JP2021069322A - Method for producing cd4-positive regulatory t-cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for probabilistically producing CD4 positive regulatory T cells via pluripotent stem cells. A method for producing CD4 positive regulatory T cells, which comprises a step of inducing iPS cells from HTLV-1-infected CD4 positive T cells and a step of differentiating the iPS cells into CD4 positive regulatory T cells. [Selection diagram] None

Description

The present invention relates to a method for producing CD4 positive regulatory T cells (Tregs) via pluripotent stem cells.

Since regulatory T cells (Treg) have an immune response-suppressing function, they can be expected to be used for the treatment of autoimmune diseases, inflammatory diseases, allergic diseases, and graft-versus-host disease (GVHD). A method of amplifying it in vitro and injecting Treg to use it for treatment is conceivable, but Treg has poor growth efficiency in vitro and is difficult to cryopreserve. It is difficult to stably obtain sufficient Tregs when needed.

Human T-cell leukemia virus type 1 (HTLV-1) ^{infects T lymphocytes (CD4 +} CD25 ⁺ T cells) and is a malignant tumor, adult T-cell leukemia (ATL), and a refractory neurological disease, HTLV-1. It is a retrovirus that causes related myelopathy (HTLV-1 associated myelopathy: HAM), and it is estimated that there are currently about 800,000 infected people in Japan. The HTLV-1 viral genes include two oncogenes, Tax and HTLV-1 bZIP factor (HBZ), and it is thought that these actions cause the infected cells to become cancerous.
In addition, ATL cells are CD4 ⁺ CD25 ⁺ CCR4 ⁺ Foxp3 ⁺ , and the origin of tumor cells is considered to be derived from regulatory T cells.

On the other hand, in recent years, a technique for inducing various T cells from pluripotent stem cells such as induced pluripotent stem (iPS) cells has been reported. Various cell-mediated immunotherapy such as controlling abnormal or excessive immune response to self becomes possible.

So far, a method of producing iPS cells from antigen-specific T cells and inducing differentiation into T cells showing the same antigen specificity as the cells from which they were derived has been reported (Patent Documents 1 and 2). However, although CD8-positive T cells can be induced by this method, it is difficult to induce CD4-positive T cells, and the actual situation is that CD4 gene cannot be induced without artificial gene transfer.

International Publication No. 2011/096482 Japanese Patent No. 6164746

The present invention relates to providing a method for probabilistically producing CD4 positive regulatory T cells via pluripotent stem cells.

When the present inventors examined to achieve the above-mentioned problems, iPS cells were produced from HTLV-1-infected CD4-positive T cells derived from adult T-cell leukemia (ATL) patients and induced to differentiate into T cells again. , Have found that CD4-positive T cells having the same expression type as Treg can be obtained, and have completed the present invention.

That is, the present invention relates to the following 1) to 2).
1) A method for producing CD4 positive regulatory T cells, which comprises a step of inducing iPS cells from HTLV-1-infected CD4 positive T cells and a step of differentiating the iPS cells into CD4 positive regulatory T cells.
2) A pharmaceutical composition containing CD4 positive regulatory T cells produced by the method of 1).

According to the present invention, it is possible to produce CD4 positive regulatory T cells without introducing the CD4 gene via iPS cells. This allows, for example, cell immunotherapy to control an abnormal or excessive immune response against oneself.

Confirmation of Tax and HBZ gene expression in CD4T-iPS cells. A phenotype of T cells induced to differentiate from HTLV-1-infected CD4T-iPS and iPS derived from CD4T cell clones. Phenotype of CD4-positive T cells induced by HTLV-1-infected CD4T-iPS. Confirmation of gene rearrangement by Southern blotting of TCRCβ1. Confirmation of Tax and HBZ gene expression in differentiation-induced CD4 T cells.

