JP2020010704A - Methods for reconstructing immune functions using pluripotent stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish an iPS cell from a human T lymphocyte, and further, the established iPS cell is functionally T while maintaining the recombinant structure of the TCR gene possessed by the original human T lymphocyte. Providing a method for inducing differentiation into lymphocytes. A method for producing human T cells, which comprises a step of inducing iPS cells from human T cells and a step of differentiating the iPS cells into T cells, and a drug containing the T cells produced by the method. The composition, and a method of immuno-cell therapy utilizing the method. [Selection diagram] None

Description

  The present invention relates to a method for restoring immune function using pluripotent stem cells, and more particularly to a method for producing human T cells, a pharmaceutical composition containing the T cells produced by the method, and an immunity utilizing the method. A method of cell therapy.

  In the case of immune function deterioration caused by various causes, if replenishment and regeneration of immune cells and the like can be carried out, it will be an extremely effective means for improving the disease state and improving the therapeutic effect of the disease. In particular, although there is a strong demand for replenishment and regeneration of T lymphocytes responsible for cell-mediated immunity, no effective treatment method has been established at present. Although immunity is characterized by specificity, it is largely due to the difficulty in exploiting this specificity.

  With the dramatic progress of gene manipulation technology in recent years, attempts have been made to introduce an antigen-specific T cell receptor (TCR) gene into various lymphoid cells to replenish or regenerate a specific immune response ( Non-patent documents 1 and 2). In these attempts, CD34-positive cells, which are bone marrow progenitor cells, naive T lymphocytes, and the like are used as the transgenic cells, but these have low self-renewal ability in Ex-vivo and low transfection efficiency. And it is difficult to regulate differentiation by gene transfer.

  In recent years, embryonic stem cells (ES cells) and induced pluripotent stem cells (inducible pluripotent stem cells, iPS cells) have been established from many animal species including humans. These pluripotent stem cells maintain the vigorous division ability while maintaining pluripotency, and can be differentiated into almost all organs by appropriately inducing differentiation. It is considered a source. In particular, since iPS cells can be established from autologous somatic cells, ethical and social problems are less likely to occur than ES cells produced by destroying embryos, and since they are autologous, Rejection, which is the biggest barrier to regenerative medicine and transplantation medicine, can be avoided.

  iPS cells can be established by reprogramming somatic cells in various ways. Since the reprogrammed iPS cells inherit the genetic information used for reprogramming as they are, the cells of the immune system, especially B lymphocytes and T lymphocytes, are reconstituted and terminally differentiated, and are also reprogrammed. Whether it was possible was of great interest. In such a situation, the establishment of iPS cells from mouse B lymphocytes was reported by Jaenisch et al. In 2008 (Non-Patent Document 3), and the establishment of iPS cells from mouse T lymphocytes was reported by Yamanaka et al. In 2009. (Non-Patent Document 4). However, there is no report that iPS cells were established from human T lymphocytes.

  In order to realize immunotherapy using T lymphocytes, in addition to establishing iPS cells from human T lymphocytes, the established iPS cells are further replaced with TCRs that were contained in the original human T lymphocytes. It is necessary to induce differentiation into functional T lymphocytes while maintaining the recombinant structure of the gene, but such a technique has not yet been established.

Gattinoni L. Et al., Nature Reviews Immunology, 2006, 6, 383-393. Morgan R.M. Et al., Science, 2006, 314, 126-129. Hanna J. et al. Et al., Cell, 2008, 133 (2), 250-264. Hong H. Et al., Nature, 2009, 460, 1132-1135.

  The present invention has been made in view of the above-mentioned problems of the prior art, and has as its object to establish iPS cells from human T lymphocytes, and to further establish the established iPS cells with the original human T lymphocytes. Another object of the present invention is to provide a method capable of inducing differentiation into functional T lymphocytes while maintaining the recombinant structure of the TCR gene. It is a further object of the present invention to provide a pharmaceutical composition containing the T cell thus produced, and a method for immune cell therapy using the T cell thus produced.

  The present inventors have conducted intensive studies to achieve the above object, and as a result, succeeded in establishing iPS cells from human T cells while maintaining the same TCR reconstituted state as the original human T lymphocytes. did. Furthermore, the present inventors have developed a function having cytokine production ability and the like from iPS cells (TiPS cells) derived from human T cells thus established, while maintaining the same TCR reconstituted state as the original human T lymphocytes. T cells were successfully induced to differentiate. Then, by transplanting the thus obtained human T cells into the body of a patient, it was found that ideal immune cell therapy can be performed, and the present invention was completed.

More specifically, the present invention provides the following inventions.
(1) A method for producing human T cells, comprising the steps of inducing iPS cells from human T cells and differentiating the iPS cells into T cells.
(2) The method according to (1), wherein the human T cell is a T cell expressing at least one molecule selected from the group consisting of CD4 and CD8.
(3) The method according to (1) or (2), wherein the T cells induced to iPS cells have antigen specificity.
(4) The method according to (3), wherein the T cells induced into the iPS cells and the T cells differentiated from the iPS cells have the same antigen specificity.
(5) A human T cell produced by the method according to any one of (1) to (4).
(6) A pharmaceutical composition comprising the human T cell according to (5).
(7) isolating T cells having the desired antigen specificity from humans;
Inducing iPS cells from the T cells having the desired antigen specificity;
Differentiating the iPS cells into T cells;
Administering the obtained iPS cell-derived T cells into a human body.

Lymphocytes that acquire structural changes in the TCR or BCR gene base sequence during development are extremely unique cells as a source of iPS cell establishment. In recent years, it has been reported that iPS cells having a TCR reconstituted from mouse T cells and B cells can be established, suggesting that the TCR reconstitution pattern is not lost by nuclear transfer or reprogramming. It had been. However, these are all cases in mice. According to the present invention, it has been shown for the first time that iPS cells can be obtained from human peripheral blood T lymphocytes, and furthermore, when a T lineage cell is derived from an iPS cell having at least one in-frame TCR rearrangement, the TCR is obtained. It has been clarified for the first time that human T lineage cells having the following appear in a monoclonal. In particular, one in-frame (in
In the induction of T lineage cells from iPSCs (iPSCs) that have only a frame) rearrangement, the TCR that appeared was identical to the nucleotide sequence conserved in the iPS cells, and was completely monoclonal. there were. This means that ES cells, CD34-positive hematopoietic stem cells must always take into account the inhibitory effect (interfere) due to the expression of endogenous TCR α chain β chain (particularly α chain which is incompletely controlled by allelic exclusion). Alternatively, it was shown that T cells having a monoclonal TCRαβ against the antigen of interest can be induced more efficiently than the method of introducing a TCR gene into naive T cells.

  Therefore, according to the present invention, it is possible to efficiently produce human T lymphocytes specific to a target antigen. By utilizing the human T lymphocytes thus produced, it is possible to provide a method of immune cell therapy, which has the least problem of rejection, which is the biggest barrier for regenerative medicine and transplantation medicine.

