regulatory T cells useful in the medical field.

  Regulatory T cells (Treg: Regulatory T cells) are generally defined as T cells that have a function of suppressing abnormal or excessive immune responses and are responsible for immune tolerance. Decrease or dysfunction of regulatory T cells in humans causes autoimmune diseases and allergies. Therefore, quantitative or qualitative control of regulatory T cells is useful for the treatment of autoimmune diseases and allergies, and also for the treatment of refractory graft-versus-host disease (GVHD), etc. It is expected to become a new cell therapy in the field of transplant immunity.

  The regulatory T cells are roughly classified into an endogenous regulatory T cell (nTreg: Natural Occurring Regulatory T cell) and an inducible regulatory T cell (iTreg: Inducible Regulatory T cell). Endogenous regulatory T cells are defined as those that differentiate in the thymus and are naturally produced independent of external antigen sensitization. On the other hand, inducible regulatory T cells are defined as those induced to differentiate from peripheral naive T cells in response to foreign antigen stimulation in an artificial tolerance induction model. These regulatory T cells are further subdivided according to the type of marker expressed in the cells.

CD4 positive (hereinafter also referred to as “CD4 ⁺ ”) CD25 positive (hereinafter also referred to as “CD25 ⁺ ”) regulatory T cells, one of the endogenous regulatory T cell groups, is removed from the living body. Then, various organ-specific autoimmune diseases spontaneously develop, and when CD4 ⁺ CD25 ⁺ regulatory T cells are transplanted, the onset of autoimmune diseases is prevented, so that endogenous CD4 ⁺ CD25 ⁺ regulatory properties are obtained. T cells are thought to play an important role in maintaining immune tolerance in the periphery. Later, the function of CD4 ⁺ CD25 ⁺ regulatory T cells was analyzed in detail, suppressing not only autoimmunity but also most immune responses such as inflammation due to foreign antigens, allergies, transplant rejection, infection immunity, tumor immunity, etc. It has become clear to get. At present, the transcription factor Foxp3 has been clarified as a master regulator of CD4 ⁺ CD25 ⁺ regulatory T cells (for example, Non-Patent Document 1).

  As a method for producing regulatory T cells from the peripheral blood of a patient by expansion culture, the following two methods are mainly conceivable. One is a method in which a T cell-containing fraction derived from peripheral blood is subjected to expansion culture, and regulatory T cells are selected during the culture process or after completion of the culture. The other is a method of expanding culture using regulatory T cells previously separated from peripheral blood as a starting material.

  However, in the former, regulatory T cells have poor proliferation ability compared with other T cells in vitro, and there is an operation of selecting regulatory T cells having a reduced abundance ratio from a wide variety of expanded cell groups. There is a problem that it is difficult. In addition, since Foxp3 expressed in regulatory T cells is an intracellular molecule, in order to confirm its expression, it is necessary to perform permeabilization of the cell membrane for staining. Therefore, the expression of Foxp3 cannot be used as an index when separating live cells.

  Even in the latter case, regulatory T cells have a poor ability to proliferate in vitro, and there is a problem that many regulatory T cells can be obtained (for example, Non-Patent Document 2).

  Therefore, in order to use the regulatory T cells for the various cell therapies described above, a method is desired that can efficiently expand the regulatory T cells from a small amount of peripheral blood.

  Regulatory T cell expansion culture method using anti-CD3 antibody that is a CD3 activator (Non-patent Document 3), and regulatory T cell expansion culture method using anti-CD3 antibody and anti-CD28 antibody in combination (Non-patent Document 3) 4) has been reported. However, these methods are not satisfactory as a method for producing Foxp3-positive regulatory T cells.

Nature Immunology, Volume 4, Pages 330-336 (2003) Annual Review of Immunology, Vol. 22, pp. 531-562 (2004) Journal of Experimental Medicine, Vol. 193, pages 1285 to 1294 (2001), Journal of Experimental Medicine. Blood, Vol. 104, pp. 453-461 (2004) Blood 108, 4260-4267 (2006) Immunology, Vol. 121, pp. 129-139 (2007) European Journal of Immunology, Vol. 37, pp. 2378-2389 (2007) Journal of Immunology, Vol. 178, pp. 320-329 (2007)

  An object of the present invention is to provide a method for producing a cell population containing regulatory T cells, a cell population produced by the method, and a medicament containing the cell population.

  The present inventors have significantly increased the cell proliferation of the CD25 positive T cell population by causing a CD3 activator to act in the presence of fibronectin or a fragment thereof or a mixture thereof, and Foxp3 positive regulatory T cells. It has been found that a cell population containing can be produced. Furthermore, also in the method for expanding regulatory T cells using a CD3 activator and a CD28 ligand together, the cell proliferation of the CD25 positive T cell population is significantly increased by coexisting fibronectin or a fragment thereof or a mixture thereof. The present inventors have found that a cell population containing Foxp3-positive regulatory T cells can be efficiently produced, and have completed the present invention.

That is, when the present invention is outlined, the first invention of the present invention relates to a method for producing a cell population containing regulatory T cells, which comprises the following steps:
(1) separating a CD25 positive T cell population from a cell population containing T cells; and (2) obtained in step (1) in the presence of a CD3 activator and fibronectin or a fragment thereof or a mixture thereof. Culturing a cell population in vitro.

  In the first aspect of the present invention, the method of culturing the cell population obtained in step (1) in vitro includes the step of culturing in the presence of CD28 ligand. It is included in a method for producing a cell population containing T cells. Further, the CD28 ligand may be an anti-CD28 antibody. Furthermore, in the first aspect of the present invention, the CD25 positive T cell population may be a CD4 positive CD25 positive T cell population, and the CD3 activator may be an anti-CD3 antibody.

  The second invention of the present invention relates to the cell population produced in the first aspect of the present invention.

  The third invention of the present invention relates to an immunosuppressive agent comprising the cell population produced in the first aspect of the present invention.

  As a first aspect of the present invention, there is a method for producing a cell population containing Foxp3-positive regulatory T cells.

  As a second aspect of the present invention, the present invention relates to a cell population containing Foxp3-positive regulatory T cells produced in the first aspect of the present invention.

  As an aspect of the third invention of the present invention, an immunosuppressive agent comprising a cell population containing Foxp3-positive regulatory T cells produced in the first aspect of the present invention can be mentioned.

  By using the method of the present invention, a cell population containing regulatory T cells can be efficiently expanded in vitro. Regulatory T cells produced by this method are useful as an immunosuppressant for the prevention and treatment of organ transplant rejection, allergic diseases, autoimmune diseases, graft-versus-host disease (GVHD) and the like.

It is a figure which shows the cell growth magnification | multiplying_factor (Fold expansion: In the figure below, it displays with an expansion factor in a figure) of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the growth inhibitory activity with respect to CD4 positive cell of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of CD4 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the CD4 positive and Foxp3 positive cell number of a cultured cell. It is a figure which shows the ratio of CD4 positive, CD25 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows the number of CD4 positive, CD25 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows the growth inhibitory activity with respect to CD4 positive cell of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the ratio of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the number of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows the growth inhibitory activity with respect to CD8 positive cell of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of CD4 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the ratio of the CD4 positive, Foxp3 positive, and CCR7 positive cell of a cultured cell. It is a figure which shows the ratio of the CD4 positive, Foxp3 positive, and CD27 positive cell of a cultured cell. It is a figure which shows the CD4 positive and Foxp3 positive cell number of a cultured cell. It is a figure which shows CD4 positive, Foxp3 positive, and CCR7 positive cell number of a cultured cell. It is a figure which shows the number of CD4 positive, Foxp3 positive, and CD27 positive cells of a cultured cell. It is a figure which shows the electrophoresis photograph of the bisulfite PCR product of cultured cell origin DNA. It is a figure which shows the frequency of DNA methylation of the regulatory T cell specific demethylation area | region of a cultured cell. It is a figure which shows the DNA methylation degree of the regulatory T cell specific demethylation area | region in each clone of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of CD4 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the CD4 positive and Foxp3 positive cell number of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the ratio of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the number of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of the cultured cell after restimulation. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the ratio of CD4 positive, CD25 positive, Foxp3 positive, and CCR7 positive cells of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the CD4 positive, CD25 positive, Foxp3 positive, and CCR7 positive cell number of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the ratio of the CD4 positive and CD25 positive and Foxp3 positive cell of a cultured cell. It is a figure which shows the ratio of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows CD4 positive, CD25 positive, and Foxp3 positive cell number of a cultured cell. It is a figure which shows the number of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 positive cells of a cultured cell. It is a figure which shows the time-dependent change of the cell growth rate of a cultured cell. It is a figure which shows the CD4 positive and Foxp3 positive cell number of a cultured cell. It is a figure which shows CD4 positive, Foxp3 positive, and CCR7 positive cell number of a cultured cell. It is a figure which shows the number of CD4 positive, Foxp3 positive, and CD27 positive cells of a cultured cell.

  T cells are also called T lymphocytes, mean cells derived from the thymus among lymphocytes involved in immune responses, and include differentiated T cells and undifferentiated T cells. T cells include helper T cells (Th1, Th2, Th9, Th17, Th22, Tfh), regulatory T cells, cytotoxic T cells, naive T cells, memory T cells, α chain Known are αβ T cells that express TCR of β chain, γδ T cells that express TCR of γ chain and δ chain, NKT cells, and the like.

Regulatory T cells mean T cells that have the ability (immunosuppressive activity) to inhibit the activation (proliferation, cytokine production, etc.) of effector T cells when stimulated via a T cell receptor. . Regulatory T cells are usually present in the CD4 ⁺ CD25 ⁺ T cell fraction. Regulatory T cells themselves do not show a substantial proliferative response to stimulation through T cell receptors and may be unresponsive. The transcription factor Foxp3 is known as a master regulator of CD4 ⁺ CD25 ⁺ regulatory T cells (where “+” indicates that the molecule is expressed on the cell surface (positive)) The same shall apply hereinafter.

Among the expanded cultured CD4 ⁺ CD25 ⁺ T cell population, cells positive for both L-selectin (CD62L) and CCR7 have a particularly strong immunosuppressive activity among regulatory T cells, and CD45RA ⁺ CD4 ⁺ It has been reported that the proportion of cells that are both positive for CD62L and CCR7 increases by expanding the CD25 ⁺ T cell (naive regulatory T cell) population [Blood, 108, 4260-4267. (2006)]. In addition, it has been reported that CD27 expression correlates with Foxp3 expression and immunosuppressive activity in an expanded cultured CD4 ⁺ CD25 ⁺ T cell population [Immology, Vol. 121, pp. 129-139 (2007)]. ]. Furthermore, it is reported that for stable expression of Foxp3 in regulatory T cells, it is important that the “regulatory T cell-specific demethylation region” (TSDR) in the Foxp3 locus is demethylated. [European Journal of Immunology, Vol. 37, pp. 2378-2389 (2007)]. Furthermore, it has been reported that the proportion of cells expressing Foxp3 and CD25 increases by expanding the regulatory T cells in the presence of rapamycin [Journal of Immunology, Vol. 178, pp. 320-329]. (2007)].

