JP6884386B2 - Immune function reconstruction method using pluripotent stem cells - Google Patents

Description

The present invention relates to a method for reconstructing immune function using pluripotent stem cells, and more specifically, a method for producing human T cells, a pharmaceutical composition containing T cells produced by the method, and immunity using the method. Regarding the method of cell therapy.

If immune cells can be replenished or regenerated in the case of decreased immune function caused by various causes, it will be an extremely effective means for improving the pathological condition of the disease and improving the therapeutic effect. In particular, there is a strong demand for functional replacement and regeneration of T lymphocytes responsible for cell-mediated immunity, but no effective treatment method has been established at present. Immune action is characterized by specificity, which is largely due to the difficulty of making good use of this specificity.

Due to the dramatic progress in 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 supplement or regenerate a specific immune response (). Non-Patent Documents 1 and 2). In these attempts, CD34-positive cells, which are bone marrow progenitor cells, and naive T lymphocytes are used as gene transfer cells, but these have low self-renewal ability in ex-vivo and low gene transfer efficiency. , It is difficult to regulate differentiation by gene transfer, and so on.

In recent years, embryonic stem cells (ES cells) and induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) have been established from many animal species including humans. These pluripotent stem cells are the most useful cells for regenerative medicine because they maintain vigorous division ability while maintaining pluripotency and can differentiate into almost all organs by appropriately inducing differentiation. It is considered a source. In particular, since iPS cells can be established from self-derived somatic cells, they are less likely to cause ethical and social problems than ES cells produced by destroying embryos, and are also self-derived. It also makes it possible to avoid rejection, which is the biggest barrier to regenerative medicine and transplantation medicine.

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 those that reconstitute genes such as B lymphocytes and T lymphocytes and are terminally differentiated, are also reprogrammed. Was of great interest to see if it was possible. Under such circumstances, the establishment of iPS cells from mouse B lymphocytes was reported by Jaenich 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 contained in the TCR that the original human T lymphocytes had. It is necessary to induce the 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, Volume 6, pp. 383-393 Morgan R. Et al., Science, 2006, Vol. 314, pp. 126-129 Hanna J. Et al., 2008, Vol. 133, No. 2, pp. 250-264 Hong H. Et al., Nature, 2009, 460, pp. 1132-1135

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

As a result of intensive studies to achieve the above object, the present inventors have succeeded in establishing iPS cells from human T cells while maintaining the same TCR rearranged state as the original human T lymphocytes. did. Furthermore, the present inventors have a function of producing cytokines and the like from the human T cell-derived iPS cells (TiPS cells) thus established while maintaining the same TCR rearranged state as the original human T lymphocytes. Succeeded in inducing differentiation of typical T cells. Then, they have found that ideal immune cell therapy can be performed by transplanting the human T cells thus obtained into the patient's body, and have completed the present invention.

More specifically, the present invention provides the following inventions.
(1) 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.
(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 to iPS cells and the T cells differentiated from iPS cells have the same antigen specificity.
(5) Human T cells produced by the method according to any one of (1) to (4).
(6) A pharmaceutical composition containing the human T cells according to (5).
(7) A step of isolating T cells having a desired antigen specificity from humans, and
A step of inducing iPS cells from T cells having the desired antigen specificity, and
The step of differentiating the iPS cells into T cells,
A method for immune cell therapy, which comprises a step of administering the obtained iPS cell-derived T cells into a human body.

Lymphocytes that acquire structural changes in the TCR and BCR gene base sequences during development are extremely specific cells as a source for the establishment of iPS cells. In recent years, it has been reported one after another that iPS cells with TCR reconstituted from mouse T cells and B cells can be established, suggesting that the TCR reconstitution mode is not lost by nuclear transfer or reprogramming. It had been. However, these are all examples in mice. INDUSTRIAL APPLICABILITY The present invention has shown for the first time that iPS cells can be obtained from human peripheral blood T lymphocytes, and further, when T lineage cells are induced from iPS cells having at least one inframe TCR rearrangement, the TCR is obtained. For the first time, it has been revealed that human T-lineage cells with the same color appear monoclonally. In particular, one in-frame (in) for both the TCR α chain and β chain.
In the induction from iPSCs (iPS cells) that have only frame) reconstruction to T lineage cells, the emerging TCRs are electronically identical to the nucleotide sequence conserved in the iPS cells and are completely monoclonal. there were. This means that ES cells, CD34-positive hematopoietic stem cells, for which the inhibitory effect (interfere) due to the expression of the endogenous TCR α chain β chain (particularly the α chain that is incompletely controlled by allelic exclusion) must be taken into consideration. Or, it was shown that T cells having monoclonal TCRαβ against the target antigen can be induced more efficiently than the method of introducing the TCR gene into naive T cells.