The method for producing a CD4 positive regulatory T cell of the present invention includes a step of inducing iPS cells from HTLV-1-infected CD4 positive T cells and a step of inducing differentiation of the iPS cells into CD4 positive T cells.
(1) Isolation of HTLV-1-infected CD4-positive T cells HTLV-1-infected CD4-positive T cells can be isolated from, for example, tissues of ATL patients by a known method. Tissues of ALT patients include tissues containing CD4-positive T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, cord blood, and lesion tissue. Among these, peripheral blood is preferable from the viewpoint of low invasiveness to humans and easy preparation.
In the present invention, examples of HTLV-1-infected CD4-positive T cells include CD4 ⁺ CD25 ⁺ T cells, preferably CD4 ⁺ CD25 ⁺ CCR4 ⁺ T cells, and more preferably CD4 ⁺ CD25 ⁺ CCR4 ⁺ Foxp3 ⁺ T cells. It is a cell.

Known methods for isolating HTLV-1-infected CD4-positive T cells include, for example, magnetic selection using magnetic beads for cell separation and flow cytometry using an anti-CD4 antibody and a cell sorter.

(2) Establishment of iPS cells (referred to as "CD4T-iPS cells") from HTLV-1-infected CD4-positive T cells The isolated HTLV-1-infected CD4-positive T cells are reprogrammed into iPS cells.
Here, the "iPS cell" is a cell also referred to as an induced pluripotent stem cell or an induced pluripotent stem cell, and a cell reprogramming factor is added to the HTLV-1-infected CD4-positive T cell. It can be induced by introducing it.
The "cell reprogramming factor" may be a factor capable of imparting pluripotency to the somatic cell alone or in cooperation with other pluripotency factors by being introduced into the CD4-positive T cell. Although not particularly limited, Oct3 / 4, c-Myc, Sox2, Klf4, Klf5, LIN28, Nanog, ECAT1, ESG1, Fbx15, Eras, ECAT7, ECAT8, Gdf3, Sox15, ECAT15-1, ECAT15- It is preferably at least one protein selected from the group consisting of 2, Fthl17, Sal14, Rex1, Utf1, Tcl1, Stella, β-catenin, Stat3, and Grb2. Further, among these proteins, it is more preferable to introduce four factors of Oct3 / 4, Sox2, Klf4 and c-Myc into the CD4 positive T cells from the viewpoint that iPS cells can be efficiently established with a small number of factors.

In the present invention, the method for introducing the cell reprogramming factor into HTLV-1-infected CD4-positive T cells is not particularly limited, and a known method can be appropriately selected and introduced in the form of protein or nucleic acid. it can. For example, when introduced into the CD4-positive T cell in the form of a nucleic acid encoding the cell reprogramming factor, the nucleic acid (eg, cDNA, RNA) is an appropriate expression vector containing a promoter that functions in the T cell. The expression vector is injected into cells by genome editing such as infection, lipofection method, liposome method, electroporation method, calcium phosphate co-precipitation method, DEAE dextran method, microinjection method, electroporation method, and CRISPR / Cas9. Can be introduced.

Examples of such expression vectors include virus vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, and Sendai virus, and animal cell expression plasmids, but insertion mutations are unlikely to occur and gene transfer is performed. From the viewpoint of high efficiency and a large number of copies of the gene to be introduced, it is preferable to introduce a nucleic acid encoding the cell reprogramming factor into the CD4-positive T cell using Sendai virus.
Examples of the promoter used in such an expression vector include SRα promoter, SV40 promoter, LTR promoter, CMV promoter, RSV promoter, HSV-TK promoter and the like. Further, such a promoter may be capable of controlling the expression of a gene inserted downstream of the promoter depending on the presence or absence of a drug (for example, tetracycline). In addition to the promoter, the expression vector may further contain an enhancer, a poly A addition signal, a selectable marker gene (for example, a neomycin resistance gene), an SV40 origin of replication, and the like.