  In the present invention, “production of a cell population containing regulatory T cells” means a concept including proliferation (expansion culture) of regulatory T cells. According to the present invention, a cell population containing a high ratio of regulatory T cells having particularly strong immunosuppressive activity can be efficiently obtained.

  In the present invention, “fibronectin” and fragments thereof may be isolated from nature, artificially prepared, or recombinantly prepared by genetic engineering, that is, non-naturally occurring. Fibronectin and fragments thereof are described, for example, in Luoslati E. [Ruoslahti E. et al. , Et al. , Journal of Biological Chemistry (Vol. 256, No. 14, pp. 7277-7281 (1981)), manufactured in a substantially pure form from a naturally occurring substance. can do. For the nucleic acid sequence encoding fibronectin and the amino acid sequence of fibronectin, which are information for preparing a fibronectin fragment, NCBI RefSeq Accession No. NM_002026 and NP_002017, respectively. As used herein, substantially pure fibronectin or fibronectin fragment means that they are essentially free of other proteins that are naturally present with fibronectin. The above fibronectin and fragments thereof can be used in the present invention alone or in combination with a plurality of types.

  The domain structure of fibronectin is divided into seven, and the amino acid sequence includes three types of similar sequences, and the whole is constituted by repetition of these sequences. Three similar sequences are called type I, type II and type III, respectively. There are 14 type III sequences in fibronectin, of which 8th, 9th and 10th (hereinafter referred to as III-8, III-9 and III-10, respectively) are cell binding domains, The 12th, 13th and 14th (hereinafter referred to as III-12, III-13 and III-14, respectively) are contained in the heparin binding domain. A region called IIICS exists on the C-terminal side of the heparin-binding domain. IIICS has a region called CS-1, which consists of 25 amino acids and has binding activity to VLA-4. The fibronectin fragment that can be used in the present invention may be a fragment containing any domain of III-7, 8, 9, 11, 12, 13 or CS-1, and is a fragment in which a plurality of domains are repeatedly linked. There may be. For example, a fragment containing a cell adhesion domain containing a ligand for VLA-5, a heparin binding domain, a CS-1 domain that is a ligand for VLA-4, and the like is used in the present invention. Examples of the recombinant fragment include CH-271 (SEQ ID NO: 1 in the Sequence Listing) described in Journal of Biochemistry, Vol. 110, pages 284 to 291 (1991), CH- Examples include 296 (SEQ ID NO: 2 in the sequence listing), H-271 (SEQ ID NO: 3 in the sequence listing), H-296 (SEQ ID NO: 4 in the sequence listing), or derivatives or modifications thereof. The aforementioned CH-296 is a recombinant fibronectin fragment having a cell adhesion domain, a heparin binding domain and a CS-1 domain, and is commercially available under the name of RetroNectin (registered trademark, manufactured by Takara Bio Inc.).

  In the present invention, the “CD25 positive T cell population” means a T cell population that is positive for CD25. For example, a CD4 positive CD25 positive T cell population that is positive for both CD4 and CD25 is a CD25 positive T cell population. include.

Hereinafter, the present invention will be specifically described.
1. Method for producing cell population containing regulatory T cells Production of a cell population containing regulatory T cells according to the method of the present invention comprises a first step of separating a CD25 ⁺ T cell population from a cell population containing T cells, The CD25 ⁺ T cell population obtained by the step is performed by a second step of culturing in the presence of a CD3 activator and fibronectin or a fragment thereof or a mixture thereof.

Already, CD4 ⁺ cells were preferentially expanded over CD8 ⁺ cells by costimulation with anti-CD3 and anti-CD28 antibodies, and further CD4 by costimulation with CH-296, an anti-CD3 antibody and fibronectin fragment. It has been reported that CD8 ⁺ cells are preferentially expanded rather than ⁺ cells [Cancer Gene Therapy, Vol. 15, pp. 508-516 (2008)].

The present inventors have found that by culturing in the presence of the CD25 + ^T cell population CD3 activator and fibronectin or a fragment or a mixture thereof that, cell proliferation rate of the CD25 + ^T cell population is significantly increased efficiency It was found that a cell population containing well Foxp3 positive regulatory T cells can be produced.

In the first step, the present invention is not particularly limited as a “cell population containing T cells”, but peripheral blood mononuclear cells (PBMC), peripheral lymphocytes, cord blood mononuclear cells and the like are exemplified. . In addition, various cell populations derived from blood cells containing T cells can be used in the present invention. These cells may be activated in vivo (in vivo) or ex vivo (ex vivo) by cytokines such as IL-2. As these cells, those collected from a living body or those obtained through in vitro culture may be used as they are, or may be used after being cryopreserved. In addition, for example, a cell population obtained from a cell population obtained from a living body through various induction operations or separation operations, for example, a CD25 ⁺ cell population separated from PBMC or the like can be used. Furthermore, in the method for producing a cell population of the present invention, a material containing the cells, for example, blood such as peripheral blood or umbilical cord blood, blood from which components such as red blood cells or plasma are removed, bone marrow fluid, or the like is used. You can also

The step of separating the CD25 ⁺ T cell population from the T cell-containing cell population can be performed using a known method such as a method using a flow cytometer or an immunomagnetic bead method. In the method using a flow cytometer, a CD25 ⁺ T cell population can be separated by selectively recovering CD25 ⁺ cells that are sorted by staining with a fluorescently labeled anti-CD25 antibody. Alternatively, the CD25 ⁺ T cell population can be separated by selectively recovering CD25 ⁺ cells that are targeted to another marker [eg, glucocorticoid-induced tumor necrosis factor receptor (GITR)]. Further, in this step, the CD25 ⁺ CD4 ⁺ T cell population is separated by selectively recovering CD25 ⁺ and CD4 ⁺ cells that are sorted by co-staining with fluorescently labeled anti-CD25 antibody and anti-CD4 antibody. Can do.

The immunomagnetic bead method is a method of separating cells by using magnetic beads combined with various antibodies and a magnet together. Examples of kits for isolating CD25 ⁺ CD4 ⁺ T cells include Dynabeads Regulatory CD4 ⁺ CD25 ⁺ T Cell Kit (manufactured by Invitrogen), CD4 ⁺ CD25 ⁺ Regulatory T Cell Isolation Kit (Milten) CD25 ⁺ CD4 ⁺ T cells can be isolated according to the instruction manual attached to.

Incidentally, from a cell population containing a T cell, by removing cells other than CD25 ⁺ CD4 ⁺ T cell, the operation of obtaining a cell population having a high content of CD25 ⁺ CD4 ⁺ T cell also the first step in the present invention It is one mode.

The CD25 ⁺ CD4 ⁺ T cell population obtained by the method exemplified above can also be used as a starting material for the method for producing a cell population containing regulatory T cells of the present invention.

In the second step, the CD25 ⁺ T cell population obtained in the first step is cultured in the presence of a CD3 activator and fibronectin or a fragment thereof or a mixture thereof.

  According to the present invention comprising these two steps, a cell population containing regulatory T cells can be produced in vitro. Furthermore, the regulatory T cells obtained by this method are useful as an immunosuppressive agent for organ transplant rejection, allergic diseases, autoimmune diseases, graft-versus-host diseases (GVHD, etc., cell therapy, etc. It is suitable for use in.

  In the method of the present invention, the second step is usually used for the culture of lymphocytes in the presence of the active ingredients characterizing the production method of the present invention, that is, a CD3 activator and fibronectin or a fragment thereof or a mixture thereof. It is carried out in a medium containing general components.

The medium used in the method for producing a cell population containing regulatory T cells of the present invention is not particularly limited as long as it contains the above-mentioned active ingredients. Components suitable for maintaining and growing the CD25 ⁺ T cell population are mixed. The produced well-known culture medium can be used, for example, a commercially available culture medium and its modification may be sufficient. These media may contain appropriate proteins, cytokines, and other components in addition to the original components. Preferably, a medium containing IL-2 is used in the present invention. The concentration of IL-2 in the medium is not particularly limited. For example, it is preferably 0.1 to 1 × 10 ⁵ IU / mL, more preferably 1 to 1 × 10 ⁴ IU / mL, and more preferably. Is 10 to 1 × 10 ³ IU / mL. In addition, other cytokines (IL-7, IL-15, IL-21, etc.), lymphocyte stimulating factors such as lectins or rapamycin which is an immunosuppressive agent can be added.

  The CD3 activator used in the present invention as an active ingredient is not particularly limited as long as it has a T cell receptor or CD3 binding activity. For example, anti-CD3 antibody, concanavalin A (ConA), phytohemagglutinin Examples include (PHA), allo MHC molecules, antigens presented by artificial antigen-presenting cells and dendritic cells, and the like. Preferably, an anti-CD3 monoclonal antibody is used in the present invention, and for example, OKT3 [Science, 206, 347-349 (1979)] is particularly suitable. The concentration of CD3 ligand in the medium is not particularly limited. For example, when an anti-CD3 monoclonal antibody is used, 0.001 to 100 μg / mL, particularly 0.01 to 50 μg / mL is preferable.

  Further, the concentration of fibronectin or a fragment thereof or a mixture thereof used in the present invention as an active ingredient is not particularly limited. For example, 0.001 to 500 μg / mL, particularly 0.01 to 100 μg / mL is preferable. .

  Furthermore, in the present invention, a CD28 ligand may be added to the medium as necessary. The CD28 ligand used in the present invention is not particularly limited as long as it is a substance having a binding activity to CD28, and examples thereof include an anti-CD28 antibody. The concentration of CD28 ligand in the medium is not particularly limited. For example, when an anti-CD28 monoclonal antibody is used, 0.001 to 100 μg / mL, particularly 0.01 to 20 μg / mL is preferable. In the present invention, the culture in the presence of CD28 ligand may be performed only in a part of the culture process, or the entire culture may be performed in the presence of the above components.

  Among these components, “CD3 activator”, “fibronectin or a fragment thereof or a mixture thereof” and “CD28 ligand” are not only dissolved and coexisting in a medium, but also an appropriate solid phase, for example, Cell culture equipment such as petri dishes, flasks, bags, etc. (including both open and closed systems), or immobilized on cell culture carriers such as beads, membranes, glass slides, etc. May be. The material of the solid phase is not particularly limited as long as it can be used for cell culture. For example, in the case where the component is solid-phased on the device, when the medium is put in the device, the device has a ratio similar to a desired concentration when the component is dissolved in the medium and used. It is preferable to solidify a certain amount of each component with respect to the amount of medium to be put in, but the amount of the solid phase of the component is not particularly limited as long as a desired effect is obtained. The carrier is used by immersing it in a culture solution in a cell culture equipment during cell culture. When the component is solid-phased on the carrier, when the carrier is placed in the medium, the medium is placed in the equipment so that the ratio is the same as the desired concentration when the component is dissolved in the medium. It is preferable to solid-phase a certain amount of each component with respect to the amount, but the amount of the solid-phased component is not particularly limited as long as a desired effect is obtained. In any case, immobilization of the components is performed according to a known method, for example, a fibronectin fragment immobilization method described in WO 97/18318 pamphlet and WO 00/09168 pamphlet. Can be done. Furthermore, an active ingredient other than “CD3 activator”, “fibronectin or a fragment thereof or a mixture thereof” and “CD28 ligand” may be immobilized on a cell culture device or a cell culture carrier. For example, anti-4-1BB antibody, anti-OX40 antibody, anti-GITR antibody, CD80, CD86, 4-1BBL, OX40L and the like are exemplified.