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

It is a figure which shows the outline of the production of the human iPS cell from the peripheral blood T lymphocyte. In the figure, "Tapering" indicates the period during which the medium was gradually replaced with a human iPS cell medium (hiPS medium, etc.). 3 is a photomicrograph showing ES cell-like colonies derived from T cells formed by the method shown in FIG. In the figure, the upper part shows the colonies obtained by introducing Yamanaka 3/4 factor into CD3 T cells, and the lower part shows the cells resown into Day6: MEF cells and Day15: ES cell-like colonies from the left side. Forming cells, Day24: Shows cells that have completed colonization (see FIG. 1 for Day). The scale bar shows 200 μm. It is a micrograph which shows the result of observing the iPS cell colony by immunostaining. 9 is an electrophoretic photograph showing the results of analysis of pluripotency marker expression in T-iPS cells, human ES cells (KhES3), and peripheral blood-derived CD3 + cells (PB CD3 +) by RT-PCR. ACTB (β-actin) and GAPDH were used as loading controls. It is a graph which shows the reprogramming efficiency to the pluripotency state by 3 factors (excluding 3 Factors, c-Myc) or 4 factors (including 4 Factors, c-Myc) in peripheral blood CD3 ^{+ T cells.} In the figure, the vertical axis ^{shows the number of ALP-positive colonies derived from 3 × 10 5} T lymphocytes on the 22nd day (day22) (results by 3 factors are mean ± SEM = 5.1). It was ± 2.4 [n = 7], and the result by the four factors was mean ± SEM = 77.0 ± 25.0 [n = 12]). Comparison results (left side) between human T-iPS cells (TkT3V1-7) and active CD4T lymphocytes (PB CD4 T-cell) in a comprehensive gene expression pattern using an oligonucleotide DNA microarray, and a comprehensive gene expression pattern. It is a scatter diagram which shows the comparison result (right side) of TkT3V1-7 and a human ES cell (KhES3) in. The two parallel lines in the figure indicate that there was a 5-fold difference between the corresponding samples. 6 is a photomicrograph showing karyotypes of two representative examples of T-iPS cells (TkT3V1-7 and TkT4V1-3). The number of passages of the T-iPS cells examined was 10, and no chromosomal abnormality was observed in any of them. 9 is a photomicrograph showing a representative HE-stained section of teratoma formed in NOD-Scid mice 8 weeks after injection of T-iPS cells. Tissues derived from three embryos (nervous tissue (neural tissue, neural plate) and retinal cells (retinocytes) as ectoderm, cartilage and muscle cells (myopathy) as mesoderm, stomach as endoblast It indicates the presence of like mucosa (gut-like mucosa) and secretory glands / ducts (lands / ducts). It is an electrophoretic photograph which shows the result of PCR analysis for detecting the gene rearrangement of TCRγ chain. FIG. 6 is an electrophoretic photograph showing typical results of PCR analysis for detecting TCRG reconstruction. The reconstituted TCRG was identified by a PCR band within the effective size range (about 200 bp). FIG. 5 is an electrophoretic photograph showing typical results of PCR analysis for detecting TCRB reconstruction. It should be noted that the PCR products in the columns of "Tube A" and "Tube B" in the figure show that they are V (D) J reconstituted (effective size range: 240 to 285 bp). On the other hand, the PCR products in the "Tube C" column show DJ reconstitution (effective size range: 170-210 bp, and 285-325 bp). 9 is an electrophoretic photograph showing typical results of PCR analysis for detecting TCRA reconstruction in TkT3V1-7. It is a figure which shows the sequence result of the PCR product derived from TkT3V1-7 shown in FIGS. 11 and 12. In the figure, the functional reconstruction of one of the alleles of the TCRA gene is shown at the top. In addition, the functional rearrangement (V (D) Jβ rearrangement) in one of the alleles of the TCRB gene is shown in the central part. The non-functional reconstruction (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 then naive T cells (Nive, CD45RA ⁺ CD62L ⁺ ), central memory T cells (Cemtorial Memory, CD45RA ^- CD62L ⁺ ), effector memory T cells. cells ^{(effector Memory, CD45RA - CD62L -} ), or terminal effector T cells (effector, CD45RA ⁺ CD62L ^-) shows the results of classification in a scatter plot. It is a graph which shows the result of having evaluated the reprogramming efficiency in each T cell subset based on the number of ^{ALP +} colonies obtained from 1 × 10 ⁵ T cell subsets reseeded on MEF. In the figure, the vertical axis is the average number ± S. ^{of ALP + colonies in three independent experiments.} D. Is shown. (A) is a diagram showing an outline of T lineage cell production from pluripotent cells by culturing on OP9 and OP9-DL1 stromal cell layers, and a micrograph showing the state of cells at each time point. In the figure, "Cytokine cocktail" indicates that hIL-7, hFlt-3L, and hSCF were added. (B) is a scatter plot showing the results of examining the expression of CD45, CD3, CD4, CD8, and TCRαβ in T lineage cells prepared from each pluripotent cell using flow cytometry. In the figure, "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 showing an outline of T lineage cell production from pluripotent cells such as T-iPS cells by culturing on OP9 and OP9-DL1 stromal cell layers. That is, pluripotent cells were cultured on the irradiated OP9 cell layer in an essential medium containing no cytokine and based on α-MEM for 10 to 14 days, and then the induced hematopoietic cells were used as OP9-DL1 cells. It is a figure which shows that it was cultured in the essential medium based on