It is a figure which shows the outline | summary of production of human iPS cell from peripheral blood T lymphocyte. In the figure, "Tapering" indicates a period during which the medium was gradually replaced with a human iPS cell culture medium (such as hiPS medium). 2 is a micrograph showing a T cell-derived ES cell-like colony formed by the method shown in FIG. 1. In the figure, the upper part shows colonies obtained by introducing Yamanaka 3/4 factor into CD3 T cells, and the lower part shows, from the left side, Day6: cells re-seeded into MEF cells and Day15: ES cell-like colonies from the left. Forming cells, Day 24: show cells that have completed colony formation (see FIG. 1 for Day). The scale bar indicates 200 μm. It is a microscope picture which shows the result of having observed the iPS cell colony by immunostaining. It is an electrophoresis photograph which shows the result of having analyzed the expression of the pluripotency marker in T-iPS cell, human ES cell (KhES3), and peripheral blood-derived CD3 + cell (PBCD3 +) by RT-PCR. ACTB (β-actin) and GAPDH were used as loading controls. FIG. 4 is a graph showing the reprogramming efficiency of peripheral blood CD3 ⁺ T cells to a pluripotent state by three factors (excluding 3 Factors and c-Myc) or four factors (including 4 Factors and c-Myc). In the figure, the vertical axis indicates the number of ALP-positive colonies derived from 3 × 10 ⁵ T lymphocytes on day 22 (day 22) (results from three factors are mean ± SEM = 5.1). ± 2.4 [n = 7], and the result with four factors was mean ± SEM = 77.0 ± 25.0 [n = 12]). Comparative results (left side) between human T-iPS cells (TkT3V1-7) and active CD4 T lymphocytes (PB CD4 T-cell) in an exhaustive gene expression pattern using an oligonucleotide DNA microarray (left side), and an exhaustive gene expression pattern FIG. 4 is a scatter diagram showing the comparison results (right side) between TkT3V1-7 and human ES cells (KhES3). Note that two parallel lines in the figure indicate that there was a 5-fold difference between the corresponding samples. It is a microscope picture which shows the karyotype (Karyotype) of two typical examples (TkT3V1-7 and TkT4V1-3) of a T-iPS cell. The number of passages of the examined T-iPS cells was 10, and no chromosomal abnormality was observed in any of them. It is a microscope picture which shows the typical HE stained section of the teratoma formed in NOD-Scid mouse 8 weeks after injecting T-iPS cell. Tissues derived from three germ layers (neural tissue, neural plate for ectoderm and retinocytes), cartilage and myocytes for mesoderm, and stomach for endoderm It shows that gut-like mucosa and secretory glands / ducts are present. It is an electrophoresis photograph which shows the result of the PCR analysis for detecting the gene rearrangement of TCR (gamma) chain. Fig. 4 is an electrophoretic photograph showing a representative result of PCR analysis for detecting TCRG reconstitution. The reconstituted TCRG was identified by a PCR band in an effective size range (about 200 bp). FIG. 4 is an electrophoretic photograph showing a representative result of PCR analysis for detecting TCRB reconstitution. In the figure, the PCR products in the “Tube A” and “Tube B” columns show V (D) J reconstitution (effective size range: 240 to 285 bp). On the other hand, the PCR product in the “Tube C” column indicates that it has been DJ-reconstructed (effective size range: 170 to 210 bp, and 285 to 325 bp). It is an electrophoresis photograph which shows the representative result of PCR analysis for detecting TCRA reconstitution in TkT3V1-7. FIG. 13 shows the results of sequencing the PCR products derived from TkT3V1-7 shown in FIGS. 11 and 12. In the figure, a functional (productive) rearrangement in one allele of the TCRA gene is shown at the top. The middle part shows the functional rearrangement (V (D) Jβ rearrangement) in one allele of the TCRB gene. The non-functional (Unproductive) rearrangement (DJβ rearrangement) in the other allele of the TCRB gene is shown below. CD3 ⁺ CD56 ^- peripheral blood T cells are first divided into CD4 expression subsets and CD8 expression subsets, and further, naive T cells (Naive, CD45RA ⁺ CD62L ⁺ ), central memory T cells (Central Memory, CD45RA ^- CD62L ⁺ ), effector memory T FIG. 9 is a scatter diagram showing the results of classification into cells (Effector Memory, CD45RA ⁻ CD62L ⁻ ) or terminal effector T cells (Effector, CD45RA ⁺ CD62L ⁻ ). Based on ALP ⁺ number of colonies obtained from 1 × 10 ⁵ cells of T cell subsets were again plated on MEF, the results of evaluation of the reprogramming efficiency in each of the T cell subset, a graph. In the figures, the vertical axis represents the average number ± S.D. Of ALP ⁺ colonies in three independent experiments. D. Is shown. (A) is a figure which shows the outline | summary of T lineage cell production from a pluripotent cell by culture | cultivation on OP9 and OP9-DL1 stromal cell layer, and the micrograph which shows the state of the cell in each time point. In the figure, “Cytokine cocktail (cytokine cocktail)” indicates that hIL-7, hFlt-3L, and hSCF were added. (B) is a scatter diagram showing the results of examining the expression of CD45, CD3, CD4, CD8, and TCRαβ using T cell lineage cells produced from each pluripotent cell using flow cytometry. In the figures, "ES" indicates T lineage cells derived from ES cells, "skin iPS" indicates T lineage cells derived from skin iPS cells, and "T-iPS" indicates T lineage cells derived from T-iPS cells. , “PB” indicates peripheral blood (control data). (A) is a diagram schematically showing the generation of T lineage cells from pluripotent cells such as T-iPS cells by culturing on OP9 and OP9-DL1 stromal cell layers. That is, pluripotent cells are cultured on an irradiated OP9 cell layer for 10 to 14 days in an essential medium containing no cytokine and based on α-MEM, and then induced hematopoietic cells are converted to OP9-DL1 cells. FIG. 3 shows that the cells were cultured for 3 or 4 weeks in an αMEM-based essential medium supplemented with hIL-7, hFlt-3L, hSCF and the like. (B) At each culture time, the suspension cells in the medium were collected, and the expression of T lineage markers CD45, CD34, CD3, CD4, CD8, and TCRαβ were examined using flow cytometry. It is a scatter diagram which shows the typical result in the experiment of 1 time. In addition, the numerical values shown in each scatter diagram indicate the ratio (%) of cells in each section. The bottom of B shows the result of analyzing fresh PBMC (control data). FIG. 4 is a scatter plot analyzing the expression of T lineage cell markers by flow cytometry and showing representative results in at least three independent experiments. In the figures, the numerical values shown in each scatter diagram indicate the percentage (%) of cells in each section, "Human ESC" indicates the results in human ES cells (KhES3), and "CB-iPS" indicates umbilical cord blood CD34 positive. The results in cell-derived iPS cells (TkCB7-4) are shown, “HDF-iPS” shows the results in human skin fibroblast-derived iPS cells (TkDA4M), and “T-iPSC” is the T-iPS cells (TkT3V1-7). ), And “PBMNC” indicates the result in peripheral blood mononuclear cells. FIG. 2 is a graph showing CD4 and CD8 expression on CD3 ⁺ T lineage cells, evaluated by flow cytometry. In addition, in the figure, the vertical axis represents the relative distribution ± S.T. D. Is shown. "Human ESC" shows the result in human ES cells, "CB-iPSC" shows the result in cord blood CD34-positive cell-derived iPS cells, and "HDF-iPSC" shows the result in human skin fibroblast-derived iPS cells. And "T-iPSC" indicates the results in T-iPS cells. The column of each cell indicates "DN", "CD8", "CD4", and "DP" in order from the left. Further, “DN” indicates CD4 ⁻ CD8 ⁻ , “CD4” indicates CD4 ⁺ CD8 ⁻ , “CD8” indicates CD4 ⁻ CD8 ⁺ , and “DP” indicates CD4 ⁺ CD8 −. ⁺ Graphs and photomicrographs showing proportional change and morphological change in redifferentiated T lineage cells after TCR stimulation with OKT-3 and hIL-2 and unstimulated redifferentiated T lineage cells. is there. FIG. 4 is a scatter diagram showing the results of analyzing the cytokine production profile of regenerated T lineage cells by flow cytometry. The analyzed redifferentiated T lineage cells (CD3 ⁺ cells from TkT3V1-7) or PBMNC (PB CD3, control) were stimulated with PMA / ionomycin together with hIL-1α overnight and stained with the antibody. The numerical values shown in the plots indicate the percentage (%) of cytokine-producing cells. It is a figure which shows the sequence and amino acid sequence of TCR (beta) mRNA in a re-differentiated T lineage cell (T lineage cell derived from TiPS cell). It is a figure which shows the sequence and amino acid sequence of TCRa mRNA in a redifferentiated T lineage cell (T lineage cell derived from TiPS cell). It is an electrophoresis photograph showing the result of amplifying the TkT3V1-7 TCR by PCR through the construction of a cDNA library by SMART and analyzing the obtained PCR product. FIG. 25 is a diagram showing the results of sequencing analysis on PCR products in the effective size range (approximately 750 bp) shown in FIG. 24, wherein the sequences of all transcribed mRNAs are the sequences of the genome of T-iPS cells (see FIG. 13). FIG. 2 shows that no offspring from the DJβ reconstituted allele were identified. Tables 8 and 9 show details and results of other samples. It is a graph which shows the result of having evaluated CD3 expression by the flow cytometry. In the figure, the vertical axis indicates the ratio of CD3 ⁺ T lineage cells in CD45 ⁺ blood cells (Averages ± SD of 3 or more independent experiments in each group). "Human ESC" indicates the results in human ES cells, "CB-iPSC" indicates the results in cord blood CD34-positive cell-derived iPS cells, and "HDF-iPSC" indicates the results in human skin fibroblast-derived iPS cells. And “T-iPSC” indicates the results in T-iPSCs. In addition, ** indicates that p <0.01 for all other samples. It is a graph which shows the average fluorescence intensity (MFI, Mean fluorescence intensity) of CD3 evaluated by the flow cytometry. The vertical axis represents the averages ± S.D. Of independent experiments of CD-iPS and HDF-iPS in each group 2 and others in each group 3 or more. D, "Human ESC" indicates the result in human ES cells, "CB-iPSC" indicates the result in cord blood CD34-positive cell-derived iPS cells, and "HDF-iPSC" indicates human skin fibroblast-derived iPS cells. , “T-iPSC” indicates the result in T-iPS cells, and “PB” indicates the result in peripheral blood mononuclear cells. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the rearrangement of a TCRG gene and the sequence and position of a primer for detecting the rearranged state. The human TCRG locus extends over 135 kb on chromosome 7q14. Further, the forward primer and the reverse primer are each designed in a region having high homology in the Vγ segment or the Jγ segment, and by detecting a PCR band of about 200 bp, the TCRG gene can be reconstituted. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the rearrangement of a TCRB gene and the sequence and position of a primer for detecting the rearranged state. The human TCRB locus extends over 685 kb on chromosome 7q34. Further, in the Vβ gene segment and the Jβ gene segment, 23 Vβ primers, 2 Dβ primers, and 13 Jβ primers can be annealed to the same conserved region. Designed. Further, the primers were divided into three tubes, each of which was mixed and used for analysis. That is, “Tube A”, “Tube B”, and “Tube C” in the figure indicate this. Also, by detecting a PCR band of about 270 bp using the primer set shown in Tube A and Tube B, V (D) Jβ rearrangement can be detected. Using the primer set shown in Tube C, DJβ rearrangement can be detected by detecting a PCR band of 170 to 210 bp or 285 to 325 bp.

<Method for producing human T cells>
The present invention provides a method for producing human T cells, comprising a step of inducing iPS cells from human T cells and a step of differentiating the iPS cells into T cells.

-Step of inducing iPS cells from human T cells-
The “human” from which T cells are isolated in the present invention is not particularly limited. It may be a healthy person, a person having a reduced immune function, or a person suffering from a malignant tumor, an infectious disease, an autoimmune disease, or the like. When the T cells obtained by the present invention are used for immune cell therapy, from the viewpoint that rejection does not occur, the human from whom the T cells are isolated is the same as the human to which the T cells obtained according to the present invention are administered. Are preferably the same, and more preferably, the T cells obtained according to the present invention are the same as the human to which they are administered.