  If the above-mentioned various components and those selected from the active ingredients of the present invention are solid-phased, the cell population and the solid phase are obtained in the process of obtaining a cell population containing regulatory T cells by the method of the present invention. The active ingredient and the cell population can be easily separated only by separation, and contamination of the active ingredient and the like into the cell population can be prevented.

The second step in the method of the present invention may include culturing in a medium containing no active ingredient, which is carried out following the cultivation of the CD25 ⁺ T cell population in the presence of the active ingredient. In this case, a medium having the same composition may be used except that it does not contain “CD3 activator”, “fibronectin or a fragment thereof, or a mixture thereof”, or both. May be used.

  In the method of the present invention, the culture period in the presence of the active ingredient may be 1 day or longer, preferably 2 days or longer, and the period may be within 8 days, preferably 6 Within days. Further, in order to increase the multiplication factor, the medium containing the active ingredient again, or the cell culture equipment or cell culture in which the active ingredient is solidified after or during the culture in the medium not containing the active ingredient Culture may be performed using a carrier for use.

  In the present invention, a medium containing no serum or plasma can be used, but serum or plasma may be added to the medium. The amount added to these media is not particularly limited, but examples include a content of more than 0 to 20% by volume, preferably more than 0 to 6% by volume, and serum or plasma used depending on the culture stage The amount of can be changed. For example, the serum or plasma concentration can be decreased in stages and used. The origin of serum or plasma may be either self (meaning that the origin is the same as the cell to be cultured) or non-self (meaning that the origin is different from the cell to be cultured). From this point of view, self-derived ones are preferably used.

Although it does not specifically limit as a cell number at the time of the culture | cultivation start used in this invention, For example, Preferably it is 10 < ² > -1 * 10 < ⁸ > cells / mL, More preferably, 10 < ³ > -5 * 10 < ⁷ > cells / mL. mL, more preferably 10 ⁴ to 1 × 10 ⁷ cells / mL are exemplified. Moreover, there is no limitation in particular in culture conditions, The conditions normally used for cell culture can be used. For example, it can be cultured in conditions such as 37 ℃, 5% CO _2. In addition, operations such as dilution by adding a fresh medium to the cell culture medium at an appropriate time interval, exchanging the medium, and exchanging the cell culture equipment can be performed.

Although it does not specifically limit as a cell culture equipment used in the manufacturing method of this invention, For example, a petri dish, a flask, a bag, a large culture tank, a bioreactor etc. can be used. As the bag, a CO ₂ gas permeable bag for cell culture can be used. Moreover, when manufacturing a large cell population industrially, a large culture tank can be used. Moreover, although culture | cultivation can be implemented in any of an open system or a closed system, it is preferable to culture by a closed system from a viewpoint of the safety | security of the cell population obtained.

  In the present invention, the total number of culture days in the culture in the presence and absence of active ingredients is preferably 5 to 40 days, more preferably 6 to 35 days, and even more preferably 7 to 30 days. It is. Within this range, the number of cells that can be used for general immunotherapy can be obtained, and the period during which cell growth is maintained well may be selected as appropriate.

  The cell population obtained by the production method of the present invention is positive for both CD4 and CD25 and has a high content of Foxp3-positive cells. Furthermore, the content rate of cells in which both CD4 and CD25 are positive and both CD62L and CCR7 are positive is high. As described above, cells positive for CD4, CD25, CD62L and CCR7 are known to be regulatory T cells with strong immunosuppressive activity.

  That is, as shown in each Example below, cells contained in a high ratio in the cell population obtained by the production method of the present invention are regulatory T cells with strong immunosuppressive activity. In addition, the production method of the present invention is an expanded culture that is superior to the conventional method using only anti-CD3 antibody and anti-CD28 antibody in that the proliferation of regulatory T cells is better.

  In the cell population obtained by the method for producing a cell population containing regulatory T cells of the present invention, cells other than regulatory T cells are usually also present. The production method of the present invention may further include a step of separating regulatory T cells from the obtained cell population. That is, in the present invention, a cell population enriched with regulatory T cells is prepared by separating cells other than regulatory T cells from regulatory T cells in a cell population containing regulatory T cells. And can be used. Isolation of regulatory T cells can be performed according to a known method. For example, using a flow cytometer, select CD4-positive, CD25-positive, CD62L-positive, and CCR7-positive cells that are differentiated by co-staining with fluorescently labeled anti-CD4 antibody, anti-CD25 antibody, anti-CD62L antibody, and anti-CCR7 antibody. The regulatory T cells having a high immunosuppressive activity can be isolated by recovering the target. Further, by removing cells other than regulatory T cells from the cell population obtained by the production method of the present invention, a cell population containing a high amount of regulatory T cells can be obtained. In addition, the cell population containing a high amount of regulatory T cells in the present invention may include a cell population containing only regulatory T cells.

  According to the present invention, there is provided a method for producing a cell population containing regulatory T cells into which a desired gene has been introduced, utilizing the above-described “method for producing a cell population containing regulatory T cells”. . The method includes a gene introduction step in addition to the first step and the second step described above.

  The step of gene introduction for introducing a desired gene into a cell population containing regulatory T cells can be performed after completion of the first step, simultaneously with the second step, or after completion of the second step. By combining the first and second steps and the gene introduction step described above, the efficiency of gene introduction into a cell population containing regulatory T cells is improved. That is, the cell population produced by the method of the present invention is a cell population containing a high percentage of regulatory T cells into which a desired gene has been introduced. The present invention is particularly useful for the production of cells for gene therapy.

  In the present invention, the desired gene to be introduced into the cell population containing regulatory T cells is not particularly limited, and any gene desired to be introduced into the cell can be selected from a self-derived gene or a foreign gene. . As such a gene, for example, a gene encoding an antisense nucleic acid, siRNA (small interfering RNA) or ribozyme can be used in addition to a gene encoding a protein (for example, an enzyme, cytokines, receptors, etc.). In addition, an appropriate marker gene that enables selection of the gene-introduced cell and a suicide gene for selectively removing the gene-introduced cell may be introduced together with the gene. That is, the number of desired genes to be introduced is not limited, and a plurality of genes can be introduced.

  The desired gene to be introduced may have been obtained from nature, or may have been genetically engineered, or may be a DNA molecule of different origin bound by a known means such as ligation. Also good. Furthermore, it may have a sequence in which a mutation is introduced into a natural sequence depending on the purpose.

  Although not particularly limited, one embodiment of the present invention includes introduction of a gene encoding a receptor that recognizes a desired antigen. The gene includes a gene encoding a T cell receptor (TCR) that recognizes the surface antigen of the target cell, or an antigen recognition site of an antibody against the surface antigen of the target cell, a transmembrane domain and an intracellular domain derived from the receptor molecule. Examples include genes encoding chimeric receptors. The cell population containing regulatory T cells into which this gene has been introduced is a cell population containing regulatory T cells that recognize a desired antigen. The cell population is a cell population having a high specificity for a desired antigen as compared to a cell population into which a gene encoding a receptor has not been introduced. It is useful for use in immunotherapy because it can react specifically with cells presenting the.

  The desired gene can be used by being inserted into a vector or a plasmid so as to be expressed under the control of an appropriate promoter, for example. In addition, in order to achieve efficient gene transcription, promoters or other regulatory elements that cooperate with the transcription initiation site, such as enhancer sequences or terminator sequences, may be present in the vector. In addition, for the purpose of inserting into the chromosome of the regulatory T cell to be introduced by homologous recombination, for example, from the base sequence having homology to the base sequences on both sides of the desired target insertion site of the gene in the chromosome The gene may be placed between flanking sequences.

  In the present invention, the means for introducing a desired gene in the gene introduction step is not particularly limited, and an appropriate one can be selected and used by a known gene introduction method. As the gene introduction method, any of a method using a viral vector and a method not using the vector can be used in the present invention. Much literature has already been published about the details of these methods.

  The viral vector is not particularly limited, and is a known viral vector commonly used in gene transfer methods, such as retroviral vectors (including lentiviral vectors and pseudotype vectors), adenoviral vectors, adeno-associated viral vectors, and simian. Viral vectors, vaccinia virus vectors, Sendai virus vectors and the like can be used. Particularly preferably, retroviral vectors, adenoviral vectors or lentiviral vectors are used. As the above-mentioned virus vector, those lacking replication ability so that they cannot self-replicate in infected cells are suitable.

  Although the present invention is not limited as a gene transfer method without using a viral vector, for example, a method using a carrier such as a liposome or a ligand-polylysine, a calcium phosphate method, an electroporation method, a particle gun method or the like is used. be able to. In this case, a foreign gene incorporated into plasmid DNA, linear DNA or RNA is introduced.

  Retroviral vectors and lentiviral vectors can stably incorporate a foreign gene inserted into the chromosomal DNA of a cell into which the vector is introduced, and are used for gene therapy and other purposes. Since the vector can infect cells that are dividing and proliferating, it is particularly suitable for gene transfer by the production method of the present invention. Many retrovirus vectors and lentivirus vectors have been published in the literature and are already commercially available. These known vectors can be used in the present invention.

  Moreover, these vectors can be prepared as virus particles in which the vector is packaged by using a known packaging cell line. In addition, retrovirus-producing cells can be prepared using 293 cells or 293T cells with high transfection efficiency.

  As described above, retroviruses and lentiviruses produced by pseudotype packaging having an envelope derived from a virus different from that from which the virus genome is derived can also be used in the present invention. For example, pseudotyped retroviruses with envelopes derived from Moloney murine leukemia virus (MoMLV), gibbon leukemia virus (GaLV), vesicular stomatitis virus (VSV) or feline endogenous virus can be used. Furthermore, retroviruses and lentiviruses having a sugar chain modified envelope or other membrane protein on their surface can also be used in the present invention.

  When gene transfer is performed using a retrovirus vector, the gene transfer efficiency can be improved by using a functional substance having a retrovirus binding activity. Furthermore, a target cell-specific gene transfer can be carried out in combination with a functional substance having target cell binding activity (for example, International Publication No. 95/26200, International Publication No. 97/18318, International Publication No. (See Publication No. 00/01836 pamphlet).

  As described above, gene transfer into a cell population containing regulatory T cells can be achieved by the method of the present invention in which gene transfer is performed after the end of the first step, simultaneously with the second step, or after the end of the second step. It becomes possible to carry out efficiently. In addition, the method of the present invention does not require any special equipment or apparatus, and is effective for various retroviral vectors and target cells. Furthermore, since the method is suitable for use in a closed system, it is very useful in clinical applications such as gene therapy.

  In the method for producing a cell population containing regulatory T cells into which the gene of the present invention has been introduced, the step of culturing the gene-transferred cells obtained in the gene transfer step when the gene transfer step is performed after the end of the second step. Further, it may be included. The culture step can be performed in the same manner as the second step.