αMEM to which hIL-7, hFlt-3L, hSCF and the like were added for 3 or 4 weeks. In (b), at each culture time, floating cells in the medium were collected, and the expression of the T lineage markers CD45, CD34, CD3, CD4, CD8, and TCRαβ was examined using flow cytometry, and at least 3 independent cells were examined. It is a scatter diagram which shows the typical result in one experiment. The numerical values shown in each scatter plot indicate the proportion (%) of cells in each category. Further, the lowermost part of B shows the result (control data) of analysis of fresh PBMC. It is a scatter plot which analyzes the expression of the T lineage cell marker by flow cytometry and shows the representative result in at least three independent experiments. In the figure, the numerical values shown in each spray diagram indicate the proportion (%) of cells in each category, "Human ESC" indicates the results in human ES cells (KhES3), and "CB-iPS" is positive for cord blood CD34. 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 T-iPS cells (TkT3V1-7). ), And "PBMNC" shows the results in peripheral blood mononuclear cells. It is a graph which shows the expression of CD4 and CD8 in the ^{CD3 +} T lineage cell evaluated by the flow cytometry. In the figure, the vertical axis represents the distribution ± S. in T lineage cells derived from each pluripotent cell. D. Is shown. In addition, "Human ESC" shows the results in human ES cells, "CB-iPSC" shows the results in cord blood CD34-positive cell-derived iPS cells, and "HDF-iPSC" shows the results in human skin fibroblast-derived iPS cells. Shown, "T-iPSC" indicates the results in T-iPS cells. The columns of each cell show "DN", "CD8", "CD4", and "DP" in order from the left. Furthermore, "DN" is CD4 ^- CD8 ^- a indicates that, "CD4" is CD4 ⁺ CD8 ^- indicates a "CD8" is CD4 ^- CD8 indicates a ⁺ "DP" is CD4 ⁺ CD8 Indicates that it is ^+. Graphs and micrographs showing proportional and morphological changes in redifferentiated T lineage cells after TCR stimulation with OKT-3 and hIL-2 and unstimulated redifferentiated T lineage cells. is there. It is a scatter plot which shows the result of having analyzed the cytokine production profile of the redifferentiated T lineage cell by flow cytometry. The analyzed redifferentiated T lineage cells (CD3 ⁺ cells from TkT3V1-7) or PBMNC (PB CD3, control) were stimulated overnight with PMA / ionomycin together with hIL-1α and stained with an antibody. The numerical values shown in the plot show the percentage of cytokine-producing cells. It is a figure which shows the sequence of TCRβ mRNA and the amino acid sequence in the redifferentiated T lineage cell (T lineage cell derived from TiPS cell). It is a figure which shows the sequence of TCRα mRNA and the amino acid sequence in the redifferentiated T lineage cell (T lineage cell derived from TiPS cell). It is a photograph of electrophoresis showing the result of amplifying the TCR of TkT3V1-7 by PCR through the construction of a cDNA library by SMART and analyzing the obtained PCR product. It is a figure which shows the result of the sequencing analysis about the PCR product in the effective size range (about 750bp) shown in FIG. 24, and the sequence of all transcription mRNA is the sequence of the genome of T-iPS cell (see FIG. 13). It is a figure showing that no descendants from the DJβ reconstituted allele were confirmed. Details and results of other samples are shown in Tables 8 and 9. It is a graph which shows the result of having evaluated the expression of CD3 by flow cytometry. In the figure, the vertical axis shows the ratio of CD3 ^{+ T lineage cells in CD45 +} blood cells (Average ± SD of 3 or more independent experiments in each group). In addition, "Human ESC" shows the results in human ES cells, "CB-iPSC" shows the results in cord blood CD34-positive cell-derived iPS cells, and "HDF-iPSC" shows the results in human skin fibroblast-derived iPS cells. "T-iPSC" indicates the result in T-iPS cells. Furthermore, ** indicates that p <0.01 for all other samples. It is a graph which shows the mean fluorescence intensity (MFI, mean fluorosense integrity) of CD3 evaluated by the flow cytometry. The vertical axis is the Averages ± S.A. of independent experiments in which CD-iPS and HDF-iPS are in each group 2 and the others are in each group 3 or more. Shows D, "Human ESC" shows results in human ES cells, "CB-iPSC" shows results in cord blood CD34-positive cell-derived iPS cells, and "HDF-iPSC" shows results in 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. It is a schematic diagram which shows the rearrangement of a TCRG gene, and the sequence and position of a primer for detecting the rearranged state. The human TCRG locus spans 135 kb on chromosome 7q14. In addition, 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 the rearrangement of the TCRG gene can be detected by detecting a PCR band of about 200 bp. It is a schematic diagram which shows the rearrangement of a TCRB gene, and the sequence and position of a primer for detecting the rearranged state. The human TCRB locus spans 685 kb on chromosome 7q34. In addition, 23 Vβ primers, 2 Dβ primers, and 13 Jβ primers are used as all Vβ and Jβ primers that can be annealed to the same storage region in the Vβ gene segment and the Jβ gene segment, respectively. Designed. In addition, 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 that. In addition, V (D) Jβ rearrangement can be detected by detecting the PCR band of about 270 bp using the primer sets shown in Tube A and Tube B, and the primer set shown in Tube C can be used. DJβ rearrangement can be detected by detecting the PCR band 170-210 bp or 285-325 bp.