In addition, when introducing such a nuclear reprogramming factor, expressing the SV40 large T antigen together with the nuclear reprogramming factor in the cells to be introduced increases the proliferation and survival rate required for the reprogramming of the cells. preferable.
Therefore, as an optimal mode for introducing a cell reprogramming factor into HTLV-1-infected CD4-positive T cells, a Sendai virus vector having a nucleic acid encoding the four factors of Oct3 / 4, Sox2, Klf4 and c-Myc inserted therein is used. Infection of the CD4 positive T cells with a Sendai virus vector having a nucleic acid encoding the SV40 rage T antigen inserted can be mentioned, but it can be established with a Sendai virus vector to which other factors have been added, or it encodes the SV40 rage T antigen. It may be established without using the Sendai virus vector into which the nucleic acid is inserted.

In addition, when establishing T-iPS cells, HTLV-1-infected CD4-positive T cells are treated with interleukin-2 (IL-2), interleukin-7 (IL-7), or interleukin-7 (IL-7) before the introduction of the gene. It may be activated by stimulating with an anti-CD3 antibody or an anti-CD28 antibody in the presence of interleukin-15 (IL-15), and expresses phytohemaglutinin (PHA), interleukin-2 (IL-2), and allogeneic antigen. It may be stimulated and activated by at least one substance selected from the group consisting of cells, anti-CD3 antibody, anti-CD28 antibody, CD3 agonist and CD28 agonist. Such stimulation can be performed, for example, by adding PHA, IL-2, anti-CD3 antibody and / or anti-CD28 antibody or the like to a medium and culturing the HTLV-1-infected CD4-positive T cells for a certain period of time. .. Further, the anti-CD3 antibody and / or the anti-CD28 antibody may be one to which magnetic beads or the like are bound, and instead of adding these antibodies to the medium, an anti-CD3 antibody and / or an anti-CD28 antibody is used. Stimulation may be given by culturing the HTLV-1-infected CD4-positive T cells on a culture dish bound to the surface for a certain period of time.

As the medium for culturing the HTLV-1-infected CD4-positive T cells, for example, a known medium suitable for culturing T cells (more specifically, other cytokines, human serum, and the RPMI 1640) (RPMI) 1640 medium, AIM ^VTM medium, NS-A2 can be used. The medium is supplemented with amino acids (eg, L-glutamine) and antibiotics (eg, streptomycin, penicillin) necessary for culturing. There may be.

Further, the conditions for introducing the cell reprogramming factor or the like into the HTLV-1-infected CD4-positive T cells are not particularly limited, but the HTLV-1-infected CD4-positive T cells into which the factor has been introduced are not particularly limited. , It is preferable to culture under feeder-free conditions. For example, laminin 511E8 fragment iMatrix-511 solution or wells coated with vitronectin or matrigel can be mentioned. It can also be established by culturing under feeder cell conditions, and examples of feeder cells include mouse embryonic fibroblasts (MEFs), STO cells, and SNL cells whose cell division has been stopped by irradiation with radiation or treatment with antibiotics. ..

Furthermore, in the process of inducing T-iPS cells from the HTLV-1-infected CD4-positive T cells, it is preferable to add the iPS cell medium from the next day. After that, it is preferable to replace the medium by half every other day and gradually replace the CTL medium with the iPS medium.

In addition, in accordance with the transition from the HTLV-1-infected CD4-positive T cells to iPS cells, the known medium suitable for culturing T cells is gradually replaced with a medium suitable for culturing iPS cells while culturing. It is preferable to do so. As a medium suitable for culturing such iPS cells, a known medium can be appropriately selected and used. For example, in the case of iMatrix coating, StemFit AK03N or in the case of coating with Vitronectin, Essential 8 Medium, Matrigel. Dalbeco modified Eagle's medium / F12 medium (human iPS) containing knockout serum substitute, L-glutamine, non-essential amino acids, 2-mercaptoethanol, b-FGF, etc. on feeder cells such as mTeSR and MEF cells. Cell culture medium) is desirable.