In the present invention, “T cell” means a cell that expresses an antigen receptor called a T cell receptor (TCR) on the surface.
In the present invention, “human T cells induced into iPS cells” and “human T cells differentiated from iPS cells” are not particularly limited, but are preferably selected from the group consisting of CD3 and CD4 and CD8. A T cell expressing at least one selected molecule. Such human T cells include, for example, helper / regulatory T cells that are CD4 positive cells, cytotoxic T cells that are CD8 positive cells, and naive T cells (CD45RA ⁺ CD62).
L ⁺ cells), central memory T cells (CD45RA ^- CD62L ⁺ cells), effector memory T cells (CD45RA ^- CD62L ^- cells), and terminal effector T cells (CD45RA ⁺ CD62L ^- cells). When performing immunotherapy described below, it is preferable that human T cells differentiated from iPS cells have the same antigen specificity as human T cells induced into iPS cells. Note that antigen specificity in T cells is provided by an antigen-specific, reconstituted TCR gene.

  In the present invention, “human T cells induced into iPS cells” can be isolated from human tissues by a known method. The human tissue is not particularly limited as long as it is a tissue containing the T cell, and examples thereof include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and diseased tissue. Among these, peripheral blood and cord blood are preferred from the viewpoint of low invasiveness to humans and easy preparation. Known techniques for isolating human T cells include, for example, flow cytometry using an antibody against a cell surface marker such as CD4 and a cell sorter as described in Examples below. Also, desired T cells can be isolated using cytokine secretion and expression of functional molecules as indices. In such a case, for example, since T cells secrete different cytokines depending on the Th1 type or Th2 type, T cells having a desired Th type can be isolated by selecting such cytokines as an index. . In addition, cytotoxic (killer) T cells can be isolated using secretion or production of granzyme, perforin, or the like as an index.

  When “human T cells having antigen specificity” are used as human T cells to be induced into iPS cells, in the isolation thereof, the desired T cells having “antigen specificity” can be obtained from human tissues containing “T cells having desired antigen specificity”. A purification method using an affinity column or the like on which an antigen is immobilized can be employed. Further, using a tetramerized MHC (major histocompatibility complex) to which a desired antigen is bound (so-called “MHC tetramer”), a THC having a desired antigen specificity is obtained from human tissue. A method for purifying "cells" can also be employed.

  The “iPS cell” in the present invention is a cell also called an induced pluripotent stem cell or an induced pluripotent stem cell, and is induced by introducing a cell reprogramming factor into the T cell. can do. The “cell reprogramming factor” is particularly a factor that, when introduced into the T cell, alone or in cooperation with another pluripotency factor, can impart pluripotency to the somatic cell. Without limitation, Oct3 / 4, c-Myc, Sox2, Klf4, Klf5, LIN28, Nanog, ECAT1, ESG1, Fbx15, ERas, ECAT7, ECAT8, Gdf3, Sox15, ECAT15-1, ECAT15-2, It is preferably at least one kind of protein selected from the group consisting of Fth117, Sal14, Rex1, Utf1, Tcl1, Stella, β-catenin, Stat3 and Grb2. Further, among these proteins, it is more preferable to introduce Oct3 / 4, c-Myc, Sox2, and Klf4 (Four factors) into the somatic cells from the viewpoint that iPS cells can be efficiently established with a small number of factors. From the viewpoint of reducing the risk of canceration of the obtained pluripotent stem cells, it is more preferable to introduce Oct3 / 4, Sox2, and Klf4 (three factors) excluding c-Myc into the somatic cells.

  When human CD8-positive T cells are induced into iPS cells, OCT4, SOX2, KLF4, C-MYC, and NANOG are introduced into human CD8-positive T cells from the viewpoint of increasing the efficiency of iPS cell induction. It is more preferable to introduce OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28 into human CD8-positive T cells.

In the present invention, the method for “introducing a cell reprogramming factor into T cells” is not particularly limited, and a known method can be appropriately selected and used. For example, when the cell reprogramming factor is introduced into the T cell in the form of a protein, a method using a protein introduction reagent, a method using a protein transduction domain (PTD) fusion protein, an electroporation method, a microinjection method Is mentioned. In the case where the nucleic acid encoding the cell reprogramming factor is introduced into the T cell in the form of a nucleic acid encoding the cell reprogramming factor, the nucleic acid (eg, cDNA) encoding the cell reprogramming factor is appropriately converted into a nucleic acid encoding a T cell. The expression vector can be introduced into cells by infection, lipofection, liposome, electroporation, calcium phosphate coprecipitation, DEAE dextran, microinjection, or electroporation. .

  The “expression vector” according to the present invention includes, for example, viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, and animal cell expression plasmid.

  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. 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). The expression vector may further contain, in addition to the promoter, an enhancer, a polyA addition signal, a selection marker gene (for example, a neomycin resistance gene), an SV40 replication origin, and the like.

  In the “step of inducing iPS cells from human T cells”, as described in Examples below, the T cells are treated with interleukin-2 (IL-2) before introducing the cell reprogramming factor. Preferably, it is stimulated and activated by an anti-CD3 antibody and an anti-CD28 antibody in the presence. Such stimulation can be performed, for example, by adding IL-2, an anti-CD3 antibody, and an anti-CD28 antibody to a medium and culturing the T cells for a certain period of time, as described in Examples below. Further, the anti-CD3 antibody and the anti-CD28 antibody may be those to which magnetic beads or the like are bound, and instead of adding these antibodies to the medium, the anti-CD3 antibody and the anti-CD28 antibody are bound to the surface. Stimulation may be provided by culturing the T cells on a culture dish for a certain period of time. Furthermore, stimulation may be provided by adding an antigenic peptide recognized by human T cells together with feeder cells to the medium.

  In order to perform such stimulation, the concentration of IL-2 added to the medium is not particularly limited, but is preferably 1 to 200 ng / ml. Further, the concentration of the anti-CD3 antibody and anti-CD28 antibody added to the medium in such stimulation is not particularly limited, but is preferably 1 to 10 times the culture amount of the T cells. The concentration of the anti-CD3 antibody and the anti-CD28 antibody bound on the surface of the culture dish in such stimulation is not particularly limited, but the concentration at the time of coating is 1 to 100 μg / ml for the anti-CD3 antibody, Is preferably 0.1 to 10 μg / ml.

  In addition, the culture period for performing such a stimulus is a period sufficient to provide the stimulus to the T cells, and the T cells can be expanded to the number of cells necessary for introducing the cell reprogramming factor. The period is not particularly limited, but is usually 2 to 14 days, and preferably 2 to 7 days from the viewpoint of gene transfer efficiency. Furthermore, it is preferable to culture on a culture dish coated with RetroNectin from the viewpoint of increasing the gene transfer efficiency.

  Examples of a medium for culturing the T cells and adding IL-2, an anti-CD3 antibody, an anti-CD28 antibody or the like include, for example, a known medium suitable for culturing the T cells (more specifically, IL-2 and the like). Roswell Park Memorial Laboratory (RPMI) 1640 medium, minimum essential medium (α-MEM), Dulbecco's modified Eagle medium (DMEM), F12 medium, etc., containing the following cytokines and fetal calf serum (FCS): . In addition to IL-2, anti-CD3 antibody and anti-CD28 antibody, the medium may be supplemented with amino acids (eg, L-glutamine) and antibiotics (eg, streptomycin, penicillin) necessary for culture. . When CD8-positive T cells are induced into iPS cells, it is preferable to add IL-7 and IL-15 to the medium from the viewpoint of suppressing apoptosis. The concentration of IL-7 or IL-15 to be added is not particularly limited, but is preferably 1 to 100 ng / ml.

  In the present invention, the conditions for “introducing a cell reprogramming factor into T cells” or thereafter are not particularly limited, and the T cells into which the cell reprogramming factor has been introduced are cultured on a feeder cell layer. Is preferred. Such feeder cells are not particularly limited, and include, for example, mouse embryonic fibroblasts (MEF), STO cells, and SNL cells whose cell division has been stopped by irradiation or treatment with antibiotics.

  Furthermore, in the process of inducing the T cells into iPS cells, from the viewpoint of suppressing cell differentiation, when introducing basic fibroblast growth factor (bFGF) into the T cells, Thereafter, it is preferable to add the medium.

  In order to further increase the establishment efficiency of iPS cells, a histone deacetylase (HDAC) inhibitor (a small molecule inhibitor such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, M344, and siRNA against HDAC Etc.), G9a histone methyltransferase inhibitors (Small molecule inhibitors such as BIX-01294, siRNA against G9a, etc.), p53 inhibitors (Small molecule inhibitors such as Pifithrin-α (PFT-α), siRNA against p53, etc.) Is preferably added to the medium when or after the cell reprogramming factor is introduced into the T cells.

  Furthermore, when a virus vector is used in "introducing a cell reprogramming factor into T cells", protamine sulfate should be added to the medium from the viewpoint that the vector is easily bound to the T cells. Is preferred.

  In addition, as shown in Examples described later, in accordance with the transition from the T cells to iPS cells, a known medium suitable for culturing the T cells was gradually replaced with a medium suitable for culturing iPS cells. It is preferable to culture the cells while they are live. As a medium suitable for such iPS cell culture, a known medium can be appropriately selected and used. For example, a knockout serum substitute, L-glutamine, a non-essential amino acid, 2-mercaptoethanol, b-FGF and the like can be used. Contained Dulbecco's modified Eagle's medium / F12 medium (human iPS cell medium).

  In the case where human CD8-positive T cells are induced into iPS cells, from the viewpoint of further increasing the efficiency of inducing iPS cells, 1 to 1000 μM after transfer onto the feeder cells, as described in Examples below. It is preferable to culture in a medium further supplemented with a ROCK inhibitor under low oxygen concentration conditions (oxygen concentration: 5%, for example), and 1 to 1000 μM MEK inhibitor (for example, PD0325901) and 1 to 1000 μM GSK3 inhibitor (for example, , CHIR99021) is preferably added to the medium until colony formation described below.