<Method of producing human T cells>
The present invention provides 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.

-Step of deriving 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 with a weakened 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, the humans from which the T cells are isolated are the humans to which the T cells obtained by the present invention are administered and HLA from the viewpoint that rejection does not occur. It is preferable that the types of T cells are the same, and it is more preferable that the T cells obtained by the present invention are the same person as the person to whom the T cells are administered.

In the present invention, the "T cell" means a cell expressing an antigen receptor called a T cell receptor (TCR) on the surface.
In the present invention, the "human T cells induced to iPS cells" and the "human T cells differentiated from iPS cells" are not particularly limited, but are preferably from the group consisting of CD3 and CD4 and CD8. A T cell expressing at least one selected molecule. Examples of such human T cells include helper / regulatory T cells which are CD4 positive cells, cytotoxic T cells which 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 later, it is preferable that the human T cells differentiated from the iPS cells have the same antigen specificity as the human T cells induced into the iPS cells. The antigen specificity in T cells is provided by the antigen-specific, reconstituted TCR gene.

In the present invention, "human T cells induced to iPS cells" can be isolated from human tissues by a known method. The human tissue is not particularly limited as long as it contains the T cells, and examples thereof include peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissue. Among these, peripheral blood and umbilical cord blood are preferable from the viewpoint of low invasiveness to humans and easy preparation. Known methods for isolating human T cells include, for example, flow cytometry using an antibody against a cell surface marker such as CD4 as shown in Examples described later and a cell sorter. It is also possible to isolate desired T cells using the secretion of cytokines and the expression of functional molecules as indicators. In such a case, for example, since T cells secrete different cytokines depending on Th1 type or Th2 type, such cytokines can be selected as an index to isolate T cells having a desired Th type. .. In addition, cytotoxic (killer) T cells can be isolated using the secretion or production of granzyme, perforin, etc. as an index.

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

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 can impart differentiation pluripotency to the somatic cell alone or in cooperation with other differentiation pluripotency factors by being introduced into the T 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 protein selected from the group consisting of Fthl17, 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 (4 factors) into the somatic cells from the viewpoint that iPS cells can be efficiently established with a small number of factors. Further, 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 (3 factors) excluding c-Myc into the somatic cells.

When inducing human CD8-positive T cells into iPS cells, OCT4, SOX2, KLF4, C-MYC, and NANOG should be introduced into human CD8-positive T cells from the viewpoint of further enhancing the efficiency of induction into iPS cells. Is preferable, and it is more preferable to introduce OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28 into human CD8-positive T cells.

Further, in the present invention, the method of "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 transfer reagent, a method using a protein transfer domain (PTD) fusion protein, an electroporation method, and a microinjection method. Can be mentioned. Further, in the case of introducing into the T cell in the form of a nucleic acid encoding the cell reprogramming factor, an appropriate nucleic acid (for example, cDNA) encoding the cell reprogramming factor contains a promoter functioning in the T cell. The expression vector can be introduced into cells by infection, lipofection method, liposome method, electroporation method, calcium phosphate co-precipitation method, DEAE dextran method, microinjection method, electroporation method. ..

Examples of the "expression vector" according to the present invention include viral vectors such as lentivirus, retrovirus, adenovirus, adeno-associated virus, and herpesvirus, and animal cell expression plasmids.

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.

Further, in the "step of inducing iPS cells from human T cells", as shown in Examples described later, the T cells were subjected to interleukin-2 (IL-2) before the introduction of the cell reprogramming factor. It is preferably activated by stimulating with an anti-CD3 antibody and an anti-CD28 antibody in the presence. Such stimulation can be performed, for example, by adding IL-2, anti-CD3 antibody and anti-CD28 antibody to the medium and culturing the T cells for a certain period of time, as shown in Examples described later. 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 given by culturing the T cells on a culture medium for a certain period of time. Furthermore, stimulation may be given by adding an antigen peptide recognized by human T cells to the medium together with the feeder cells.

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

In addition, the culture period for performing such stimulation is a period sufficient to give such stimulation to the T cells, and the T cells can be proliferated to the number of cells required for the introduction of the cell reprogramming factor. The period is not particularly limited, but is usually 2 to 14 days, preferably 2 to 7 days from the viewpoint of gene transfer efficiency. Further, from the viewpoint of increasing the gene transfer efficiency, it is preferable to culture on a culture dish coated with retronectin.

As a medium for culturing the T cells and adding IL-2, anti-CD3 antibody, anti-CD28 antibody and the like, 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), Dalveco modified Eagle's medium (DMEM), F12 medium, etc.) containing the cytokines of the above, bovine fetal serum (FCS) can be used. .. In addition to IL-2 and anti-CD3 antibody and anti-CD28 antibody, amino acids (eg, L-glutamine) and antibiotics (eg, streptomycin, penicillin) necessary for culturing may be added to the medium. .. In addition, when inducing CD8-positive T cells to 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 and IL-15 added is not particularly limited, but is preferably 1 to 100 ng / ml.

Further, the conditions for "introducing the cell reprogramming factor into T cells" in the present invention or thereafter are not particularly limited, but the T cells into which the cell reprogramming factor has been introduced are cultured on the feeder cell layer. It is preferable to do so. The feeder cells are not particularly limited, and examples thereof 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 the T cells to iPS cells, from the viewpoint of suppressing cell differentiation, when introducing basic fibroblast growth factor (bFGF) into the T cells or when introducing the cell reprogramming factor into the T cells, or After that, it is preferable to add it to the medium.