  The total number of culture days in the method for producing a cell population containing regulatory T cells into which the gene of the present invention has been introduced may be the same as that in which no gene introduction is performed.

2. Medicament comprising a cell population containing regulatory T cells produced by the method of the present invention The cell population produced by the method of the present invention has a strong immunosuppressive activity, so an immune reaction in vivo due to some cause In the patient's body, such as by administering the cell population to the patient when an undesired immune response is occurring abnormally or an undesirable immune response is occurring or when an undesirable immune response is predicted in the future. Abnormally enhanced undesirable immune reactions can be suppressed or avoided.

  The present invention provides an immunosuppressive agent comprising a cell population that can be produced by the above method. The immunosuppressive agent of the present invention includes rejection in organ transplantation, autoimmune disease, allergic disease (hay fever, food allergy, drug allergy, asthma, atopic dermatitis, eczema, food sensitivity, hives, allergic rhinitis, Allergic conjunctivitis) and graft-versus-host disease (GVHD) are useful for prevention and treatment.

  The immunosuppressive agent of the present invention can be produced as an oral / parenteral preparation by mixing a cell population containing an effective amount of the regulatory T cells with a pharmaceutically acceptable carrier according to conventional means. . The immunosuppressive agent of the present invention is usually produced as a parenteral preparation such as an injection, a suspension, an infusion. Examples of carriers that can be included in the parenteral preparation include aqueous solutions for injection such as physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). A liquid can be mentioned. The immunosuppressant of the present invention includes, for example, a buffer (for example, phosphate buffer, sodium acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine, etc.), a stabilizer (for example, human serum). Albumin, polyethylene glycol, etc.), preservatives, antioxidants, immunosuppressants (such as rapamycin), antibody preparations, cell preparations (regulatory dendritic cells) and the like.

  Since the preparation thus obtained is safe and has low toxicity, it can be administered to mammals such as humans.

The dose of the cell population containing the regulatory T cells of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like, but usually in an adult patient (with a body weight of 60 kg), for example, parenterally In the case of administration, it is convenient to administer an effective amount up to about 2.6 × 10 ⁹ cells per day. In the case of other animals, an amount converted per 60 kg can be administered.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.

Example 1 Regulatory T Cell Culture Method Using Cell Culture Plate (1) Preparation of Fibronectin Fragment / Anti-CD3 Antibody Immobilized Plate and Anti-CD3 Antibody Immobilized Plate Fibronectin Fragment [RetroNectin (registered trademark), Takara Biotechnology] was dissolved in PBS to a concentration of 25 μg / mL, and anti-CD3 antibody (OKT3, manufactured by Janssen Pharma) to a concentration of 5 μg / mL, and the amount was 0.1 mL / well on a 96-well flat plate for cell culture. In addition, it was left at 37 ° C. for 3.5 hours. As a control, only the anti-CD3 antibody was dissolved in PBS so as to be 5 μg / mL, added to a 96-well flat plate for cell culture at 0.1 mL / well, and allowed to stand at 37 ° C. for 3.5 hours. After standing, each lysate was removed and each plate was washed twice with PBS at a volume of 0.2 mL / well to prepare fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate. .

(2) Culture of regulatory T cells CD4 ⁺ CD25 ⁺ Т Cell Kit (manufactured by Invitrogen) from human peripheral blood mononuclear cells (PBMC) prepared from healthy individuals from whom informed consent was obtained. A cell population rich in positive and CD25 positive regulatory T cells was obtained. These cells were mixed with 1.5% autologous plasma, 600 IU / mL IL-2, 0.2% human serum albumin (HSA), 2.5 μg / mL fungizone (Bristol-Myers) to 4 × 10 ⁵ cells / mL. The product was suspended in a GT-T503 medium (manufactured by Takara Bio Inc.) (this is used as a culture medium). Furthermore, a group in which an anti-CD28 antibody (CD28.2, manufactured by Biolegend) is added to the cell suspension to a final concentration of 1 μg / mL and a group in which the anti-CD28 antibody is not added are prepared, and a fibronectin fragment / anti-CD3 antibody solid phase is prepared. The plate was added to an immobilized plate or an anti-CD3 antibody-immobilized plate at 0.2 mL / well, and culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ). On the second day of culture, 0.1 mL of the above culture medium was added. Cells were collected on the 4th day of culture, 0.6 mL of culture medium was added, and the whole amount was re-wound into a 48-well plate for cell culture. On the fifth day of culture, 2.5 mL of the culture medium was added, and the entire amount was re-wound into a 12-well plate for cell culture. Cells were collected on the seventh day of culture, and the cell growth rate and Foxp3-positive cell rate were measured.

  The cell growth rate is shown in FIG. In FIG. 1, the vertical axis represents the cell growth rate (hereinafter referred to as “fold expansion” in each figure). In each figure of the Examples, “CD3” indicates a cell cultured on an anti-CD3 antibody-immobilized plate, and “RNCD3” indicates a cell cultured on a fibronectin fragment / anti-CD3 antibody-immobilized plate. “CD28” represents a group to which an anti-CD28 antibody was added, and “−” represents a group to which no anti-CD28 antibody was added. As shown in FIG. 1, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed higher cell growth rate than the cells cultured on the anti-CD3 antibody-immobilized plate. Even in the presence of the anti-CD28 antibody, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed higher cell growth rate than the cells cultured on the anti-CD3 antibody-immobilized plate.

  The ratio of cultured cells expressing Foxp3, which is a master regulator of regulatory T cells, is shown in FIG. FIG. 2 shows that PE-Cy7-labeled anti-human CD4 antibody (Becton Dickinson) and FITC-labeled anti-human CD25 antibody (Becton Dickinson) were added to the cultured cells, reacted at 4 ° C. for 20 minutes, stained, and then treated with PE anti. -The result of having measured FoxP3 positive cell rate by MACSQuant (made by Miltenyi Biotech) using human FoxP3 Staining Set (made by eBioscience) is shown. In FIG. 2, the vertical axis represents the ratio of CD4 positive, CD25 positive and Foxp3 positive cells. FIG. 2 shows that the proportion of CD4-positive, CD25-positive, and Foxp3-positive cells in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared to the cell population cultured on the anti-CD3 antibody-immobilized plate. Was expensive.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 3, the vertical axis represents the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 1 and the positive rate in FIG. From FIG. 3, the number of CD4-positive, CD25-positive, and Foxp3-positive cells increased in the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared to the condition cultured on the anti-CD3 antibody-immobilized plate. Similarly, in the presence of anti-CD28 antibody, conditions cultured on fibronectin fragment / anti-CD3 antibody-immobilized plate were more CD4-positive, CD25-positive, and Foxp3-positive than those cultured on anti-CD3 antibody-immobilized plate. The number of cells increased significantly.

Example 2 Regulatory T Cell Culture Method Using Surface Untreated Plate (1) Preparation of Fibronectin Fragment / Anti-CD3 Antibody Immobilized Plate and Anti-CD3 Antibody Immobilized Plate As in Example 1- (1) Then, an anti-CD3 antibody solution and a mixed solution of anti-CD3 antibody and retronectin were prepared, added to a surface-untreated 96-well flat plate at 0.1 mL / well, and left at 37 ° C. for 4 hours. After standing, each lysate is removed, and each plate is washed twice with PBS at a volume of 0.2 mL / well to prepare fibronectin fragment / anti-CD3 antibody immobilized plate and anti-CD3 antibody immobilized plate. did.

(2) Culture of regulatory T cells In the same manner as in Example 1- (2), a cell population containing many CD4-positive and CD25-positive cells was obtained. These cells were mixed with GT-T503 medium containing 5% autologous plasma, 600 IU / mL IL-2, 0.2% HSA, 2.5 μg / mL fungizone (hereinafter referred to as 3.2 × 10 ⁵ cells / mL). This was suspended in a culture medium). Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the second day of culture, 0.1 mL of the above culture medium was added. On the fourth day of culture, the cells were collected, 0.2 mL of culture medium was added, and the whole amount was re-wound in a 48-well plate for cell culture. On the 7th day of culture, the cells were collected, suspended in a culture medium so as to be 4.0 × 10 ⁵ cells / mL, and re-spread so as to be 1 mL / well in a 24-well plate for cell culture. On the ninth day of culture, 1 mL of culture medium was added and re-wound. On the 11th day from the start of the culture, the cells were collected, and the cell growth rate and Foxp3-positive cell rate were measured.

  FIG. 4 shows changes with time in the cell growth rate. In FIG. 4, the horizontal axis represents the number of days from the start of culture, and the vertical axis represents the cell growth rate. In each figure of the Examples, “CD3 + CD28” is a cell cultured on an anti-CD3 antibody-immobilized surface-untreated plate in the presence of anti-CD28 antibody, and “RNCD3 + CD28” is a fibronectin fragment / anti-CD3 antibody solid phase in the presence of anti-CD28 antibody. The cells cultured on the non-treated surface untreated plate are shown. From FIG. 4, even when immobilized on a surface untreated plate, the conditions cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate are higher than those cultured on the anti-CD3 antibody immobilized plate. The cell growth rate was shown.

  The ratio of cultured cells expressing Foxp3 was measured in the same manner as in Example 1- (2). The result is shown in FIG. In FIG. 5, the vertical axis represents the ratio of CD4 positive, CD25 positive and Foxp3 positive cells. From FIG. 5, even when cultured on the surface untreated plate, the cell population cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate is more in comparison with the cell population cultured on the anti-CD3 antibody immobilized plate. The proportion of CD4 positive, CD25 positive and Foxp3 positive cells was high.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 6, the vertical axis represents the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 4 and the positive rate in FIG. FIG. 6 shows that even when cultured on a surface-untreated plate, cells cultured on a fibronectin fragment / anti-CD3 antibody-immobilized plate are more CD4-positive and CD25 than those cultured on an anti-CD3 antibody-immobilized plate. The number of positive and Foxp3-positive cells was significantly increased.

Example 3 Measurement of growth inhibitory activity of cultured cells on CD4-positive cells (1) Preparation of fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate According to the procedure described in Example 2- (1), Fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate were prepared using a surface-untreated 96-well flat plate.

(2) Culture of regulatory T cells In the same manner as in Example 1- (2), a cell population containing many CD4-positive and CD25-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 6 × 10 ⁵ cells / mL. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the third day of culture, 0.1 mL of the above culture medium was added. On the fourth day of culture, the cells were collected, suspended in a culture medium so as to be 4 × 10 ⁵ cells / mL, and re-spread so as to be 0.5 mL / well in a 48-well plate for cell culture. On the fifth day of culture, the cells were collected, 2.5 mL of culture medium was added, and the whole amount was re-wound in a 12-well plate for cell culture. On the 7th day of culture, the cells were collected, suspended in a culture medium so as to be 4.32 × 10 ⁵ cells / mL, and re-spread so as to be 5.0 mL / well in a 12-well plate for cell culture. On the 10th day from the start of the culture, the cells were collected, and the cell growth rate, Foxp3-positive cell rate, and proliferation inhibitory activity against CD4-positive cells were measured.