  The selection of iPS cells (hereinafter also referred to as “TiPS cells”) derived from the T cells in this manner can be performed by appropriately selecting a known method. Examples of such known techniques include, for example, a method of observing the morphology of ES cells / iPS cell-like colonies under a microscope as described in Examples below, and a method of detecting the specific expression in iPS cells. Using a recombinant T cell in which a drug resistance gene or a reporter gene (such as a GFP gene) is targeted at the gene locus of the gene (eg, the cell reprogramming factor), and selecting the drug resistance or reporter activity as an index. Method.

Confirmation that the cells selected in this way are iPS cells can be performed, for example, by confirming the undifferentiated cell-specific markers (ALP, SSEA-4, Tra- 1-60, Tra-1-81, etc.) by immunostaining, RT-PCR, or the like, or by transplanting selected cells into mice and observing teratoma formation. .

  In addition, confirmation that the cells selected in this way are derived from the T cells can be performed, for example, by detecting the state of TCR gene rearrangement by genomic PCR, as described in Examples below. it can.

  The time to select and collect these cells can be appropriately determined while observing the growth state of the colony, and is generally 14 to 28 days after the cell reprogramming factor is introduced into the T cells. .

-Step of differentiating TiPS cells into T cells-
In order to differentiate the TiPS cells obtained as described above into T cells, from the viewpoint of facilitating the induction of mesodermal differentiation, first, the TiPS cells are made to contain cytokines on stromal cells. Preferably, the cells are cultured in an α-MEM medium containing FCS or the like. The stromal cells used are preferably OP9 cells and 10T1 / 2 cells that have been subjected to treatment such as irradiation, from the viewpoint of facilitating the induction of differentiation into hematopoietic systems. However, when efficiently inducing TiPS cells to CD34-positive hematopoietic stem cells, it is preferable to culture in a medium containing at least one cytokine selected from VEGF, SCF, TPO, SCF, and FLT3L groups, More preferably, the cells are cultured in a medium containing VEGF, SCF, and TPO, or in a medium containing VEGF, SCF, and FLT3L. The culturing period is preferably a period until a bag-like structure (also referred to as ES-sac (sac)) containing blood cells and the like is formed. Preferably it is days.

  The cells (hematopoietic progenitor cells, CD34-positive cells, blood cells, etc.) contained in the sac obtained as described above are cultured on stromal cells in an α-MEM medium containing cytokines, FCS, etc. Is preferred. The cells existing inside the sac-like structure can be separated by physical means, for example, by passing the cells through a sterilized sieving device (for example, a cell strainer or the like). As stromal cells used for this culture, OP9-DL1 cells, OP9-DL4 cells, and 10T1 / 2 / cells that have been subjected to treatment such as irradiation from the viewpoint of inducing differentiation into T lymphocytes via a notch signal. Preferably, it is a DL4 cell. The cytokine to be added to the medium includes, for example, IL-7, FLT3L, VEGF, SCF, TPO, IL-2, IL-15. Among these, IL-7 and FLT3L are preferable from the viewpoint of assisting the differentiation of early T cells. From the viewpoint of suppressing the differentiation of TiPS cells into NKT cells, SCF is preferably not contained in the medium. The concentration of IL-7 added to the medium is 0 from the viewpoint that CD3 positive CD56 negative T lineage cells are easily obtained and differentiation into CD4 single positive (SP) T cells or CD8 SPT cells is easily induced. It is preferably from 0.1 to 4 ng / ml.

  It was confirmed that the cells induced to differentiate in this manner were derived from TiPS cells and derived from the T cells from which the TiPS cells were derived, for example, by reconstituting the TCR gene as described in Examples below. Can be carried out by detecting the above state by genomic PCR.

The T cells thus obtained can be isolated by appropriately selecting a known method. Such a known technique includes, for example, flow cytometry using an antibody against a cell surface marker such as CD4 and a cell sorter as described in Examples below. When "T cells having desired antigen specificity" are isolated from humans, a method of purifying using an affinity column or the like on which a desired antigen is immobilized can be adopted. In addition, a method of purifying “T cells having desired antigen specificity” using MHC tetramer to which a desired antigen is bound can also be adopted.

<Human T cell, pharmaceutical composition, method of immune cell therapy>
Since human T cells produced by the method of the present invention have an antigen-specific immune function, they can be used, for example, for treating or preventing diseases such as tumors, infectious diseases, and autoimmune deficiencies.

  Accordingly, the present invention provides a human T cell produced by the method of the present invention, a pharmaceutical composition containing the human T cell, and a method for treating an immune cell using the human T cell.

  The pharmaceutical composition of the present invention can be prepared by formulating human T cells produced by the method of the present invention by known pharmaceutical methods. For example, it can be mainly used parenterally as capsules, solutions, film coatings, suspensions, emulsions, injections (intravenous injections, drip injections, etc.).

  In these formulations, pharmacologically acceptable carriers or vehicles, specifically, sterile water or physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, vehicles, Preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffers, coatings, lubricants, coloring agents, solubilizing agents or other additives as appropriate Can be. Further, it may be used in combination with a known pharmaceutical composition or immunostimulant used for treatment or prevention of the above-mentioned disease.

  When administering the pharmaceutical composition of the present invention, the dose is appropriately selected according to the age, weight, symptom, health condition, type of the composition (medicine, food and drink, and the like) of the subject.

  The product (pharmaceutical product) of the composition of the present invention or a description thereof may be provided with an indication that it is used for treating or preventing a decrease in immune function. Here, "the label is attached to the product or instruction" means that the label is attached to the product body, container, packaging, etc., or the instruction, package insert, advertisement, or other printed matter that discloses the product information. It means that the display is attached to the like.

  The method for treating an immune cell of the present invention comprises the steps of isolating T cells having a desired antigen specificity from humans, inducing iPS cells from the T cells having the desired antigen specificity, To a T cell, and a step of administering the obtained iPS cell-derived T cell into a human body. When performing the method of the present invention for treating an immune cell, from the viewpoint that rejection does not occur, the human from whom the T cells obtained by the present invention are administered has the same type of HLA as the human to which the T cells obtained by the present invention are administered. It is preferable that the T cells obtained according to the present invention are the same as the human to which the T cells are administered. As the human T cells to be administered, the human T cells produced by the method of the present invention may be administered as they are, or may be administered in the form of a pharmaceutical composition formulated as described above.

  Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples. In this example, the following materials were used in accordance with the following method and the like.

<Flow cytometry>
Flow cytometry analysis was performed using MoFlo (manufactured by Dako Cytomation), FACSAria (registered trademark, manufactured by BD Bioscience), or FACSCanto II (registered trademark, manufactured by BD Bioscience). Analysis of the obtained data was performed using FlowJo software (manufactured by Treestar). The antibodies used for flow cytometry analysis are as follows.
Anti-human CD3-APC antibody (manufactured by BD Bioscience), anti-human CD4-FITC antibody (manufactured by BD Bioscience), anti-human CD8-PerCP / Cy5.5 antibody (manufactured by BD Bioscience), anti-human CD56-PE antibody (manufactured by BD Bioscience) BD Bioscience), anti-human CD45RA-Pacific Blue antibody (Caltag
Laboratories), anti-human CD62L-PE-Cy7 antibody (Bioleend), anti-human CD45-Alexa 405 antibody (Molecular Probes-Invitrogen, Carlsbad, CA, USA), anti-human CD34-PE antibody (BD Bioc) ), Anti-human CD38-PerCP / Cy5.5 antibody (Bioleend), anti-human CD1a-APC antibody (Bioleend), anti-human CD5-PE / Cy7, -CD7-FITC antibody (Bioleend), anti-human Human CD5-PE / Cy7 antibody (Bioleend), anti-human CD7-FITC antibody (Bioleend), anti-human TCRaβ-FITC antibody (Bioleend). Furthermore, in the flow cytometry analysis, the cells were incubated at 4 ° C. for 30 minutes with an antibody cocktail adjusted to an appropriate concentration, and then washed with phosphate buffered saline. Also, propidium iodide was added to exclude dead cells.

<Preparation of retrovirus>
The pMXs retrovirus vector encoding human OCT4, SOX2, KLF4, c-MYC, and NANOG (Yamanaka factor, YFs) was provided by Professor Shinya Yamanaka (affiliated with iPS Cell Research Institute, Kyoto University). These YFs were introduced into 293GPG packaging cells having a Gag-Pol gene and a VSV-G pseudo-type envelope gene controlled by Tet-OFF. Three days after the virus was induced in the medium excluding tetracycline, culture supernatants of these cells were collected every day for a total of four times. The collected culture supernatant was passed through a 0.45 μm cellulose acetate filter and centrifuged at 6000 g for 16 hours. Then, the obtained supernatant was suspended in α-MEM (α-minimum essential medium) so that the concentration of the obtained supernatant was 0.5%, and stored at −80 ° C. until use.

<Preparation of T cells, retrovirus infection, and preparation of iPS cells>
The experimental protocol was approved by the Human Ethics Review Committee of the Institute of Medical Science, The University of Tokyo (Approval number: 20-6-0826). All studies were conducted in accordance with the Helsinki Declaration.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood from healthy subjects by Ficoll density gradient centrifugation (Ficoll-Paque PLUS (registered trademark), 17-1440-02, manufactured by GE Healthcare), and MoFlo ( (Manufactured by DAKO Cytomation). T cells were isolated by gating the CD3 ⁺ CD56 ^- cell population to avoid contamination of natural killer T (NKT) cells. T cell subsets were further gated and separated into CD4 (CD4 ⁺ CD8 ⁻ ) and CD8 (CD4 ⁻ CD8 ⁺ ) cohorts. CD4 and / or CD8 cells can also be na ナ イ ve (CD45RA ⁺ CD62
L ⁺ ), central memory (CD45RA ^- CD62L ⁺ ), effector memory (C
D45RA ^- CD62L ^- were classified into) ^-), or terminal effector (CD45RA ⁺ CD62L.