In addition, in order to further enhance the establishment efficiency of iPS cells, histone deacetylase (HDAC) inhibitors (small molecule inhibitors such as valproic acid (VPA), tricostatin A, sodium butyrate, MC 1293, 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 Piffithrin-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 viral vector is used when "introducing a cell reprogramming factor into T cells", protamine sulfate should be added to the medium from the viewpoint that the vector can easily bind to the T cells. Is preferable.

Further, as shown in Examples described later, in accordance with the transition from the T cells to the iPS cells, the known medium suitable for culturing the T cells is gradually replaced with a medium suitable for culturing the iPS cells. It is preferable to incubate while living. As a medium suitable for culturing such iPS cells, a known medium can be appropriately selected and used, and for example, knockout serum substitute, L-glutamine, non-essential amino acid, 2-mercaptoethanol, b-FGF and the like can be used. Examples thereof include Dalveco modified Eagle's medium / F12 medium (human iPS cell medium) contained therein.

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

The iPS cells derived from the T cells in this manner (hereinafter, also referred to as "TiPS cells") can be selected by appropriately selecting a known method. Such known methods include, for example, a method of observing and selecting the morphology of ES cells / iPS cell-like colonies under a microscope as shown in Examples described later, and knowing that they are specifically expressed in iPS cells. Using recombinant T cells in which a drug resistance gene or a reporter gene (GFP gene, etc.) is targeted at the locus of the gene (for example, the cell reprogramming factor), drug resistance or reporter activity is selected as an index. The method can be mentioned.

Confirmation that the cells selected in this way are iPS cells can be confirmed by, for example, undifferentiated cell-specific markers (ALP, SSEA-4, Tra-) in the selected cells, as shown in Examples described later. The expression of 1-60, Tra-1-81, etc.) can be detected by immunostaining, RT-PCR, etc., or by transplanting selected cells into mice and observing their teratoma formation. ..

Further, confirmation that the cells selected in this manner are derived from the T cells can be performed, for example, by detecting the state of TCR gene rearrangement by genomic PCR, as shown in Examples described later. it can.

The time for selecting and collecting 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, first, from the viewpoint of facilitating the induction of differentiation into the mesoderm system, the TiPS cells are first contained with cytokines on the stromal cells. Instead, it is preferable to culture in α-MEM medium containing FCS and the like. The stromal cells to be used are preferably OP9 cells and 10T1 / 2 cells that have been treated with irradiation or the like from the viewpoint of facilitating differentiation into a hematopoietic system. However, in order to efficiently induce TiPS cells into CD34-positive hematopoietic stem cells, it is preferable to culture them in a medium containing at least one cytokine selected from the 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-shaped structure (also referred to as ES-sac) containing blood cell cells or the like is formed, and 10 to 14 after the start of culturing TiPS cells. It is preferably days.

The cells contained in the sack obtained as described above (hematopoietic progenitor cells, CD34-positive cells, blood cell cells, etc.) should be cultured on stromal cells in an α-MEM medium containing cytokines, FCS, etc. Is preferable. 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 stromal cells used in this culture include OP9-DL1 cells, OP9-DL4 cells, and 10T1 / 2 / that have been subjected to treatment such as irradiation from the viewpoint of inducing differentiation into T lymphocytes via notch signaling. It is preferably DL4 cells. Examples of cytokines added to the medium include IL-7, FLT3L, VEGF, SCF, TPO, IL-2, and 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, it is preferable that SCF is not contained in the medium. The concentration of IL-7 added to the medium is 0 from the viewpoint that CD3-positive and CD56-negative T lineage cells can be easily obtained, and differentiation into CD4 single positive (SP) T cells or CD8 SPT cells can be easily induced. .1 to 4 ng / ml is preferable.

Confirmation that the cells induced to differentiate in this way are derived from TiPS cells and derived from the T cells that are the source of the TiPS cells is confirmed, for example, by rearranging the TCR gene, as shown in Examples described later. The state can be determined by detecting the state by genomic PCR.

The 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 as shown in Examples described later and a cell sorter. When "T cells having a desired antigen specificity" are isolated from humans, a method of purifying using an affinity column or the like on which the desired antigen is immobilized can be adopted. It is also possible to adopt a method of purifying "T cells having a desired antigen specificity" using an MHC tetramer to which a desired antigen is bound.

<Human T cells, pharmaceutical compositions, methods 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 the treatment or prevention of diseases such as tumors, infectious diseases, and autoimmune disorders.

Therefore, the present invention provides human T cells produced by the method of the present invention, a pharmaceutical composition containing the human T cells, and a method for immune cell therapy using the human T cells.

The pharmaceutical composition of the present invention can be prepared by formulating human T cells produced by the method of the present invention by a known pharmaceutical method. For example, it can be mainly used parenterally as a capsule, a liquid, a film coating, a suspension, an emulsion, an injection (intravenous injection, drip injection, etc.) and 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 product (pharmaceutical product) of the composition of the present invention or a description thereof may be labeled as being used for treating or preventing a decrease in immune function. Here, "marked on a product or instruction manual" means that a label is attached to the main body, container, packaging, etc. of the product, or a manual, package insert, promotional material, or other printed matter that discloses product information. It means that the display is attached to.