  FIG. 7 shows changes with time in the cell growth rate. In FIG. 7, the horizontal axis indicates the number of days from the start of culture, and the vertical axis indicates the cell growth rate. As shown in FIG. 7, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed higher cell growth rate than the cells cultured on the anti-CD3 antibody-immobilized plate.

  The ratio of cultured cells expressing Foxp3 was measured in the same manner as in Example 1- (2). The result is shown in FIG. In FIG. 8, the vertical axis represents the ratio of CD4 positive, CD25 positive and Foxp3 positive cells. FIG. 8 shows that the proportion of CD4-positive, CD25-positive, and Foxp3-positive cells in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate is higher than that on the cell population cultured on the anti-CD3 antibody-immobilized plate. Was expensive.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 9, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 7 and the positive rate in FIG. FIG. 9 shows that the number of CD4-positive, CD25-positive, and Foxp3-positive cells is significantly higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate than on the condition cultured on the anti-CD3 antibody immobilized plate. Increased.

(3) Measurement of growth inhibitory activity against CD4 positive cells CD4 positive cells and CD4 negative cells were obtained from PBMC of the same donor as the cells used in this Example (2) using CD4 microbeads (Miltenyi Biotech). It was. After irradiating 30 Gy X-rays to CD4 negative cells, GT-T503 medium (hereinafter referred to as this) containing 5% autologous plasma, 0.2% HSA, 2.5 μg / mL fungizone to 1 × 10 ⁶ cells / mL. Was used as a medium for measuring inhibitory activity). CD4 positive cells are suspended in a suppressive activity measurement medium containing CFSE (manufactured by Sigma Aldrich) having a final concentration of 2 μM, treated with a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 8 minutes, and the cells are cultured in the suppressive activity measurement medium After washing 4 times, the suspension was suspended in a medium for measuring inhibitory activity so as to be 1 × 10 ⁶ cells / mL. This is the responder cell. X4-irradiated CD4 negative cells and CFSE-treated responder cells were mixed 1: 1 (hereinafter referred to as Tres), and anti-CD3 antibody (OKT3) was added to a final concentration of 200 ng / mL. 100 μL of this mixed cell solution was added to a 98-well round-bottom plate for cell culture, and the cultured cells obtained in this Example (2) were prepared to 5 × 10 ⁵ cells / mL with a medium for measuring inhibitory activity. 100 μL was added and cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 3 days. Thereafter, the cells were collected and the CFSE fluorescence intensity of the responder cells was measured with MACSQuant. The growth inhibitory activity against CD4 positive cells is shown in FIG. In FIG. 10, the vertical axis indicates the ratio of CFSE weakly positive cells in all CFSE positive cells, that is, the proportion of divided cells in responder cells. As a positive control, a sample obtained by culturing only Tres to which 100 μL of a suppressive activity measuring medium was added without adding cultured cells (“Positive” in the figure), and as a negative control, a sample culturing only responder cells treated with CFSE ( “Negative” in the figure was produced. As a result, the cell proliferation rate of the responder cells was suppressed to 5% or less in the samples to which the cultured cells were added (“CD3 + CD28”, “RNCD3 + CD28”), compared to the “Positive” sample containing only Tres. Therefore, it was clarified that the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate are a cell population that suppresses the cell proliferation of the responder cells, similar to the cells cultured on the anti-CD3 antibody-immobilized plate. It was.

Example 4 Measurement of growth inhibitory activity of cultured cells on CD4-positive cells (1) Preparation of fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate According to the procedure described in Example 2- (1), Fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate were prepared using a surface-untreated 96-well flat plate.

(2) Culture of regulatory T cells In the same manner as in Example 1- (2), a cell population containing many CD4-positive and CD25-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 4.05 × 10 ⁵ cells / mL. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the first day of culture, 0.05 mL of the above culture medium was added. On the 4th day of culture, the cells were collected, suspended in a culture medium so as to be 2 × 10 ⁵ cells / mL, and re-spread so as to be 1.0 mL / well in a 48-well plate for cell culture. On the 6th day of culture, the cells were collected, suspended in a culture medium so as to be 4 × 10 ⁵ cells / mL, and re-spread so as to be 2.0 mL / well in a 12-well plate for cell culture. On the 8th day of culture, cells were collected, 1 mL of culture medium was added, and the whole amount was re-wound in a 12-well plate for cell culture. On the 11th day from the start of the culture, the cells were collected, and the cell growth rate, Foxp3-positive cell rate, CD62L-positive and CCR7-positive cell rate, and growth inhibitory activity against CD4-positive cells were measured.

  FIG. 11 shows changes with time in the cell growth rate. In FIG. 11, the horizontal axis indicates the number of days from the start of culture, and the vertical axis indicates the cell growth rate. From FIG. 11, the cell growth rate was higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate than on the condition cultured on the anti-CD3 antibody immobilized plate.

  The ratio of the cultured cells expressing Foxp3 is shown in FIG. FIG. 12 shows that APC-Cy7-labeled anti-human CD4 antibody (Becton Dickinson) was added to cultured cells, reacted at 4 ° C. for 20 minutes, stained, and then subjected to PE anti-human FoxP3 Staining Set to determine the FoxP3 positive cell rate. The result measured by FACSCanto (made by Becton Dickinson) is shown. In FIG. 12, the vertical axis represents the ratio of CD4 positive and Foxp3 positive cells. From FIG. 12, the proportion of CD4-positive and Foxp3-positive cells was higher in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate than on the cell population cultured on the anti-CD3 antibody-immobilized plate. .

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 13, the vertical axis represents the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 11 and the positive rate in FIG. From FIG. 13, the number of CD4-positive and Foxp3-positive cells was significantly increased in the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared with the condition cultured on the anti-CD3 antibody-immobilized plate.

(3) Measurement of CD62L-positive and CCR7-positive cell ratio The ratio of cultured cells expressing CD62L and CCR7 is shown in FIG. FIG. 14 shows APC-Cy7-labeled anti-human CD4 antibody, PE-Cy7-labeled anti-human CD62L antibody (Becton Dickinson), PE-labeled anti-human CD25 antibody (Becton Dickinson) and FITC-labeled anti-human CCR7 antibody. (R & D Systems Co., Ltd.) is added and reacted at 4 ° C. for 20 minutes. After staining, the results measured with FACSCanto are shown. In FIG. 14, the vertical axis represents the ratio of CD4 positive, CD25 positive, CD62L positive, and CCR7 positive cells. FIG. 14 shows that the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared with the cell population cultured on the anti-CD3 antibody-immobilized plate was CD4 positive, CD25 positive, CD62L positive, and CCR7 positive cells. The rate was high.

  The absolute number of cultured cells expressing CD62L and CCR7 is shown in FIG. In FIG. 15, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 11 and the positive rate in FIG. 14. FIG. 15 shows that the number of CD4-positive, CD25-positive, CD62L-positive, and CCR7-positive cells is higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate than on the condition cultured on the anti-CD3 antibody-immobilized plate. Increased significantly.

(4) Measurement of growth inhibitory activity against CD4 positive cells
In the same manner as in Example 3- (3), the growth inhibitory activity of the cells obtained in this Example (2) on the responder cells was measured with FACSCanto. The growth inhibitory activity against CD4 positive cells is shown in FIG. In FIG. 16, the vertical axis indicates the ratio of CFSE weakly positive cells among all CFSE positive cells, that is, the proportion of divided cells in responder cells, and the horizontal axis indicates the mixing ratio of responder cells and cultured cells. From FIG. 16, in the sample added with the cultured cells (“CD3 + CD28” or “RNCD3 + CD28”), the cell proliferation of the responder cells increases as the mixed ratio of the cultured cells increases compared to the sample “Tres” that does not include the cultured cells. Suppressed. Therefore, it was clarified that the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate are a cell population that suppresses the cell proliferation of the responder cells, similar to the cells cultured on the anti-CD3 antibody-immobilized plate. It was.

Example 5 Measurement of growth inhibitory activity of cultured cells on CD8 positive cells (1) Preparation of fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate According to the procedure described in Example 2- (1), Fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate were prepared using a surface-untreated 96-well flat plate.

(2) Culture of regulatory T cells Using CD4 ⁺ CD25 ⁺ CD45RA ⁺ Regulatory T Cell Isolation Kit from PBMCs prepared from healthy individuals with informed consent, CD4 positive, CD25 positive and CD45RA positive cells are included A cell population was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 1.8 × 10 ⁵ cells / mL. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the first day of culture, 0.1 mL of the above culture medium was added. On the fourth day of culture, 0.05 mL of culture medium was added. On the fifth day of culture, the cells were collected, suspended in a culture medium so as to be 3 × 10 ⁵ cells / mL, and re-spread so as to be 0.5 mL / well in a 48-well plate for cell culture. On the 6th day of culture, 0.5 mL of the culture medium was added, and on the 7th day of culture, 0.5 mL of the culture medium was added, and the entire amount was re-wound into a 24-well plate for cell culture. On the 8th day of culture, the cells were collected, suspended in a culture medium so as to be 8 × 10 ⁵ cells / mL, and re-spread so as to be 1.0 mL / well in a 24-well plate for cell culture. The cells were collected on the 11th day from the start of the culture, and the cell growth rate, Foxp3-positive cell rate, CD62L-positive and CCR7-positive cell rate, and growth inhibitory activity against CD8-positive cells were measured.

  FIG. 17 shows changes with time in the cell growth rate. In FIG. 17, the horizontal axis indicates the number of days from the start of culture, and the vertical axis indicates the cell growth rate. From FIG. 17, the cell growth rate was higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate than on the condition cultured on the anti-CD3 antibody immobilized plate.

  The ratio of the cultured cells expressing Foxp3 is shown in FIG. FIG. 18 shows APC-Cy7-labeled anti-human CD4 antibody, APC-labeled anti-human CCR7 antibody (manufactured by R & D Systems), PE-Cy7-labeled anti-human CD62L antibody and FITC-labeled anti-human CD25 antibody (Becton Dickinson). And the reaction was carried out at 4 ° C. for 20 minutes, followed by staining, and the rate of Foxp3-positive cells was measured with FACSCanto using PE anti-human FoxP3 Staining Set. In FIG. 18, the vertical axis represents the ratio of CD4 positive, CD25 positive, and Foxp3 positive cells. FIG. 18 shows that the proportion of CD4-positive, CD25-positive, and Foxp3-positive cells in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate is higher than that on the cell population cultured on the anti-CD3 antibody-immobilized plate. Was high.

  In FIG. 19, the vertical axis represents the ratio of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive and CCR7 positive cells. FIG. 19 shows that the cell population cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate is more CD4 positive, CD25 positive, Foxp3 positive and CD62L positive than the cell population cultured on the anti-CD3 antibody immobilized plate. And the ratio of CCR7 positive cells was high.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 20, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 17 and the positive rate in FIG. FIG. 20 shows that the number of CD4-positive, CD25-positive, and Foxp3-positive cells is significantly higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate than on the condition cultured on the anti-CD3 antibody-immobilized plate. Increased.