The selection of T-iPS cells derived from the HTLV-1-infected CD4-positive T cells in this way can be performed by appropriately selecting a known method. As such a known method, for example, a method of observing and selecting the morphology of iPS cell-like colonies under a microscope can be mentioned. Alternatively, a method may be used in which all the colonies that have been established are subcultured as they are without selecting each colony of T-iPS cells.

Confirmation that the selected cells are T-iPS cells can be determined, for example, by using undifferentiated cell-specific markers (SSEA-4, Tra-1-60, and Tra-1-81, etc.) in the selected cells. It can be carried out by a method of detecting expression by q-PCR or the like, a method of confirming by ALP staining, or a method of transplanting selected cells into mice and observing their teratoma formation. Further, confirmation that the cells selected in this manner are derived from the HTLV-1-infected CD4-positive T cells can be performed by detecting the quantification of Tax and HBZ genes by qPCR.

The timing for selecting and collecting these cells can be appropriately determined while observing the growth state of the colony, and is generally after the cell reprogramming factor is introduced into the HTLV-1-infected CD4-positive T cells. It is 10 to 40 days, preferably 14 to 28 days. Unless otherwise specified, the culture environment is preferably 5% CO ₂ , 35 to 38 ° C, and more preferably 37 ° C. Efficient growth of colonies can be observed even in a hypoxic culture environment (oxygen concentration: for example, 5%).

(3) Induction of differentiation of T-iPS cells into CD4 positive regulatory T cells In order to differentiate established CD4T-iPS cells into CD4 positive regulatory T cells, it is easy to induce differentiation into the mesophyll lineage. From this point of view, first, T-iPS cells are placed on feeder cells (preferably stromal cells, more preferably human stromal cells), VEGF, serum (for example, bovine fetal serum (FBS)), insulin, transferase, L-. It is preferable to culture in a medium containing glutamine, α-monothioglycerol, ascorbic acid and the like. The stromal cells used are preferably OP9 cells and 10T1 / 2 cells (C3H10T1 / 2 cells) from the viewpoint of facilitating the induction of differentiation into the hematopoietic system.
Examples of the medium include X-VIVO medium, Iskoff-modified Dalveco medium (IMDM medium), α-MEM, and DMEM, and a bag-shaped structure containing induced cells such as hematopoietic progenitor cells (“T”). IMDM medium is preferable from the viewpoint of high formation efficiency (also referred to as "iPS sack"). The culture period of the T-iPS cells is preferably a period until the formation of the T-iPS sack, preferably 8 to 14 days after the start of the culture of the T-iPS cells, and more preferably 10 to 10 days. 14 days. The culture environment is not particularly limited, but is preferably 5% CO ₂ , 35 to 38 ° C, and more preferably 37 ° C. Further, from the viewpoint that the formation efficiency of the T-iPS sack is high and the number of blood cells contained in the T-iPS sack is large, the cells are cultured under a low oxygen concentration condition (oxygen concentration: for example, 5%) for 7 days. Is more preferable.

Next, the induced cells (hematopoietic progenitor cells and the like) contained in the obtained sack are cultured on stromal cells in α-MEM medium containing cytokines, FCS and the like. The cells existing inside the sack-like structure can be separated by passing them through a physical means, for example, a sterilized sieve device (for example, a cell strainer). The feeder cells used for this culture are preferably OP9 cells and 10T1 / 2 cells expressing Notch receptor ligand. As the cytokine to be added to the medium, IL-7, FLT3L, SCF, and TPO are preferable.

The culture period of the induced cells contained in the T-iPS sack is preferably about 28 days after the start of culturing the cells contained in the T-iPS sack. The culture environment is not particularly limited, but is preferably 5% CO ₂ , 35 to 38 ° C, and more preferably 37 ° C.
Whether or not Treg-like flow cytometry is expressed is determined after intracellular staining with anti-CD34 antibody, anti-CD45 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD25 antibody, FOXP-3, anti-CCR4 antibody and the like. It can be evaluated by the flow cytometry of.