The method of immuno-cell therapy of the present invention includes a step of isolating T cells having a desired antigen specificity from humans, a step of inducing iPS cells from the T cells having the desired antigen specificity, and the iPS cells. Is a method including a step of differentiating T cells into T cells and a step of administering the obtained iPS cell-derived T cells into a human body. When the method of immuno-cell therapy of the present invention is carried out, from the viewpoint that rejection does not occur, the humans from which T cells are isolated have the same HLA type as the humans to which the T cells obtained by the present invention are administered. It is preferable that the T cells obtained by the present invention are administered to the same person as the human being to whom 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 in more detail based on Examples, but the present invention is not limited to the following Examples. In this example, the following materials were used and the following methods and the like were followed.

<Flow cytometry>
Flow cytometry analysis was performed using MoFlo (registered trademark, manufactured by Dako Cytomy), 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 in the flow cytological 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 (Biolegend), anti-human CD45-Alexa 405 antibody (Molecular Probes-Invitrogen, Carlsbad, CA, USA), anti-human CD34-PE antibody (BD Bisc) ), Anti-human CD38-PerCP / Cy5.5 antibody (manufactured by BioLegend), anti-human CD1a-APC antibody (manufactured by Biolegend), anti-human CD5-PE / Cy7, -CD7-FITC antibody (manufactured by BioLegend), anti-human Human CD5-PE / Cy7 antibody (manufactured by BioLegend), anti-human CD7-FITC antibody (manufactured by Biolegend), anti-human TCRαβ-FITC antibody (manufactured by BioLegend). In addition, in flow cytological analysis, cells were incubated with an antibody cocktail adjusted to an appropriate concentration at 4 ° C. for 30 minutes and then washed with phosphate buffered saline. In addition, propidium iodide was added to eliminate dead cells.

<Preparation of retrovirus>
The pMXs retrovirus vector encoding human OCT4, SOX2, KLF4, c-MYC, and NANOG (Yamanaka factor, YFs) was donated by Professor Shinya Yamanaka (belonging to the Center for iPS Cell Research and Application, Kyoto University). These YFs were introduced into 293GPG packaging cells having the Gag-Pol gene and the Tet-OFF controlled VSV-G pseudo-enveloped gene. Three days after the virus was induced in the medium without tetracycline, the culture supernatants of these cells were collected four times daily. The collected culture supernatant was passed through a 0.45 μm cellulose acetate filter and centrifuged at 6000 g for 16 hours. Then, it was suspended in α-MEM (α-minimum essential medium) so that the concentration of the obtained supernatant became 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 according to the Declaration of Helsinki.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood from healthy subjects by Ficoll-Paque PLUS®, 17-1440-02, GE Healthcare, and MoFlo ( Purified by DAKO Cytomation). ^{T cells were isolated using a CD3 +} CD56 ^- cell population as a gate to avoid contamination of natural killer T (NKT) cells. Subset of T cells, sets the further ^{gate, CD4 (CD4 + CD8 -)} , and CD8 (CD4 ^- CD8 ⁺⁾ was separated into cohorts. CD4 and / or CD8 cells are further naive (CD45RA ⁺ CD62).
L ⁺ ), central memory (CD45RA ^- CD62L ⁺ ), effector memory (C
D45RA ^- CD62L ^- were classified into) ^-), or terminal effector (CD45RA ⁺ CD62L.

The cells sorted in this manner were 10% fetal bovine serum (manufactured by GIBCO-Invitrogen), 100 U / ml penicillin, 100 ng / ml streptomycin, 2 mM L-glutamine, and 20 ng / ml human interleukin 2 (hIL-2, First cultivated in Roswell Park Memorial Institute medium (RPMI) 1640 medium (manufactured by GIBCO-Invitrogen) supplemented with Novartis Vaccines & Diagnostics). Then, these cells were activated by adding 3 times the amount of anti-CD3 / CD28-binding magnetic beads (Dynabeads (registered trademark), ClinExVivo (registered trademark) CD3 / CD28, manufactured by Invitrogen) of the T cells. .. In the production of iPS cells (T-iPS cells) derived from T cells, the day of activation is defined as day0 (see FIG. 1 below). In some experiments, anti-CD3 / CD28-bound magnetic beads were used to isolate and stimulate ^{magnetically captured CD3 + cells from PBMCs.} Cells of retrovirus by spinoculation on a retronectin® coating plate (Takara) supplemented with 10 μg / ml protamine sulfate (Sigma-Aldrich) on days 2 and 3 (See Kaneko, S. et al., Blood, 2009, Vol. 113, pp. 1006-1015). The fully synthetic medium for T cell culture was changed daily. On day6, infected cells were harvested ^{and transferred onto 3 × 10 5} irradiated MEF cell layers on a 6 cm dish. Then, for 4 days (day6 to day10), half of the medium was replaced with human iPS cell medium daily. The composition of the human iPS cell medium is as follows (see Takayama, N. et al., Blood, 2008, Vol. 111, pp. 5298-5306).

20% knockout serum substitute (registered trademark), GIBCO-Invitrogen, 200 μM L-glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), 10 μM 2-mercaptoethanol (GIBCO) -Dalbeco modified Eagle's medium / F12 medium (manufactured by Sigma-Aldrich) to which 5 ng / ml b-FGF (manufactured by Wako) was added (manufactured by Invitrogen).