Cells sorted in this way were 10% fetal bovine serum (GIBCO-Invitrogen), 100 U / ml penicillin, 100 ng / ml streptomycin, 2 mM L-glutamine, and 20 ng / ml human interleukin 2 (hIL-2, The cells were first cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (GIBCO-Invitrogen) supplemented with Novartis Vaccines & Diagnostics. Then, these cells were activated by adding three times the amount of anti-CD3 / CD28-bound magnetic beads (Dynabeads (registered trademark), ClinExVivo (registered trademark) CD3 / CD28, manufactured by Invitrogen) to these T cells. . In the production of iPS cells (T-iPS cells) derived from T cells, the day of activation is defined as day 0 (hereinafter, see FIG. 1). In some experiments, CD3 ⁺ cells magnetically captured from PBMCs were isolated and stimulated simultaneously with anti-CD3 / CD28-coupled magnetic beads. On day2 and day3, retrovirus cells were cultured by spinoculation on a RetroNectin (registered trademark) coating plate (manufactured by Takara) to which 10 μg / ml protamine sulfate (manufactured by Sigma-Aldrich) was added. (Kaneko, S. et al., Blood, 2009, 113, 1006-1015). The complete synthetic medium for T cell culture was changed daily. At day 6, infected cells were harvested and transferred onto 3 × 10 ⁵ irradiated MEF cell layers on a 6 cm dish. For the next four days (day 6 to day 10), half of the medium was replaced with human iPS cell medium every day. The composition of the human iPS cell culture medium is as follows (see Takayama, N. et al., Blood, 2008, 111, 5298-5306).

20% Knockout Serum Replacement (Knockout Serum Replacement (registered trademark), GIBCO-Invitrogen), 200 μM L-glutamine (Invitrogen), 1% Non-essential amino acids (Invitrogen), 10 μM 2-mercaptoethanol (GIBCO) -Dulbecco's modified Eagle medium / F12 medium (Sigma-Aldrich) to which 5 ng / ml b-FGF (Wako) was added.

Before picking up iPS cell colonies, 0.5 mM VPA (HDAC inhibitor valproic acid) was added to the human iPS cell medium. At day 10, the whole medium was replaced with a human iPS cell medium containing VPA. At approximately day 24, when they could be identified with human ES / iPS cell-like colonies, they were mechanically isolated, dissociated into small nodules by pipetting, and plated on fresh MEF cell layers. Human ES / iPS cell-like colonies were grown on fresh MEF cell layers using trypsin solution (phosphate buffered saline with 0.25% trypsin, 1 mM CaCl ₂ , and 20% knockout serum replacement). Transferred every four days.

<Chromosome analysis>
The chromosome G band analysis was outsourced to the Japan Genetic Institute, and was performed according to a predetermined method.

<Alkaline phosphatase (ALP) staining and immunocytochemical staining>
For ALP staining, human ES / iPS cell-like colonies were fixed with an ice-cold fixative (90% methanol, 10% formaldehyde), using an ALP staining kit (manufactured by Vector Laboratories) according to the manufacturer's instructions. Stained. For immunocytochemical staining, human ES / iPS cell-like colonies were fixed with 5% paraformaldehyde and permeabilized with 0.1% Triton X-100.

The colonies thus pretreated were incubated with the primary antibody. The primary antibody used and the dilution ratio are as follows.
PE-conjugated anti-SSEA-4 (FAB1435P, manufactured by R & D Systems, dilution ratio: 1/50), anti-Tra-1-60 (MAB4360, manufactured by Millipore, dilution ratio: 1/100), anti-Tra- 1-81 (MA
B4381, manufactured by Millipore, dilution ratio: 1/100). For detection of Tra-1-60 and Tra-1-81, Alexa Fluor 488-conjugated goat anti-mouse antibody (dilution ratio: 1/500, A11029, manufactured by Molecular Probes-Invitrogen) was used as a secondary antibody. Using. Furthermore, counterstaining of the nucleus was performed using 4 ', 6-diamidino-2-phenylindol (dilution ratio: 1/1000, manufactured by Roche Diagnostics). In addition, the micrograph is a fluorescence microscope Axio Observer. Photographs were taken using Z1 (Carl Zeiss Japan).

<Teratoma formation>
Human ES / iPS cell-like colonies were aggregated, and the testis medulla on the left side of the NOD-Scid mouse was found in Masaki, H .; Et al., Stem Cell Res, 2007, vol. 1, pages 105-115. The number of cells injected was 1.0 × 10 ⁶ per mouse. Eight weeks after the injection, the tumor formed in the testis was excised, fixed with 5% paraformaldehyde, embedded in paraffin, and sectioned. Then, the obtained section was stained by a hematoxylin / eosin method, and examined with an optical microscope to determine whether or not the iPS cells had the ability to differentiate into three germ layers.

<Expression analysis of pluripotency gene and T cell-related gene>
ES cells, iPS cells (cells about 50 days after cloning), their progeny, and freshly isolated peripheral blood CD3 ⁺ T cells were obtained from the RNeasy micro kit (Q
iagen) was used to extract total RNA. Then, using the obtained total RNA as a template, a reverse transcription reaction was performed using PrimeScript II 1st Strand Synthesis Kit (manufactured by Takara).
In addition, PCR was performed using ExTaq HS (manufactured by Takara) at 30 cycles for the housekeeping gene (GAPDH or ACTB) and at 35 cycles for all pluripotency genes or T cell-related genes.
The target gene and the sequences of the PCR primers used for the analysis are shown in Tables 1 and 2.

<Microarray analysis>
Expression analysis of whole genome genes was performed on human ES cells (KhES3), T cell-derived iPS cells (TkT3V1-7), and CD3 / CD28-stimulated CD3 ⁺ CD4 ⁺ CD8 ^- cells. First, total RNA (2 μg) was extracted from each cell and used for probe preparation. Then, the fluorescently labeled complementary RNA was hybridized with Whole Human Genome Microarray 4 × 44K (G4112F, manufactured by Agilent Technologies) by a one-color method (consignment: Takara Bio Inc.). Next, the array was scanned and a spot image was detected with an Agilent Feature Extraction apparatus (manufactured by Agilent Technologies). Then, the obtained signal data is stored in GeneSpring software (Agi
Lent Technologies). Two normalization procedures were applied for normalization of the obtained data. That is, signal intensity less than 0.01 was set to 0.01. In addition, each chip was normalized to 50 ^th percentile of the measured value obtained from the chip. Then, in all three samples, analysis was performed on the gene with the present flag (36275 genes).

<Detection of TCR reconstitution in genomic DNA of T-iPS cells>
Genomic DNA was extracted from about 5 × 10 ⁶ T-iPS cells using a QIAamp DNA kit (Qiagen). The extracted DNA (40 ng) was used to detect each of TCRG, TCRB, and TCRA gene rearrangements by PCR.

PCR to detect TCRG rearrangement (Benhattar, J. et al., Diagn.
Mol Pathol, 1995, Vol. 4, pp. 108-112) was performed using ExTaq HS (manufactured by Takara). The PCR reaction conditions were 95 ° C. for 5 minutes, followed by a reaction having a relatively high annealing temperature, ie, 95 ° C. for 45 seconds, 65 ° C. for 45 seconds, 72 ° C. for 45 seconds, and 5 cycles. The reaction having a relatively low annealing temperature, that is, a reaction of 95 ° C. for 45 seconds, 55 ° C. for 45 seconds, and 72 ° C. for 30 seconds was performed in 30 cycles.
To detect TCRB rearrangement, PCR and heteroduplex analysis were performed according to the BIOMED-2 protocol with minor modifications (see van Dongen, J. Leukemia, 2003, Vol. 17, pages 2257-2317). I went.

  Primer constructs for detecting TCRA reconstitution are described in Han et al. (See Han, M. et al., J Immunol, 1999, 163, 301-3111). The PCR reaction conditions were three cycles of an amplification reaction of 95 ° C. for 30 seconds, 68 ° C. for 45 seconds, 72 ° C. for 6 minutes, 95 ° C. for 30 seconds, 62 ° C. for 45 seconds, and 72 ° C. The amplification reaction was performed at 95 ° C. for 15 seconds, at 62 ° C. for 30 seconds, and at 72 ° C. for 6 minutes, for 12 cycles.

  The PCR was performed by LATaq HS (manufactured by Takara). Then, the obtained PCR product was fractionated by gel based on the size. Next, a main band within the expected size range was cut out, purified using a QIAquick gel extraction kit (manufactured by Qiagen), and sequenced. In the sequencing reaction, BigDye terminator kit v3.1 (manufactured by Applied Biosystems) was used together with a set of Vα, Jα, Vβ, Dβ, or Jβ primers to amplify PCR and add a fluorescent dye by a multifaceted approach. . Then, the obtained reaction product was analyzed with an ABI PRISM 3100 automatic sequencer (manufactured by Applied Biosystems).

  In addition, the obtained results show that the V, D, and J segments involved in the construction of TCRA or TCRB were published sequences and ImmunGeneGenetics (IMGT) database (http://www.cines.fr/). For comparison, identification was performed by using a web tool such as v-quest (Lefranc, MP, "IMGT databases, web resources and tools for immunoglobulin and T cell receptor s./recessor seq. Leukemia, 2003, Vol. 17, pages 260-266). Gene fragments (segments) were named according to the IMGT nomenclature. In addition, Tables 3 and 4 show the primer sequences used for detection of TCR reconstitution.