  The absolute number of cultured cells expressing CD62L and CCR7 is shown in FIG. In FIG. 21, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 17 and the positive rate in FIG. FIG. 21 shows that the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate were more CD4-positive, CD25-positive, Foxp3-positive, CD62L-positive, and CCR7 than the conditions cultured on the anti-CD3 antibody-immobilized plate. There was a marked increase in the number of positive cells.

(3) Measurement of growth inhibitory activity against CD8 positive cells CD8 positive cells and CD8 negative cells were obtained from PBMC of the same donor as the cells used in this Example (2) using CD8 microbeads (Miltenyi Biotech). It was. CD8 negative cells and CD8 positive cells were subjected to the same operations as the CD4 negative cells and CD4 positive cells of Example 3- (3), respectively, to obtain a mixed cell solution containing CD8 positive responder cells. The growth inhibitory activity of the cultured cells obtained in this Example (2) against responder cells was measured with FACSCanto. The growth inhibitory activity against CD8 positive cells is shown in FIG. In FIG. 22, the vertical axis indicates the ratio of CFSE weakly positive cells among all CFSE positive cells, that is, the ratio of divided cells in the responder cells, and the horizontal axis indicates the mixing ratio of the responder cells and the cultured cells. As a result, in the samples to which the cultured cells were added (“CD3 + CD28”, “RNCD3 + CD28”), the cell proliferation of the responder cells was suppressed as the mixed ratio of the cultured cells increased. Therefore, it was clarified that the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate are a cell population that suppresses the cell proliferation of the responder cells, similar to the cells cultured on the anti-CD3 antibody-immobilized plate. It was.

Example 6 Measurement of DNA methylation degree in TSDR of cultured cells (1) Preparation of fibronectin fragment / anti-CD3 antibody immobilized plate and anti-CD3 antibody immobilized plate As in Example 1- (1), anti-CD3 An antibody solution and a mixed solution of anti-CD3 antibody and retronectin were prepared, added to a surface-untreated 96-well flat plate in an amount of 0.1 mL / well, and left overnight at 4 ° C. Then, after each plate was left at 37 ° C. for 4 hours, the lysate was removed, each plate was washed twice with PBS at a volume of 0.2 mL / well, and a fibronectin fragment / anti-CD3 antibody-immobilized plate and An anti-CD3 antibody-immobilized plate was prepared.

(2) Culture of regulatory T cells In the same manner as in Example 5- (2), a cell population containing many CD4-positive, CD25-positive, and CD45RA-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 1.0 × 10 ⁵ cells / mL. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the first day of culture, 0.05 mL of the above culture medium was added. On the fourth day of culture, 0.05 mL of the medium was removed, and 0.1 mL of the culture medium was added. On the fifth day of culture, the cells were collected, suspended in a culture medium so as to be 3.5 × 10 ⁵ cells / mL, and re-spread so as to be 1.0 mL / well in a 24-well plate for cell culture. On the seventh day of culture, 0.4 mL of the medium was removed, and 0.4 mL of the above culture medium was added. On the 8th day from the start of the culture, the cells were collected and the cell growth rate was measured. On the 11th day from the start of the culture, the cells were collected, and the rate of Foxp3 positive cells, the rate of CCR7 and CD27 positive cells, and the frequency of DNA methylation of TSDR were measured.

  FIG. 23 shows changes with time in the cell growth rate. In FIG. 23, the horizontal axis indicates the number of days from the start of culture, and the vertical axis indicates the cell growth rate. As shown in FIG. 23, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed higher cell growth rate than the cells cultured on the anti-CD3 antibody-immobilized plate.

  The ratio of the cultured cells expressing Foxp3 is shown in FIG. FIG. 24 shows APC-Cy7-labeled anti-human CD4 antibody, PE-Cy7-labeled anti-human CD27 antibody (manufactured by Becton Dickinson) and FITC-labeled anti-human CCR7 antibody added to the cultured cells, reacted at 4 ° C. for 20 minutes, and stained. The rate of FoxP3-positive cells was measured with FACSCanto using PE anti-human FoxP3 Staining Set. In FIG. 24, the vertical axis represents the ratio of CD4 positive and Foxp3 positive cells. From FIG. 24, the proportion of CD4-positive and Foxp3-positive cells was higher in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate than on the cell population cultured on the anti-CD3 antibody-immobilized plate. .

  In FIG. 25, the vertical axis represents the ratio of CD4 positive, Foxp3 positive and CCR7 positive cells. FIG. 25 shows that the proportion of CD4-positive and Foxp3-positive or CCR7-positive cells in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared to the cell population cultured on the anti-CD3 antibody-immobilized plate. Was high.

  In FIG. 26, the vertical axis represents the ratio of CD4 positive, Foxp3 positive and CD27 positive cells. FIG. 26 shows that the proportion of CD4-positive and Foxp3-positive or CD27-positive cells in the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared to the cell population cultured on the anti-CD3 antibody-immobilized plate. Was high.

  The absolute number of cultured cells that express Foxp3 is shown in FIG. In FIG. 27, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 23 and the positive rate in FIG. From FIG. 27, the number of CD4-positive and Foxp3-positive cells was significantly increased in the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared to the condition cultured on the anti-CD3 antibody-immobilized plate.

  The absolute number of cultured cells expressing CCR7 is shown in FIG. In FIG. 28, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 23 and the positive rate in FIG. FIG. 28 shows that the number of CD4-positive, Foxp3-positive, and CCR7-positive cells is significantly higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate than on the condition cultured on the anti-CD3 antibody-immobilized plate. Increased.

  The absolute number of cultured cells expressing CD27 is shown in FIG. In FIG. 29, the vertical axis indicates the value obtained by multiplying the number of cells at the start of the culture by the cell growth rate in FIG. 23 and the positive rate in FIG. FIG. 29 shows that the number of CD4-positive, Foxp3-positive, and CD27-positive cells is significantly higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate than on the condition cultured on the anti-CD3 antibody immobilized plate. Increased.

(3) Measurement of DNA methylation degree of TSDR Using NucleoSpin Tissue (manufactured by Takara Bio Inc.) from cells obtained from this Example (2) and cells cultured as PBC on an anti-CD3 antibody-immobilized plate as a control. DNA was extracted to obtain 5.1-5.6 μg of DNA from 1 × 10 ⁶ cultured cells. Next, by using Methyl Easy Xceed Rapid DNA Bisulfite Modification Kit (manufactured by Takara Bio Inc.), 2.0-2.2 μg of the DNA was bisulfite treated, and 0.5-1.0 μg of DNA after bisulfite treatment Got. Next, 20 ng of the DNA was used, and bisulfite-PCR was performed using TaKaRa EpiTaq HS (for bisulfite-treated DNA) (manufactured by Takara Bio Inc.). As the primer, 5′-TGTTTGGGGGTAGAGGATTTT-3 ′ on the forward side and 5′-TATCACCCCACCTAAAACCAA-3 ′ on the reverse side were used. PCR reaction solution was 50 μL [10X EpiTaq PCR Buffer (Mg2 + free) 5 μL, dNTP Mixture (2.5 mM each) ₆ μL, 25 mM MgCl ₂ 5 μL, TaKaRa EpiTaq HS (5 U / mL) 1 μL each 10 μL, each primer 10 μL The PCR reaction conditions were (98 ° C. 10 seconds) − (55 ° C. 30 seconds) − (72 ° C. 30 seconds) (40 cycles), and finally (72 ° C. 7 minutes), and TaKaRa PCR Thermal Cycler Dice (Takara) PCR reaction was carried out using Biotech). FIG. 30 shows the results of agarose electrophoresis using 2 μL of the reaction solution after the reaction. 30 to 32, “Tconv” indicates a cell obtained by culturing PBMC on an anti-CD3 antibody-immobilized plate. From FIG. 30, the single amplification and size (336 bp) of the PCR product of each sample were confirmed. Next, 2 μL of the PCR product was used, and TA cloning was performed using T-vector pMD20 (manufactured by Takara Bio Inc.) and DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio Inc.). Thereafter, 5 μL of the above ligation solution was used. E. coli HST08 Premium Competent Cells (manufactured by Takara Bio Inc.) were reacted at 42 ° C. for 45 seconds for transformation. After culturing at 37 ° C. for 1 hour in an SOC medium, the LB medium plate containing ampicillin coated with X-gal (manufactured by Takara Bio Inc.) was inoculated and cultured at 37 ° C. overnight. Thereafter, 24 clones of white colonies were picked up and cultured overnight in LB medium at 37 ° C., and glycerol was added to a final concentration of 17% to prepare a glycerol stock. For the sequence reaction, base sequence information was obtained by entrusting plate unit base sequence analysis (Takara Bio Inc.) using M13-47 primer.

  The obtained base sequence was analyzed using QUMA (RIKEN) software. As exclusion conditions, the lower limit of bisulfite conversion efficiency was 95%, and the upper limit of the number of alignment mismatches was 15. The frequency of DNA methylation of TSDR is shown in FIG. In FIG. 31, the horizontal axis represents the DNA methylation site of TSDR, and the vertical axis represents the sample name. From FIG. 31, the frequency of methylation at any DNA methylation site was lower in cells in which regulatory T cells were isolated and cultured than in cells in which normal T cells were cultured.

  The result of DNA methylation of each clone is shown in FIG. In FIG. 32, the horizontal axis indicates the DNA methylation site, the vertical axis indicates each clone, the black circle indicates that the site is “methylated”, and the white circle indicates that the site is “demethylated”. It shows that. As shown in FIG. 32, almost all clones in which normal T cells were cultured were “methylated”, whereas in cells in which regulatory T cells were isolated and cultured, all DNA methylation sites were “de-methylated”. The majority of the clones were “methylated”. Therefore, the cells obtained by culturing the fibronectin fragment / anti-CD3 antibody-immobilized plate can stably express Foxp3 similarly to the cells obtained by culturing the anti-CD3 antibody-immobilized plate. It became clear that the cell population was rich in cells.

Example 7 Method of culturing regulatory T cells using immobilized plate of fibronectin fragment / anti-CD3 antibody / anti-CD28 antibody (1) Fibronectin fragment / anti-CD3 antibody immobilized plate and fibronectin fragment / anti-CD3 antibody / anti-antibody Preparation of CD28 antibody-immobilized plate In the same manner as in Example 1- (1), an anti-CD3 antibody solution and a mixed solution of anti-CD3 antibody and retronectin were prepared, and 0.1 mL / well was added to a surface-untreated 96-well flat plate. And left at 4 ° C. overnight. For the preparation of fibronectin fragment / anti-CD3 antibody / anti-CD28 antibody-immobilized plate, fibronectin fragment (retronectin) was 25 μg / mL or 5 μg / mL, and anti-CD3 antibody 5 μg / mL and anti-CD28 antibody (CD28 2) was dissolved in PBS to 5 μg / mL, added to a surface-untreated 96-well flat plate in an amount of 0.1 mL / well, and allowed to stand at 4 ° C. overnight. Then, after each plate was left at 37 ° C. for 4 hours, the lysate was removed, each plate was washed twice with PBS at a volume of 0.2 mL / well, and a fibronectin fragment / anti-CD3 antibody-immobilized plate and A fibronectin fragment / anti-CD3 antibody / anti-CD28 antibody-immobilized plate was prepared.