It then stimulates the T cell receptors of the induced cells. The method of stimulating the T cell receptor can be carried out by adding PHA or the like to anti-CD3 antibody, anti-CD28 antibody, X-ray-irradiated peripheral blood mononuclear cells, and culturing the induced cells for a certain period of time. ..

The cells thus induced to differentiate are the CD4-positive T cells (CD4-positive T cells) that are the source of the T-iPS cells by detecting the state of TCR gene rearrangement by genomic PCR and by flow cytometry using intracellular staining. For example, it can be confirmed that it is derived from ^{CD4 +} CD25 ⁺ CCR4 ⁺ Foxp3 ^{+ T cells).}

The CD4 positive regulatory T cells thus obtained can be isolated by appropriately selecting a known method. Examples of such a known method include flow cytometry using an antibody against a cell surface marker such as CD4 and a cell sorter.

Since the CD4 positive regulatory T cells produced by the method of the present invention have an immune response inhibitory function, for example, for the treatment or prevention of autoimmune diseases, inflammatory diseases, allergic diseases, and graft-versus-host diseases (GVHD). Can be used for. Therefore, the present invention provides a pharmaceutical composition containing CD4 positive regulatory T cells produced by the method of the present invention.

The pharmaceutical composition of the present invention can be prepared by formulating CD4 positive regulatory T cells produced by the method of the present invention by a known pharmaceutical method. For example, it can be mainly used parenterally as an injection (intravenous injection, drip injection, etc.), capsule, liquid, film coating, or the like.

In these formulations, pharmacologically acceptable carriers or vehicles, specifically sterile water or saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, etc. Appropriately combine with preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, solubilizers or other additives. Can be done. In addition, it may be used in combination with a known pharmaceutical composition or immunostimulatory agent used for the treatment or prevention of the disease.

When the pharmaceutical composition of the present invention is administered, the dose thereof is appropriately selected according to the age, weight, symptom, health condition, type of composition (pharmaceutical product, food and drink, etc.) of the subject.

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Production of CD4-positive T cells (Treg) from HTLV-1-infected CD4-positive T cells (1) Isolation of HTLV-1-infected CD4-positive T cells After isolation of peripheral blood mononuclear cells from the peripheral blood of ATL patients, CD4 positive T cells were isolated by positive selection with CD4 beads. The phenotype of the obtained T cells was analyzed by flow cytometry using various cell surface markers, and it was confirmed that they were strongly positive for CD3CD4CD25.

(2) Establishment of T-iPS cells Sendai virus (SeV) vector (SeVp) containing nuclear reprogramming factors (Oct4, Sox2, Klf4, c-Myc) in HTLV-1-infected CD4-positive T cells obtained in (1). [KOSM302L]) and a SeV vector containing a nucleic acid encoding the SV40 rage T antigen were infected and the gene was introduced.
T cells after gene transfer were transferred to a 6-well plate coated with iMatrix and cultured in a _{CO 2} incubator in a T cell medium containing IL-2 (medium composition: RPMI, 10% human AB serum).
The day after the gene transfer, an equal amount of iPS medium (StemFitAK03N) was added, and thereafter, half the amount was replaced with StemFitAK03N every other day.
After 21 days, colonies of T-iPS cells could be observed, and then colony pickup was performed.

(3) Induction of differentiation of CD4T-iPS cells into CD4-positive cells VEGF, FBS, insulin, transferase, transferase, L-glutamine, on 10T1 / 2, which is a feeder cell, by finely crushing the T-iPS cells obtained in (2). The first week was hypoxic cultured in a medium containing α-monothioglycerol, ascorbic acid and the like, and the next week was cultured in an incubator with a 20% oxygen concentration. After that, the cells were cultured on 10T1 / 2 feeder cells expressing Notch Ligand together with cytokines (IL-7, FLT3L, SCF) for another 4 weeks while being transferred to new feeder cells once a week, and the floating cells were cultured after 4 weeks. All were harvested and stimulated with T cell receptor (TCR). TCR stimulation was stimulated with X-ray-irradiated allogeneic peripheral blood mononuclear cells and PHA, or anti-CD3 / CD28 antibody approximately every 2 weeks.