<Induction from T-iPS cells to T-lineage cells>
T-iPS cells or other pluripotent stem cells were obtained from Takayama, N.J., with some modifications so as to adopt an embryonic stem cell-derived sac (ES-sac) -like structure. Et al., Blood, 2008, 111, 5298-5306.

  Pluripotent cells were irradiated on the OP9 cell layer and supplemented with α-MEM based medium without cytokine (20% fetal calf serum, 100 U / ml penicillin, 100 ng / ml streptomycin, and 2 mM L-glutamine). αMEM) for 10 to 14 days (about Day 12).

The floating cells packed in the sack were transferred onto the OP9-DL1 cell layer, and co-cultured to Day 40 in a medium based on αMEM and supplemented with 10 ng / ml hIL-7 and hFlt-3L. The day when the pluripotent cells were transferred onto the OP9 cell layer was designated as Day0. The culture medium was changed every three days. T lineage cells floating on the OP9-DL1 cell layer and expressing CD45, CD3, and TCRαβ were sorted every week by flow cytometry and subjected to gene expression analysis. The OP9 cells and OP9-DL1 cells were obtained from the RIKEN BioResource Center through the National BioResource Project (Japan).

<Response to TCR stimulation and cytokine expression profile of T lineage cells re-differentiated from T-iPS cells>
At the end of induction into T cells (after culturing on the OP9-DL1 cell layer for about 4 weeks, Day 40), suspended cells were collected, and 30 ng / ml OKT-3 (manufactured by Janssen Pharmaceutical) and 600 IU / ml hIL -2 (Novartis Vaccines & Diagnostics). Five days later, the number of cells was counted, and the expression of CD3 was evaluated. Then, a micrograph was taken to obtain a morphological image.

In addition, the cytokine production (expression) profile of T lineage cells was slightly modified in Uckert, W. et al. Et al., Cancer Immunol Immunother, 2008, Vol. 58, pp. 809-822. That is, 1 × 10 ⁵ floating cells were treated with 10 ng / ml phorbol 12-myristate 13-acetate (PMA, manufactured by Sigma-Aldrich), 0.4 μM A23187 calcium ionophore (ionomycin, manufactured by Sigma-Aldrich). ) And 20 IU / ml hIL-1α (Peprotech) for 12 hours. In the last 3 hours, a protein secretion inhibitor brefeldin A (1 mM, manufactured by Sigma-Aldrich) was added and cultured.

  The cells after such stimulation were labeled with anti-CD3 binding magnetic microbeads (Miltenyi Biotec) and retained in a magnetic column. On the other hand, using an intracellular staining kit (Inside stain kit, manufactured by Miltenyi Biotec), solid-state intracellular cytokine staining was applied to these cells according to the manufacturer's instructions. The intracellular levels of hIFN-γ and hIL-2 were measured.

<Detection of TCR rearrangement in mRNA of T lineage cells derived from T-iPS cells>
On day 14 (day 26), day 21 (day 33), and day 28 (day 40) after the culture on the OP9-DL1 cell layer was started, CD3 ⁺ derived from T-iPS cells was used ^.
Total RNA was extracted from TCRαβ ⁺ T lineage cells.
Then, a method based on switch mechanism at the 5'-end of the reverse transcript at the 5 'end of the reverse transcript (SMART method, Du, G. et al., J Immunol Methods, 2006, Vol. 308, 19- CDNA was synthesized using Super SMART (registered trademark) cDNA synthesis kit (manufactured by Clontech Laboratories) according to the manufacturer's instructions. That is, reverse transcription was performed for 90 minutes at 42 ° C. using 3 ′ SMART (registered trademark) CDS primer and SMART II A oligo (Super SMART (registered trademark) cDNA synthesis kit) and PrimeScript Reverse Reverse Transcriptase (manufactured by Takara). The reaction was performed. The synthesis and amplification of the double-stranded cDNA is performed using the reagents contained in 5 'PCR Primer II A (Super SMART (registered trademark) cDNA synthesis kit) and Advantage2 PCR Kit (manufactured by BD Clontech). Was. The reaction conditions of PCR were 20 cycles of 95 ° C. for 5 seconds, 65 ° C. for 5 seconds, and 68 ° C. for 3 minutes.

  Then, the amplified cDNA was used as a template in a TCRA-specific amplification reaction or a TCRB-specific amplification reaction. That is, amplification was carried out using a forward primer (2nd 5'-SMART) and a reverse primer in 25 cycles of a reaction at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 3 minutes for 25 cycles. As the reverse primer, 3'-TRAC was used in TCRA amplification, and 3'-TRBC was used in TCRB amplification. The obtained PCR product was inserted into a pGEM-T-Easy vector (Promega, manufactured by Madison) and sequenced.

<Statistical analysis>
All statistical analyzes in this example were performed by ANOVA or Student's t-test using Excel (manufactured by Microsoft), Prism (manufactured by Graphpad Software), and Statcel2 (manufactured by OMS Publishing). And P <0.05 was considered significant.

(Example 1)
<Establishment of iPS cells from human peripheral blood T cells>
First, iPS cells were established using T cells in human peripheral blood as shown in FIG. That is, peripheral blood mononuclear cells (PBMC) or CD3-positive cells are isolated from peripheral blood of healthy persons (aged persons (aged from 24 to 56) and a plurality of healthy persons having different genders) and stimulated with CD3 / CD28 ClinExVivo beads. did. Then, the cells were cultured for 2 days in RPMI1640 + 10% FBS + PSG (penicillin, streptomycin, and L-glutamine) containing 20 ng / ml of hIL-2, and the VSV-G pseudotype retroviral virus vectors pMX OCT4, pMX SOX2, pMX KLF4, and The gene was transfected using pMXc-MYCm or pMXOCT4, pMXSOX2, and pMXKLF4. After the gene transfer operation for 2 consecutive days, 1 × 10 ⁵ cells were scattered on 6 cm dish of irradiated MEF (mouse fetal fibroblasts) on the 6th day from the separation day, and replaced with iPS medium (human iPS cell culture medium) in half daily amount. 4 days after seeding, the T cell medium was completely replaced with iPS medium. From around 11 days after the separation (5 days after the seeding), a cobblestone-like cell cluster was observed, and after several days (18 to 24 days after the start of the culture), the cells appeared like human ES cells (FIG. 2). : See photo of colony (human ES cell-like colony))). These cells were positive for ALP staining, positive for undifferentiated markers such as SSEA-4, TRA-1-60, and TRA-1-81 (see FIG. 3: Stained photographs of colonies). A similar gene expression pattern was observed (see FIG. 4: electrophoresis photograph). In addition, alkaline phosphatase (ALP) -positive human ES cell-like colonies were respectively obtained from 3 × 10 ⁵ cells into which c-MYC was introduced or not, 77 ± 25 (0.03%) and 5.1. It was obtained at a rate of ± 2.4 (0.001%) (see FIG. 5).

  In addition, microarray analysis was used to compare the gene expression profiles of CD3-derived pluripotent cells with human ES cells (KhES3) and peripheral blood T cells. The overall pattern of gene expression in CD3-derived iPS cells was similar to that in human ES cells, but different from that in T cells (see FIG. 6).

Furthermore, the cell had a normal karyotype (karyotype) (see FIG. 7: Karyotype analysis photograph), and formed a teratoma (teratoma) showing differentiation into three germ layers by transplantation into NOD / SCID mouse testis (see FIG. 7). 8: Teratoma formation pathological findings).
Therefore, it was revealed that iPS cells established from human peripheral blood T cells had pluripotency.

(Example 2)
To confirm that the established iPS cells were derived from T cells, reconstitution of the TCR gene was confirmed. The three groups of TCR genes, TCRδ, TCRγ, and TCRβ, start rearranging early (in this order). Rearrangement of the TCRγ gene was observed in 2 out of 4 clones derived from 4 factors and 6 out of 9 clones derived from 3 factors (see FIG. 9: electrophoresis). Analysis of the rearrangement of the TCRβ and TCRa genes of those clones revealed that all clones had rearranged the TCRβ and TCRa genes in frame (Table 5: Inframe of TCRa chain region). in frame gene rearrangement, see Table 6: in frame gene rearrangement of TCR β chain region). In all of them, there was no NKT cell type combination in which TRAV24 and TRBV11 were specifically used, and it was confirmed that the iPS cells were derived from peripheral blood T cells. In Tables 5 and 6, “iPS Clone” indicates an established T cell-derived iPS cell line, “Rearrangement” indicates a combination of segments used for reconstitution, and “Sequence of junctional region” indicates a binding region. Shows the base sequence.

(Example 3)
Further, as in Example 2, reconstitution of the TCR gene was confirmed in order to confirm that the established iPS cells were derived from T cells. That is, humans have four TCR genes (TCRA, TCRB, TCRG, and TCRD), and it is known that their DNA rearrangements are involved in the development of normal T lymphocytes in the thymus. I have. In addition, the rearrangement of the TCR gene is an irreversible genetic phenomenon specific to T lineage cells, and this genetic phenomenon gives T cells a genetic signature and is characterized. Therefore, by examining these traces, it can be retroactively confirmed whether the iPS cells obtained in Example 1 are derived from mature peripheral T lymphocytes of healthy donors.