(2) Culture of regulatory T cells In the same manner as in Example 5- (2), a cell population containing many CD4-positive, CD25-positive, and CD45RA-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 0.85 × 10 ⁵ cells / mL. For those cultured on a fibronectin fragment / anti-CD3 antibody-immobilized plate, anti-CD28 antibody (CD28.2) was added to the cell suspension to a final concentration of 2 μg / mL. The cell suspension was added to each plate at 0.1 mL / well, and culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ). On the first day of culture, 0.1 mL of the above culture medium was added. On the fourth day of culture, 0.05 mL of the medium was removed, and 0.05 mL of the culture medium was added. On the fifth day of culture, cells were collected, 0.2 mL of culture medium was added, and the whole amount was re-wound in a 48-well plate for cell culture. On the sixth day of culture, 0.5 mL of the culture medium was added, and the entire amount was re-wound into a 24-well plate for cell culture. On the 8th day from the start of the culture, the cells were collected and the cell growth rate was measured. On the 11th day from the start of the culture, the cells were collected and the Foxp3-positive cell rate was measured.

  FIG. 33 shows changes with time in the cell growth rate. In FIG. 33, the horizontal axis represents the number of days from the start of culture, and the vertical axis represents the cell growth rate. In each of FIGS. 33 to 35, “RNhiCD3 + CD28” is a cell cultured on a plate on which 25 μg / mL of a fibronectin fragment and an anti-CD3 antibody are immobilized in the presence of an anti-CD28 antibody, and “RNloCD3 + CD28” is an anti-CD28 antibody. Below, cells cultured on a plate on which 5 μg / mL fibronectin fragment and anti-CD3 antibody were immobilized, “RNhiCD3CD28” was cultured on a plate on which 25 μg / mL fibronectin fragment, anti-CD3 antibody and anti-CD28 antibody were immobilized. “RNloCD3CD28” indicates a cell cultured on a plate on which 5 μg / mL fibronectin fragment, anti-CD3 antibody and anti-CD28 antibody are immobilized. From FIG. 33, the conditions under which the fibronectin fragment was cultured at a low concentration (5 μg / mL) showed a high cell growth rate, similar to the conditions cultured at a high concentration (25 μg / mL). Similarly, a high cell growth rate was exhibited even under conditions in which the anti-CD28 antibody was cultured on a solid-phased plate.

  The ratio of the cultured cells expressing Foxp3 is shown in FIG. In FIG. 34, APC-Cy7-labeled anti-human CD4 antibody was added to cultured cells, reacted at 4 ° C. for 20 minutes, stained, and then the rate of Foxp3-positive cells was measured with FACSCanto using PE anti-human FoxP3 Staining Set. In FIG. 34, the vertical axis represents the ratio of CD4 positive and Foxp3 positive cells. From FIG. 34, the ratio of CD4 positive and Foxp3 positive cells was high in the cell population cultured under any condition.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 35, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 33 and the positive rate in FIG. From FIG. 35, the number of CD4-positive and Foxp3-positive cells was remarkably increased under any condition.

Example 8 Regulatory T cell culture method by restimulation (1) Preparation of fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate According to the procedure described in Example 6- (1) A fibronectin fragment / anti-CD3 antibody-immobilized plate and an anti-CD3 antibody-immobilized plate were prepared using a treated 96-well flat plate.

(2) Culture of regulatory T cells In the same manner as in Example 5- (2), a cell population containing many CD4-positive, CD25-positive, and CD45RA-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 1.5 × 10 ⁵ cells / mL. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the first day of culture, 0.05 mL of the above culture medium was added. On the fourth day of culture, 0.05 mL of the medium was removed, and 0.05 mL of the culture medium was added. On the fifth day of culture, the cells were collected, suspended in the culture medium so as to be 4 × 10 ⁵ cells / mL, and re-spread so as to be 0.5 mL / well in a surface untreated 48-well plate. On the 6th day of culture, cells were collected, 0.5 mL of culture medium was added, and the whole amount was re-wound into a surface untreated 24-well plate. On the 8th day from the start of the culture, the cells were collected and the cell growth rate was measured. On the 11th day from the start of the culture, the cells were collected, and the Foxp3-positive cell rate, CD62L-positive and CCR7-positive cell rate were measured.

  FIG. 36 shows changes with time in the cell growth rate. In FIG. 36, the horizontal axis indicates the number of days from the start of culture, and the vertical axis indicates the cell growth rate. From FIG. 36, the cell culture magnification was higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate than on the condition cultured on the anti-CD3 antibody immobilized plate.

  The ratio of cultured cells expressing Foxp3 was measured in the same manner as in Example 5- (2). The result is shown in FIG. In FIG. 37, the vertical axis represents the ratio of CD4 positive and Foxp3 positive cells. From FIG. 37, compared to cells cultured on the anti-CD3 antibody-immobilized plate, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate have the same proportion of CD4 positive, CD25 positive and Foxp3 positive cells. It was a little expensive.

  In FIG. 38, the vertical axis represents the ratio of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive and CCR7 positive cells. FIG. 38 shows that the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate is more CD4 positive, CD25 positive, Foxp3 positive, CD62L positive, and CCR7 than the cell population cultured on the anti-CD3 antibody immobilized plate. The percentage of positive cells was similar or slightly higher.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 39, the vertical axis represents the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 36 and the positive rate in FIG. From FIG. 39, the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate has a remarkable number of CD4-positive, CD25-positive and Foxp3-positive cells compared to the cell population cultured on the anti-CD3 antibody-immobilized plate. Increased.

  The absolute number of cultured cells expressing CD62L and CCR7 is shown in FIG. In FIG. 40, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 36 and the positive rate in FIG. As shown in FIG. 40, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate were more CD4-positive, CD25-positive, Foxp3-positive, CD62L-positive, and CCR7-positive cells than the cells cultured on the anti-CD3 antibody-immobilized plate. The number increased significantly.

(3) Culture of regulatory T cells by restimulation culture The same composition as that described in Example 2- (2) so that the cells prepared in this Example (2) are 2.0 × 10 ⁵ cells / mL. In the culture medium. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and the fibronectin fragment / anti-CD3 antibody-immobilized plate prepared in this Example (1) It added to a 0.2 mL / well CD3 antibody immobilized plate, CO ₂ incubator _{(37 ℃, 5% CO 2} ) was initiated restimulated cultured. On day 3 of the re-stimulation culture, the cells were collected and suspended in a culture medium equivalent to the culture solution, and the whole amount was reapplied to a surface untreated 48-well plate. On the 4th day of the re-stimulation culture, 0.2 mL of the culture medium was added, and on the 5th day of the re-stimulation culture, 0.5 mL of the culture medium was added. On day 7 after the start of restimulation culture, the cells were collected, and the cell growth rate, Foxp3-positive cell rate, and CCR7-positive cell rate were measured.

  FIG. 41 shows changes with time in the cell growth rate after restimulation. In FIG. 41, the horizontal axis indicates the number of days from the start of restimulation culture, and the vertical axis indicates the cell growth rate. From FIG. 41, compared with the conditions cultured on the anti-CD3 antibody-immobilized plate, the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed a higher cell growth rate.

  FIG. 42 shows changes with time in the cell growth rate. In FIG. 42, the horizontal axis indicates the number of days from the start of culture, and the vertical axis indicates the cell growth rate. From FIG. 42, the cells cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed higher cell growth rate than the cells cultured on the anti-CD3 antibody-immobilized plate.

  The proportion of cultured cells expressing Foxp3 is shown in FIG. FIG. 43 shows that after adding APC-Cy7-labeled anti-human CD4 antibody, APC-labeled anti-human CCR7 antibody and FITC-labeled anti-human CD25 antibody to cultured cells and reacting at 4 ° C. for 20 minutes, staining was performed, and then PE anti-human FoxP3 Staining Set Was used to measure the Foxp3-positive cell rate with FACSCanto. In FIG. 43, the vertical axis indicates the ratio of CD4 positive, CD25 positive, and Foxp3 positive cells. From FIG. 43, the proportion of CD4 positive, CD25 positive and Foxp3 positive cells in the cell population cultured on the fibronectin fragment / anti-CD3 antibody solid phase plate compared to the cell population cultured on the anti-CD3 antibody solid phase plate. Was comparable or slightly higher.

  In FIG. 44, the vertical axis represents the ratio of CD4 positive, CD25 positive, Foxp3 positive and CCR7 positive cells. From FIG. 44, the cell population cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate is more CD4 positive, CD25 positive, Foxp3 positive and CCR7 positive than the cell population cultured on the anti-CD3 antibody immobilized plate. The percentage of cells was high.

  The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 45, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 42 and the positive rate in FIG. FIG. 45 shows that the number of CD4-positive, CD25-positive, and Foxp3-positive cells is significantly higher in the condition cultured on the fibronectin fragment / anti-CD3 antibody immobilized plate than on the condition cultured on the anti-CD3 antibody immobilized plate. Increased.

  The absolute number of cultured cells expressing CCR7 is shown in FIG. In FIG. 46, the vertical axis represents the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 42 and the positive rate in FIG. FIG. 46 shows that the number of CD4-positive, CD25-positive, Foxp3-positive, and CCR7-positive cells is more marked in the cells cultured in the fibronectin fragment / anti-CD3 antibody-immobilized plate than in the cells cultured on the anti-CD3 antibody-immobilized plate. Increased to.

Example 9 Method of culturing regulatory T cells in the presence of rapamycin (1) Preparation of fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate In the same manner as in Example 1- (1), A CD3 antibody solution and a mixed solution of anti-CD3 antibody and retronectin were prepared, each added to a surface-untreated 96-well flat plate in an amount of 0.1 mL / well, and left at 4 ° C. overnight. Thereafter, each plate was allowed to stand at 37 ° C. for 2.5 hours, and then the lysate was removed, each plate was washed twice with PBS at a volume of 0.2 mL / well, and the anti-CD3 antibody-immobilized plate and fibronectin were washed. Fragment / anti-CD3 antibody immobilized plates were prepared.

(2) Culture of regulatory T cells In the same manner as in Example 5- (2), a cell population containing many CD4-positive, CD25-positive, and CD45RA-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 0.6 × 10 ⁵ cells / mL. Further, anti-CD28 antibody (CD28.2) was added to the cell suspension so as to have a final concentration of 2 μg / mL, and 0. 5 was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate. The culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ) at ₂ mL / well. On the first day of culture, 0.1 mL of the above culture medium was added. On the fourth day of culture, 0.05 mL of the culture medium was added, and on the fifth day of culture, 0.05 mL of the culture medium was added. On day 7 of culture, 0.1 mL of the medium was removed, and 0.1 mL of culture medium was added. Cells were collected on the 8th day from the start of the culture.