(4) Results and discussion The expression of Tax and HBZ genes in CD4T-iPS cells was confirmed by qPCR (Fig. 1). Compared with the original HTLV-1-infected CD4-positive T cells (ATL PBMC), the expression of the Tax gene and the HBZ gene in the CD4T-iPS cells (CD4T-iPS) was reduced to 1/1000 or less.

FIG. 2 shows the results of intracellular staining and analysis of the phenotype of T cells obtained from CD4T-iPS by induction of differentiation by flow cytometry. As a control, we are trying to induce the differentiation of CD4 T cells from ^{CD4 +} T cell cellone-iPS established from healthy human-derived CD4 T cell clones at the same time. When differentiation induction was performed from HTLV-1-infected CD4T cell-derived CD4T-iPS cells, a large number of CD4T cells were induced (No. 1: 20%, No. 2: 20.9%). Furthermore, when the expression of FOXP3 and CD25 was confirmed by gated to CD4 positive cells, double positive cells were confirmed. On the other hand, only 1.57% of CD4-positive T cells were induced from T-iPS cells derived from CD4 T cells not infected with HTLV-1, and 0% of FOXP3 and CD25-positive cells. As shown in FIG. 3, the CD4-positive T cells induced by HTLV-1-infected CD4T-iPS were FOXP3-positive, CCR4-positive, and CD25-positive, and showed a Treg-like phenotype. When the rearrangement of TCRCβ1 was confirmed by Southern blotting as shown in FIG. 4, the original ATL cells showed neoplastic clonal proliferation, but the established iPS cells and the CD4T cells induced to differentiate were neoplastic clones. No sexual proliferation was observed. In addition, as shown in FIG. 5, the expression of Tax and HBZ was also abolished in the differentiation-induced CD4T cells.

From this, it was extremely difficult to induce CD4T cells from normal iPS cells, and the phenotype of Treg could not be confirmed, but CD4 positive regulatory T cells were found from CD4T-iPS cells established from HTLV-1-infected CD4T cells. It was confirmed that it could be induced. Differentiation-induced CD4T cells do not show neoplastic clonal proliferation, and the expression of Tax and HBZ, which are positive for HTLV-1 infection, has disappeared, so that they can be expected to be used for cell therapy.

Since regulatory T cells (Treg) have an immune response-suppressing function, they can be expected to be used for the treatment of autoimmune diseases, inflammatory diseases, allergic diseases, and graft-versus-host disease (GVHD). A method of amplifying it in vitro and injecting Treg to use it for treatment is conceivable, but Treg has poor growth efficiency in vitro and is difficult to cryopreserve. It is difficult to stably obtain sufficient Tregs when needed. So far, a method of producing iPS cells from antigen-specific T cells and inducing differentiation into T cells showing the same antigen specificity as the cells from which they were derived again has been reported (Patent Documents 1 and 2). .. However, although CD8-positive T cells can be induced by this method, it is difficult to induce CD4-positive T cells, and the actual situation is that CD4 genes cannot be induced without artificial gene transfer. According to the present invention, it is possible to induce CD4 positive regulatory T cells from CD4T-iPS cells established from HTLV-1-infected CD4T cells without introducing the CD4 gene. Differentiation-induced CD4 positive regulatory T cells do not show neoplastic clonal proliferation, and the expression of Tax and HBZ, which are positive for HTLV-1 infection, has disappeared, so they are used for cell therapy that suppresses the immune response. You can expect to be able to do it.

Claims (2)

A method for producing CD4 positive regulatory T cells, which comprises a step of inducing iPS cells from HTLV-1-infected CD4 positive T cells and a step of differentiating the iPS cells into CD4 positive regulatory T cells. A pharmaceutical composition containing CD4 positive regulatory T cells produced by the method according to claim 1.

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