Further, before picking up the iPS cell colonies, 0.5 mM VPA (HDAC inhibitor valproic acid) was added to the human iPS cell medium. On day 10, the whole medium was replaced with VPA-containing human iPS cell medium. When they became identifiable with human ES / iPS cell-like colonies, they were mechanically isolated at approximately day24, dissociated into small masses by pipetting, and seeded on a fresh MEF cell layer. Human ES / iPS cell-like colonies were subjected to 3 to 3 to fresh MEF cell layers using trypsin solution (0.25% trypsin, 1 mM CaCl ₂ , and 20% knockout serum substitute-added phosphate buffered saline). Transferred every 4 days.

<Chromosome analysis>
Chromosome G band analysis was outsourced to the Japan Genetic Research Institute and 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 ice-cold fixative (90% methanol, 10% formaldehyde), using an ALP staining kit (manufactured by Vector Laboratories), and 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 then 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 the detection of Tra-1-60 and Tra-1-81, Alexa Fluor 488-conjugated goat anti-mouse antibody (dilution rate: 1/500, A11029, manufactured by Molecular Probes-Invitrogen) was used as a secondary antibody. Using. Furthermore, counterstaining of nuclei was performed using 4', 6-diamidino-2-phenyllindol (dilution ratio: 1/1000, manufactured by Roche Diagnostics). In addition, the photomicrograph is taken from the fluorescence microscope Axio Observer. Photographed using Z1 (manufactured by Carl Zeiss Japan).

<Teratoma formation>
Human ES / iPS cell-like colonies were aggregated and Masaki, H. et al. Et al., Stem Cell Res, 2007, Volume 1, pp. 105-115. Incidentally, the injection cell number per mouse is 1.0 × 10 ⁶ cells. Eight weeks after the injection, the tumor formed in the testis was excised, fixed with 5% paraformaldehyde, embedded in paraffin, and then cut into sections. Then, the obtained sections were stained by the hematoxylin / eosin method, and it was examined with an optical microscope whether or not the iPS cells had the ability to differentiate into three germ layers.

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

<Microarray analysis>
Whole-genome gene expression analysis 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 and Whole Human Genome Microarray 4x44K (G4112F, manufactured by Agilent Technologies) were hybridized by a one-color method (contractor: Takara Bio Inc.). The array was then scanned and spot images were detected on an Agilent Feature Extraction device (manufactured by Agilent Technologies). Then, the obtained signal data is used in the GeneSpring software (Agi).
Analysis was performed using lent Technologies (manufactured by Lent Technologies). In addition, Two normalization processes were applied to the normalization of the obtained data. That is, the signal intensity less than 0.01 was set to 0.01. Further, each chip was normalized to 50 ^th percentile of the measurements obtained from the chip. Then, in all three samples, the genes with the present flag (36275 genes) were analyzed.

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

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

Primer constructs for detecting TCRA rearrangements are described in Han et al. (See Han, M. et al., J Immunol, 1999, Vol. 163, pp. 301-11). The PCR reaction conditions were an amplification reaction of 95 ° C. for 30 seconds, 68 ° C. for 45 seconds, 72 ° C. for 6 minutes for 3 cycles, 95 ° C. for 30 seconds, 62 ° C. for 45 seconds, and 72 ° C. The amplification reaction of 6 minutes was defined as 15 cycles, the amplification reaction at 95 ° C. for 15 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 6 minutes was defined as 12 cycles.

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

The results obtained show that the V, D, and J segments involved in the construction of the TCRA or TCRB are the published sequences and the ImMunoGeneTics (IMGT) database (http://www.cines.fr/). They were compared and identified by using a web tool such as v-quest (Lefranc, MP "IMGT database, web resources and tools for cinemaglobulin and T cell receptor. , Leukemia, 2003, Vol. 17, pp. 260-266). The gene fragments (segments) were named according to the IMGT nomenclature. The primer sequences used to detect TCR rearrangements are shown in Tables 3-4.

<Induction of T-iPS cells to T-lineage cells>
Takayama, N. et al., In which T-iPS cells or other pluripotent stem cells have been slightly modified to adopt an embryonic stem cell-derived sac (ES-sac) -like structure. , 2008, Vol. 111, pp. 5298-5306.

Pluripotent cells were added with cytokine-free α-MEM-based medium (20% fetal bovine serum, 100 U / ml penicillin, 100 ng / ml streptomycin, and 2 mM L-glutamine) on the irradiated OP9 cell layer. It was co-cultured in αMEM) for 10 to 14 days (about Day 12).

The floating cells clogged in the sack were transferred onto the OP9-DL1 cell layer and co-cultured up to Day 40 in a medium supplemented with 10 ng / ml hIL-7 and hFlt-3L based on αMEM. The day when the pluripotent cells were transferred onto the OP9 cell layer is defined as Day 0. The culture medium was changed every 3 days. T lineage cells floating on the OP9-DL1 cell layer and expressing CD45, CD3, and TCRαβ were sorted weekly by flow cytometry and gene expression analysis was performed. OP9 cells and OP9-DL1 cells were obtained from RIKEN BioResource Center through the National BioResource Project (Japan).