First, TCRG or TCRB rearrangement was detected by PCR analysis using a TCR primer set designed by Benhatter et al. And the BIOMED-2 Consortium (see FIGS. 28 and 29, Benhattar, J. et al., Diagn Mol Pathol, 1995). 4, pp. 108-112, van Dongen, JJ, Leukemia, 2003, 17, 2257-2317). As a result, TCRG or TCRB rearrangement was identified in all the examined iPS cell lines, and it was confirmed that all the examined iPS cell lines were derived from peripheral blood T lymphocytes (FIGS. 10 and 11). .
The TCRβ chain forms a heterodimer with the TCRα chain, and most peripheral blood T cells express TCRαβ (Davis, MM, et al., Nature, 1998, 334, 395-395). See page 402). For TCRG and TCRB, the TCRA locus is complex. The TCRA locus is a region that extends over 1000 kb, not only containing the TCRD locus, but also including 103 Vα segments, 61 Jα segments, and Cα segments. Therefore, in order to detect TCRA gene rearrangement in genomic DNA, a set of 34 forward primers for the Vα segment and 12 reverse primers for the Jα segment was designed. When these were used in the analysis of the multiplex structure, the number was reduced to such an extent that a huge number of combinations of primers could be used. Then, all the Jα primers were mixed in one tube, and 34 PCR reactions were performed on one sample using this Jα primer mix and each Vα primer. As a result, TCRA reconstitution could be identified in TCRB reconstituted iPS cells by this method (see FIG. 12).

  Furthermore, the binding of the TCR to the HLA-peptide complex is necessarily determined by the three-dimensional structure of the antigen recognition site, which consists of three types of complementarity determining regions (CDRs 1, 2, and 3). In these three regions, CDR3 is the most versatile because it spans the V (D) J binding region where various random nucleotides (N- or P-nucleotides) are inserted. Therefore, sequences encoding individual TCR chains in the CDR3 region of the genome of the established iPS cells were identified. As a result, it was revealed that the functional rearrangement (Rearranged (Productive), a configuration linked in frame and without a stop codon) in the original T cells was conserved, and in all T-iPS cells. It was found to have only one functional TCRA and TCRB rearrangement on one allele. The other allele was found to have a complete or non-functional rearrangement (intact or Rearranged (Unproductive)), including DJβ rearrangement (see Table 7 and FIG. 13). . In Table 7, "iPS Clone" indicates an established T cell-derived iPS cell line, and "Productivity" indicates whether or not the reconstituted T cell receptor has a function, that is, a complex with a β receptor. Indicates whether the antigen is recognized (functional: Productive) or not (non-functional: Unproductive), and "Sequence of junctional region" indicates the base sequence of the binding region. In addition, there were no human NKT lymphocytes in which the TRAV24 and TRBV11 segments were specifically confirmed (see Godfrey, DI, et al., Semin Immunol, Vol. 22, pages 61-67). Therefore, it became clear that the iPS cell was produced from one peripheral blood T cell without changing the TCR production ability engraved in the CDR3 sequence.

(Example 4)
<Reprogramming of CD4 ⁺ lymphocytes or CD8 ⁺ lymphocytes into iPS cells>
Peripheral T lymphocytes are derived primarily from two functional subsets: CD4 ⁺ helper / regulatory cells and CD8 ⁺ cytotoxic cells. It is also known that there is no significant difference between CD4 ⁺ lymphocytes and CD8 ⁺ lymphocytes in the efficiency of gene transfer by virus (Berger, C. et al., Blood, 2003, 101, 476- 484, see Kaneko, S. et al., Blood, 2009, Volume 113, pp. 1006-1015). Therefore, peripheral T lymphocytes are divided into CD4 ⁺ T cells and CD8 ⁺ T cells, and reprogramming to iPS cells is performed. Is there a difference in the efficiency of reprogramming to iPS cells depending on the source T cells? I checked whether. The induction conditions for each cell are shown below.

<Induction conditions for CD4 ⁺ T cells>
A CD3-positive CD56-negative CD4-positive T cell fraction of peripheral blood mononuclear cells (hereinafter referred to as CD4-positive T cells) obtained by blood collection is fractionated at a flow site, and an anti-CD3 antibody, an anti-CD28 antibody (bead binding, solidification) Irrespective of the addition to the culture medium). About 20 ng / ml of human IL-2 was added to the medium, and the Yamanaka factor gene was transduced a plurality of times on a 24-well plate coated with RetroNectin 48 to 96 hours after the stimulation. The Yamanaka factors used at that time are OCT4, SOX2, KLF4, and C-MYC, or OCT4, SOX2, and KLF4. About 2 days after the last gene transfer, 1 to ⁵ × 10 ⁵ transgenic T cells were placed on the irradiated MEF on a 6 cm dish. When CD4 positive T cells were not sorted and used at the flow site on the first day, CD4 positive T cells were sorted at the flow site and placed on the MEF at this timing. For four days from the next day after the transfer, half of the medium was replaced with a medium for ES cells containing 0.5 μM valproic acid, and thereafter, the medium was replaced with a medium for ES cells containing 0.5 μM valproic acid every day or every other day, and culture was continued. Continued. From around 10 days after the transfer, cell-stone-like aggregates were observed, and completed iPS cell colonies were observed within a few days.

<Induction conditions for CD8 ⁺ T cells>
A CD3-positive CD56-negative CD8-positive T cell fraction (hereinafter, CD8-positive T cell) of peripheral blood mononuclear cells obtained by blood collection is fractionated at a flow site, and an anti-CD3 antibody, an anti-CD28 antibody (bead binding, solidification) Irrespective of the addition to the culture medium). About 20 ng / ml of human IL-2, about 10 ng / ml of IL-7 and about 10 ng / ml of IL-15 were added to the medium, and 48 to 96 hours after the stimulation, the cells were treated on a 24-well plate coated with RetroNectin several times. Of Yamanaka factor gene. The Yamanaka factors used at that time are OCT4, SOX2, KLF4, C-MYC, NANOG and LIN28, or OCT4, SOX2, KLF4, C-MYC and NANOG. About 2 days after the last gene transfer, 1 to ⁵ × 10 ⁵ transgenic T cells were placed on the irradiated MEF on a 6 cm dish. When CD8-positive T cells were not sorted and used at the flow site on the first day, CD8-positive T cells were sorted at the timing and placed on the MEF at this timing. For four days from the next day after the transfer, half of the medium was replaced with a medium for ES cells containing 0.5 μM valproic acid, and thereafter, the medium was replaced with a medium for ES cells containing 0.5 μM valproic acid every day or every other day, and culture was continued. Continued. Cobblestone-like cell aggregation was observed from about 2 weeks after transfer, and completed iPS cell colonies were observed within 1 to 2 weeks.

As a result of the examination under the above induction conditions, in this example, the success rate of inducing CD4 ⁺ T lymphocytes into T-iPS cells under the conditions in which NANOG was not introduced was determined by the conditions under which the reprogramming factor NANOG was further introduced. Was twice as successful as the induction of T-iPS cells from CD8 ⁺ T lymphocytes in. In this example, CD4 ⁺ T lymphocytes
Without introducing ANOG, T-iPS cells can be obtained in 7 or 8 trials.

Also, although not shown in the figure, when CD8 ⁺ T lymphocytes were induced into T-iPS cells, M-
After transfer onto EF, T was added by hypoxic culture in the presence of 10 μM ROCK inhibitor under 5% O ₂ or by adding 1 μM MEK inhibitor (PD0325901) and 3 μM GSK3 inhibitor (CHIR99021) to the medium until colony formation. -It was confirmed that the induction efficiency to iPS cells tends to slightly increase.

In response to the above results, CD4 ⁺ T lymphocytes were further converted to CD45RA and CD62L /
Based on the expression of CCR7, it was divided into naive (CD45RA + CD62L +), central memory (CD45RA-CD62L +), effector memory (CD45RA-CD62L-), and a subset of terminal effector T cells (CD45RA + CD62L-) and induced into iPS cells (reprogramming). (See Sallusto, F. et al., Nature, 1999, vol. 401, pages 708-712, see FIG. 14). As a result, the average reprogramming efficiency in three healthy donors was 0.0041% for naive T cells, 0.017% for central memory T cells, 0.0036% for effector memory T cells, and terminal effector. It was 0.0008% in T cells, and it was revealed that T-iPS cells were established relatively better than central memory T cells in particular (see FIG. 15). The high reprogramming efficiency of CD4 ⁺ central memory T cells may reflect the sensitivity to anti-CD3 / CD28 stimulation.

(Example 5)
<Functional analysis of T lineage cells differentiated from TiPS cells>
By transplanting CD34-positive cells obtained from human ES cells into SCID-hu mice, T cell lineage differentiation is induced in the transplanted human fetal thymus (Galic, Z et al., PNAS, 2006, 103, 31). No. 11742-7). In vivo, ES cells are induced by OP9-DL1 cells into CD3-positive and TCR-positive cells, mainly CD4 / CD8 double positive (see Timermans et al., JI, 2009, 182, 6879-6888). ).

  Therefore, using the obtained T cell-derived iPS cells (T-iPS cells), an attempt was made to induce differentiation into T cells. Since T cells do not undergo high frequency somatic mutation after undergoing TCR gene recombination during development, the T cell lineage derived from T-iPS cells retains the TCR information of the original T-iPS cells, Some allele exclusion status was expected to have a monoclonal TCR. However, whether T cells can be derived from T-iPS cells and whether the derived T cells are functional, the reconstitution state of the TCR is the same as the T cells from which the T-iPS cells were derived. Since it is unclear whether or not, these points were examined as follows.

  First, from the established T-iPS cells, one allele of both the TCR α chain and the TCR β chain undergoes in-frame (in frame) rearrangement, and the opposite allele forms an out-frame (out frame) rearrangement of the α chain. In, a clone stopped by DJ reconstitution was selected (TkT3V1-7), and a T cell lineage was induced in vitro. T-iPS cells cultured on OP9 in the presence of IL-7, Flt3L (human FMS-like tyrosine kinase 3 ligand) or SCF (human stem cell factor, hSCF) were ES-sac (Sac) (Takayama et al., Blood, 2008). Year, Vol. 111, No. 11, p. 5298-5306), and CD45-positive blood cells were induced therein by day 14 (see FIG. 16 (a)). On the 12th to 14th days, blood cells were transferred to OP9-DL1 and medium exchange was repeated every 3 days to continue the culture. On the 21st and 40th days, CD3-positive cells were collected by FACS, and T cell-related genes were collected. Was evaluated. Although the CD3 positive cells on the 40th day were mainly CD4 / 8 negative cells, CD8 single positive cells were partially observed (see FIG. 16 (b)).