(3) Culture of regulatory T cells by addition of rapamycin The cells prepared in Example (2) have the same composition as that described in Example 2- (2) so as to be 2.0 × 10 ⁵ cells / mL. It was suspended in the culture medium. Furthermore, anti-CD28 antibody (CD28.2) was added to the cell suspension to a final concentration of 2 μg / mL, and rapamycin (manufactured by Sigma Aldrich) was added to a final concentration of 2 nM. In addition, about the cell liquid culture | cultivated by the fibronectin fragment / anti-CD3 antibody solid-phase plate, the conditions which add CD28 antibody but not rapamycin were set. It was added to the fibronectin fragment / anti-CD3 antibody-immobilized plate or anti-CD3 antibody-immobilized plate prepared in this Example (1) at 0.2 mL / well, and a CO ₂ incubator (37 ° C., 5% CO ₂ ) To start the culture with rapamycin. On day 3 of the rapamycin-added culture, the group not added with rapamycin collected the cells, suspended them in the culture medium to 2 × 10 ⁵ cells / mL, and added 1.0 mL / well to the surface untreated 24-well plate. It was re-rolled to become. In the group to which rapamycin was added, 0.05 mL of the medium was removed and 0.1 mL of the culture medium was added. On day 5 of rapamycin-added culture, 0.5 mL of culture medium was added to the group not added with rapamycin. In the group to which rapamycin was added, the cells were collected, suspended in a culture medium containing 2 nM rapamycin so as to be 4 × 10 ⁵ cells / mL, and 0.5 mL / well in a surface-untreated 48-well plate. Re-rolled. On day 7 after the start of the culture with rapamycin, the cells were collected, and the cell growth rate, Foxp3-positive cell rate, CD62L-positive and CCR7-positive cell rate were measured.

  FIG. 47 shows changes with time in the cell growth rate. In FIG. 47, the horizontal axis indicates the number of days from the start of culture, the vertical axis indicates the cell growth rate, and the arrow indicates the timing at which rapamycin is added. 47 to 51, “RNCD3 + CD28” is a cell cultured on a fibronectin fragment / anti-CD3 antibody-immobilized plate in the presence of an anti-CD28 antibody, and “CD3 + CD28 + Rapa” is a rapamycin added in the presence of an anti-CD28 antibody. “RNCD3 + CD28 + Rapa” indicates a cell cultured on a fibronectin fragment / anti-CD3 antibody-immobilized plate with rapamycin added in the presence of the anti-CD28 antibody. As shown in FIG. 47, the conditions of culturing on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed a higher cell proliferation rate than the condition of adding rapamycin. In addition, among the conditions in which rapamycin was added, the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed a higher cell growth rate than the conditions cultured on the anti-CD3 antibody-immobilized plate.

  The ratio of cultured cells expressing Foxp3 was measured in the same manner as in Example 5- (2). The results are shown in FIG. In FIG. 48, the vertical axis represents the ratio of CD4 positive, CD25 positive and Foxp3 positive cells. From FIG. 48, the ratio of CD4 positive, CD25 positive, and Foxp3 positive cells was higher in the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate than on the condition where the culture was performed on the anti-CD3 antibody-immobilized plate and rapamycin was added. It was. Moreover, the ratio of CD4-positive, CD25-positive, and Foxp3-positive cells was even higher when cultured on a fibronectin fragment / anti-CD3 antibody-immobilized plate and added with rapamycin. This result shows that high-purity regulatory T cells can be obtained by stimulation with anti-CD3 antibody, anti-CD28 antibody and retronectin without addition of rapamycin, and by adding rapamycin to this condition, even higher purity can be obtained. It shows that regulatory T cells can be obtained.

  In FIG. 49, the vertical axis represents the ratio of CD4 positive, CD25 positive, Foxp3 positive, CD62L positive and CCR7 positive cells. FIG. 49 shows that the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate has a higher proportion of CD4-positive, CD25-positive, Foxp3-positive, CD62L-positive, and CCR7-positive cells than the cell population to which rapamycin was added. It was. Compared to the cell population cultured on the anti-CD3 antibody-immobilized plate, the cell population cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate between the conditions added with rapamycin was CD4 positive, CD25 positive, Foxp3 positive and The proportion of CD62L positive and CCR7 positive cells was high.

  The absolute number of cultured cells that express Foxp3 is shown in FIG. In FIG. 50, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 47 and the positive rate in FIG. From FIG. 50, the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate showed a marked increase in the number of CD4-positive, CD25-positive, and Foxp3-positive cells compared to the condition where rapamycin was added. Among the conditions where rapamycin was added, the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate were more CD4-positive, CD25-positive and Foxp3-positive cells than the conditions cultured on the anti-CD3 antibody-immobilized plate. The number increased significantly.

  The absolute number of cultured cells expressing CD62L and CCR7 is shown in FIG. In FIG. 51, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate in FIG. 47 and the positive rate in FIG. As shown in FIG. 51, the number of CD4-positive, CD25-positive, Foxp3-positive, CD62L-positive, and CCR7-positive cells was significantly increased in the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate compared to the conditions in which rapamycin was added. Compared with the conditions cultured on the anti-CD3 antibody-immobilized plate, the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate were more CD4-positive, CD25-positive and Foxp3-positive between the conditions where rapamycin was added. The number of CD62L positive and CCR7 positive cells was remarkably increased.

Example 10 Comparison with CD3 / 28 beads (1) Preparation of fibronectin fragment / anti-CD3 antibody-immobilized plate and anti-CD3 antibody-immobilized plate According to the procedure described in Example 6- (1), surface untreated 96 wells A fibronectin fragment / anti-CD3 antibody-immobilized plate and an anti-CD3 antibody-immobilized plate were prepared using a flat plate.

(2) Culture of regulatory T cells In the same manner as in Example 5- (2), a cell population containing many CD4-positive, CD25-positive, and CD45RA-positive cells was obtained. These cells were suspended in a culture medium having the same composition as that described in Example 2- (2) so as to be 1.0 × 10 ⁵ cells / mL. In the group cultured on a solid phased plate, an anti-CD28 antibody (CD28.2) was added to the cell suspension to a final concentration of 2 μg / mL, and a fibronectin fragment / anti-CD3 antibody solid phased plate or anti-CD3 was added. It added to a antibody immobilized plate in 0.2 mL / well, CO ₂ incubator _{(37 ℃, 5% CO 2} ) and culturing was started. On the other hand, in the group cultured with growth stimulating beads, Dynabeads Human Treg Expander (manufactured by Invitrogen) (hereinafter referred to as CD3 / 28 beads) after washing into the cell suspension has a cell to CD3 / 28 bead ratio of 1 pair. 4 was added to a surface untreated 96-well flat plate at 0.2 mL / well, and culture was started in a CO ₂ incubator (37 ° C., 5% CO ₂ ). On the first day of culture, 0.05 mL of the above culture medium was added. On the fourth day of culture, 0.05 mL of the medium was removed, and 0.1 mL of the culture medium was added. On the fifth day of culture, the cells were collected, suspended in a culture medium so as to be 3.5 × 10 ⁵ cells / mL, and re-spread so as to be 1.0 mL / well in a 24-well plate for cell culture. On day 7 of culture, 0.4 mL of the medium was removed, and 0.4 mL of culture medium was added. On the 8th day from the start of the culture, the cells were collected and the cell growth rate was measured. In the group cultured with CD3 / 28 beads, the CD3 / 28 beads were removed with a magnet, and the cells were collected, suspended in a culture medium so as to be 8.7 × 10 ⁵ cells / mL, and 12 wells for cell culture. The plate was rewound to 2.0 mL / well. On the 8th day from the start of the culture, the cells were collected and the cell growth rate was measured. On the 11th day from the start of the culture, the cells were collected, and the Foxp3-positive cell rate, the CCR7-positive cell rate, and the CD27-positive cell rate were measured.

  FIG. 52 shows changes with time in the cell growth rate. In FIG. 52, the horizontal axis represents the number of days from the start of culture, and the vertical axis represents the cell growth rate. As shown in FIG. 52, the conditions of culturing with the fibronectin fragment / anti-CD3 antibody-immobilized plate showed higher cell proliferation rate than the conditions of culturing with the anti-CD3 antibody-immobilized plate and the condition of culturing with CD3 / 28 beads.

  The ratio of cultured cells expressing Foxp3 was measured in the same manner as in Example 6- (2). The absolute number of cultured cells expressing Foxp3 is shown in FIG. In FIG. 53, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate and the positive rate of Foxp3 in FIG. The number of CD4-positive and Foxp3-positive cells is more prominent when cultured on fibronectin fragment / anti-CD3 antibody-immobilized plate than when cultured on anti-CD3 antibody-immobilized plate and CD3 / 28 beads. Increased to.

  The absolute number of cultured cells expressing CCR7 is shown in FIG. In FIG. 54, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate and the ratio of CD4 positive, Foxp3 positive and CCR7 positive cells in FIG. From FIG. 54, compared with the conditions cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate, the conditions cultured on the anti-CD3 antibody-immobilized plate and the conditions cultured on CD3 / 28 beads were CD4. The number of positive, Foxp3-positive and CCR7-positive cells was significantly increased.

  The absolute number of cultured cells expressing CD27 is shown in FIG. In FIG. 55, the vertical axis indicates the value obtained by multiplying the number of cells at the start of culture by the cell growth rate and the ratio of CD4 positive, Foxp3 positive and CD27 positive cells in FIG. As compared with FIG. 55, the condition cultured on the fibronectin fragment / anti-CD3 antibody-immobilized plate is more CD4 positive than the condition cultured on the anti-CD3 antibody-immobilized plate and the condition cultured on CD3 / 28 beads. And the number of Foxp3-positive and CD27-positive cells was remarkably increased.

  As described above, the method of the present invention provides a method capable of efficiently expanding and culturing a cell population containing a large amount of regulatory T cells having strong immunosuppressive ability.

  By using the method of the present invention, a cell population containing regulatory T cells can be efficiently expanded in vitro. Regulatory T cells produced by this method are useful as an immunosuppressant for the prevention and treatment of organ transplant rejection, allergic diseases, autoimmune diseases, graft-versus-host disease (GVHD) and the like.

SEQ ID NO: 1; Fibronectin fragment named CH-271.
SEQ ID NO: 2; Fibronectin fragment named CH-296.
SEQ ID NO: 3; Fibronectin fragment named H-271.
SEQ ID NO: 4; Fibronectin fragment named H-296.
SEQ ID NO: 5; TSDR forward primer.
SEQ ID NO: 6; TSDR reverse primer.

Claims (3)

A method for producing a cell population containing regulatory T cells, comprising the following steps:
(1) separating a CD4 positive CD25 positive T cell population from a T cell-containing cell population, and (2) in the presence of an anti-CD3 antibody, an anti-CD28 antibody and fibronectin or a fragment thereof or a mixture thereof ( A step of culturing the cell population obtained in 1) in vitro.   The method according to claim 1, wherein the fibronectin fragment is a fragment containing a cell adhesion domain containing a ligand for VLA-5, a heparin binding domain, and a CS-1 domain which is a ligand for VLA-4.   The method according to claim 1 or 2, wherein the fibronectin fragment is a fragment having the amino acid sequence of any one of SEQ ID NOs: 1 to 4 in the sequence listing.

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