After the sac was transferred onto the OP9-DL1 cell layer, the floating cells coming out of the sac were collected and analyzed weekly using flow cytometry (see FIG. 17 (a)). Consequently, in the first week, most floating cells express CD45 weakly, and in some cells, CD34, CD7, and CD1a, while cells expressing CD3-TCRaβ complex and CD5. There was no. In the second week, CD45 in most cells
Was upregulated, and in some cells the CD3-TCRaβ complex was also starting to be expressed. By the third week, CD34 cells had completely disappeared, and CD3-TCRaβ-positive cells were the cell group with the largest number of floating cells. There was no TCRaβ ^- cell among the CD3 + cells (see FIG. 17 (b)). Also, even after the third week, most redifferentiated CD3 ⁺ TCRaβ ⁺ cells were of the co-negative (DN) stage, but in some cell populations, only CD4 positive cells (8. 5 ± 8.2%) and CD8 only positive cells (15.5 ± 15.9%) were also detected (see FIGS. 18 and 19).

It is generally known that even at the thymocyte stage, TCR-expressing T lineage cells can respond to TCR stimulation and can also produce cytokines (Fischer, M. et al., J Immunol, 1991, 146, See pages 3452-3456). Therefore, the floating cells were stimulated with OKT-3 and IL-2, and the activation and number of CD3-expressing cells were examined. That is, as a result of stimulating floating cells with 30 ng / ml OKT-3 and 600 IU / ml hIL-2 for 5 days, the number of CD3 ⁺ cells increased, and the CD3 expressing cells were activated by morphological criteria. (See FIG. 20). However, it was confirmed that the actual CD3 ⁺ cells did not change.

In addition, in order to clarify that the induced CD3 ⁺ TCRαβ ⁺ cells functionally contribute to the induction of differentiation into T lineage cells, the cytokine producing ability was evaluated as follows. That is, as a result of stimulation with PMA and a calcium ionophore (ionomycin), 12.5% of T lineage cells produce IFN-γ, 1.0% of T lineage cells produce IL-2, and 6.2% It was revealed that T lineage cells produced both of them (see FIG. 21). Therefore, it became clear that CD3 ⁺ TCRαβ ⁺ cells derived from T-iPS cells contributed to the induction of T lineage cells both morphologically and functionally.

  In addition, as described above, since the TCR gene generally does not undergo somatic hyper-variable mutations, the re-differentiated T lineage cells derived from T-iPS cells are frequently located at the origin of T-iPS cells. It is expected that TCRαβ encoded in the genome of the transformed cell is expressed. However, it is not known whether the reconstituted state of the TCR is the same as the T cell from which the T-iPS cells were derived, and this point was examined as follows.

  As a result, when the TCR expression of the induced CD3 cells was analyzed, only one type of TCR β chain appeared in TkT3V1-3 and TkT3V1-7, and the sequence centered on the CDR3 region was the same as that of the iPS cells before induction. (See FIG. 22). Most of the TCR α-chains that showed diversity were derived from the reconstituted α-chain (see FIG. 23). It was revealed that the reprogrammed T cells clonally express the TCR when re-induced to T cells.

In addition, CD3 ^high TCRαβ ^high T lineage cells regenerated from 4 T-iPS cells derived from two donors were analyzed. That is, cDNA libraries of these cells were constructed by SMART-mediated reverse transcription reaction (Du, G., et al., J Immunol Methods, 2006, Vol. 308, pp. 19-35), and the TCR gene was amplified. Then, the amplified TCR gene was inserted into a cloning vector, cloned and analyzed.

As a result, the sequence of the analyzed clone was consistent with the TCR sequence encoded in the genome of the cell before T-iPS cell differentiation (see FIGS. 13, 23 and 25). On the other hand, most of the transcripts were derived from a functional chain (productive chain), and transcripts from a non-functional chain (unproductive chain) were also present (see Table 8).
. However, no transcripts were found that differed from sequences from functional or non-functional rearrangements (see Table 9). In Table 8, “iPS Clone” indicates an established T cell-derived iPS cell line, and “Productivity” indicates whether or not the reconstituted T cell receptor has a function, ie, a complex with β receptor. Indicates whether the antigen is recognized (functional: Productive) or not (non-functional: Unproductive), and "Sequence of junctional region" indicates the base sequence of the binding region. In Table 9, “Productivity” indicates whether the reconstituted T cell receptor has a function, that is, whether it forms a complex with a β receptor and recognizes an antigen (functional: Productive) or not (Functional: Productive). Non-functional: Unproductive), “No. of Sequenced Samples” indicates the number of samples analyzed in each TCR gene, and “Sequence alignment with T-iPSC's genome” indicates the number of samples analyzed before regeneration. The numbers and proportions (Identical) that match the genome of the T-iPS cells or the numbers and proportions (Different) different from the genome are shown.

Furthermore, the induction of CD34-positive cells from other pluripotent cells was compared with the method of the present invention. As a result, ES cells (human ES cells, Human ESC), skin-derived iPS cells (human skin fibroblast-derived iPS cells, HDF-iPS), CD34-positive cell-derived iPS cells (umbilical cord blood CD34-positive cell-derived iPS cells, CB -IPSC), T-iPS cells (T-i
PSC) induced CD3-positive cells, but T-iPS cells were clearly more likely to be induced into T cells than iPS and ES cells derived from skin cells and CD34-positive cells. (See FIGS. 16, 17, 26 and 27).

(Example 6)
<Induction of functional T cells from iPS cells established from human T cells 2>
Using T-iPSC clones (6 clones) derived from CD4 T cells or CD8 T cells of multiple donors, induction of a T cell population having a monoclonal TCR was attempted. That is, irradiation OP9 in which 3 × 10 ⁵ T-iPS cells were spread on a 10 cm dish on the induction start day.
Cells or 10T1 / 2 cells were seeded and co-cultured for about 14 days using a differentiation medium. Blood cells in bag-like structures appeared during co-culture on days 12-14. At this time, blood cells appeared even without the use of cytokines, but it was revealed that the addition of VEGF improved the recovery of CD34-positive hematopoietic stem cells (hereinafter, CD34 cells). It was also found that the addition of VEGF, SCF, and TPO, or VEGF, SCF, and FLT3L further improved the recovery of CD34 cells. As will be described later, these trends were also reflected in the recovery of T lineage cells, which are final products.

On any of the 12th to 14th days, 3 × 10 ⁵ cells were irradiated on irradiated OP9 / DL1 cells, OP9 / DL4 cells, or 10T1 / 2 / DL4 cells in which blood cells containing the CD34 cells were spread on a 10 cm dish. Planting, 20% FBS in the presence of IL-7 and Flt3L
The cells were co-cultured in an α-MEM containing medium. SCF was not used because it strongly promotes differentiation into NK cells. As described above, some of the suspended cells become CD3-positive TCRαβ-positive T lineage cells at week 2 and week 3 at co-culture, but those cells are double-negative in CD4 and CD8 expression ( DN), double positive (DP), and single positive (SP), and were not uniform populations (see FIG. 17). Therefore, as a result of further detailed examination, it was found that the structure of DN, DP, CD4SP, and CD8SP depends on the concentration of IL-7. Specifically, although not shown in the figure, when IL-7 was not added, the number of CD3-positive CD56-negative T cells was small and most of them were DN. At IL-7 of 5 to 10 ng / ml, a CD3-positive CD56-positive NKT-like cell population appeared, and again most of the cells were DN. On the other hand, at 1 ng / ml of IL-7, CD3 positive CD56 negative T lineage cells occupy the majority, and differentiation from DN to DP, CD4SP, and CD8SP was confirmed. When these SP cells were analyzed using peripheral blood T cell differentiation markers, about 80% of CD8SP was CD45RA ⁺ CD62L ⁺ naive T cells, and the majority of CD4SP was CD45RA ^- CD62L ⁺ central memory T cells. That is, it was suggested that T cells that had progressed to the effector memory stage could rejuvenate into naive T cells (for CD8SP) or central memory T cells (for CD4) via T-iPS cells.

  As described above, the method for producing human T lymphocytes of the present invention is particularly suitable for the production of human T lymphocytes that are specific to an antigen of interest, and thus, the specificity of the antigen of interest and the desired human histocompatibility. It is excellent for the production of CD4 only positive cells or CD8 only positive cells restricted by sex antigen (HLA). Therefore, the present invention can greatly contribute to the treatment or prevention of various intractable diseases such as chronic intractable infections, malignant tumors, and autoimmune diseases.

SEQ ID NOs: 1 to 109
<223> Base sequence of artificially synthesized primer

Claims (7)

A method for producing human T cells, comprising:
Inducing iPS cells from human T cells;
Differentiating said iPS cells into T cells.   The method according to claim 1, wherein the human T cell is a T cell expressing at least one molecule selected from the group consisting of CD4 and CD8.   The method according to claim 1 or 2, wherein the T cells induced to iPS cells have antigen specificity.   The method according to claim 3, wherein the T cells induced into the iPS cells and the T cells differentiated from the iPS cells have the same antigen specificity.   A human T cell produced by the method according to claim 1.   A pharmaceutical composition comprising the human T cell according to claim 5. Isolating T cells having the desired antigen specificity from humans;
Inducing iPS cells from the T cells having the desired antigen specificity;
Differentiating the iPS cells into T cells;
Administering the obtained iPS cell-derived T cells into a human body.