<Responsiveness to TCR stimulation and cytokine expression profile of T lineage cells redifferentiated from T-iPS cells>
At the end of induction into T cells (cultured on OP9-DL1 cell layer for about 4 weeks, then Day40), suspended cells were collected, 30 ng / ml OKT-3 (manufactured by Janssen Pharmaceuticals) and 600 IU / ml hIL. -2 (Novartis Vaccines & Diagnostics) was stimulated. 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 by Uckert, W. et al. Et al., Cancer Immunol Immunother, 2008, Vol. 58, pp. 809-822. That is, 1 × 10 ⁵ floating cells were mixed with 10 ng / ml phorbol 12-millistart 13-acetylate (PMA, manufactured by Sigma-Aldrich), 0.4 μM A23187 calcium ionohore (ionomycin, manufactured by Sigma-Aldrich). ), And 20 IU / ml hIL-1α (manufactured by Peprotech) for 12 hours. Further, in the last 3 hours, the protein secretion inhibitor Breferdin A (1 mM, manufactured by Sigma-Aldrich) was added and cultured.

The cells after such stimulation were labeled with anti-CD3-bound magnetic microbeads (manufactured by 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-phase intracellular staining (solid-state intracellular cytokine staining) was performed on these cells according to the instructions of the manufacturer. Intracellular levels of hIFN-γ and hIL-2 were measured.

<Detection of TCR rearrangement in T-lineage cell mRNA derived from T-iPS cells>
^{CD3 +} derived from T-iPS cells on the 14th day (day26), 21st day (day33), and 28th day (day40) after the start of culturing on the OP9-DL1 cell layer.
Total RNA was extracted from TCRαβ ^{+ T lineage cells.}
Then, a method (SMART method, Du, G. et al., J Immunol Methods, 2006, 308, 19-) based on the transfer mechanism at the 5'end of the reverse transcriptase (switch mechanism at the 5'-end of the reverse transcript). In (see page 35), cDNA was synthesized using the Super SMART® cDNA synthesis kit (manufactured by Clontech Laboratories) according to the manufacturer's instructions. That is, using 3'SMART (registered trademark) CDS primer and SMART II A oligo (Super SMART (registered trademark) cDNA synthesis kit), and PrimeScript Reverse Transcriptase (manufactured by Takara) for 90 minutes at 42 ° C. The reaction was carried out. In addition, the synthesis and amplification of double-stranded cDNA is performed using reagents contained in 5'PCR Primer II A (Super SMART® cDNA synthesis kit) and Advantage 2 PCR Kit (manufactured by BD Clontech). It was. The PCR reaction conditions 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 the TCRA-specific amplification reaction or the TCRB-specific amplification reaction. That is, using a forward primer (2nd 5'-SMART) and a reverse primer, a reaction of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 3 minutes was carried out for 25 cycles for amplification. As the reverse primer, 3'-TRAC was used for TCRA amplification and 3'-TRBC was used for TCRB amplification. Then, 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 Statuel2 (manufactured by OMS Publishing). Then, P <0.05 was considered significant.

(Example 1)
<Establishment of iPS cells from human peripheral blood T cells>
First, iPS cells were established as shown in FIG. 1 using T cells in human peripheral blood. That is, peripheral blood mononuclear cells (PBMC) or CD3-positive cells were isolated from the peripheral blood of healthy subjects (age (24-56 years old) and multiple healthy subjects of different genders) and stimulated with CD3 / CD28 ClinExVivo beads. did. Then, the cells were cultured in RPMI1640 + 10% FBS + PSG (penicillin, streptomycin, and L-glutamine) containing 20 ng / ml of hIL-2 for 2 days, and VSV-G pseudo-type retroviral viral vectors pMX OCT4, pMX SOX2, pMX KLF4, and Genes were introduced using pMX c-MYCm or pMX OCT4, pMX SOX2, pMX KLF4. ^{After the gene transfer operation for 2 consecutive days, 1 × 10 5} cells were sprinkled on 6 cm dish irradiated MEF (mouse fetal fibroblasts) on the 6th day from the day of isolation, and replaced with iPS medium (human iPS cell medium) in half daily. Then, 4 days after seeding, the T cell medium was completely replaced with the iPS medium. From about 11 days after separation (5 days after sowing), paving stone-like cell collection elephants were observed, and after a few days (18 to 24 days after the start of culture), the appearance of human ES cells began to appear (Fig. 2). : See photo of colonies (human ES cell-like colonies),). 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: colony staining photograph), and silencing of foreign genes and ES cells. A similar gene expression pattern was observed (see Fig. 4: Electrophoretic photograph). Alkaline phosphatase (ALP) -positive human ES cell-like colonies were generated from ^{3 × 10 5} cells into which c-MYC was introduced and not introduced, respectively, at 77 ± 25 (0.03%) and 5.1. Obtained at a rate of ± 2.4 (0.001%) (see FIG. 5).

In addition, the expression gene profiles of CD3-derived pluripotent cells and human ES cells (KhES3) and peripheral blood T cells were compared by microarray analysis. 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 Figure 6).

In addition, the cariotype (karyotype) of the cells was normal (see Figure 7: Cariotype analysis photo), and transplantation into the testis of NOD / SCID mice formed a teratoma showing differentiation into three germ layers (Fig. 7). 8: See teratoma-forming pathological findings).
Therefore, it was revealed that iPS cells established from human peripheral blood T cells have pluripotency.

(Example 2)
In order to confirm that the established iPS cells are derived from T cells, the rearrangement of the TCR gene was confirmed. As for the TCR gene, the three groups of TCRδ, TCRγ, and TCRβ begin to rearrange from an early stage (in almost this order). Reconstitution 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 TCRβ gene and TCRα gene rearrangements in these clones revealed inframe TCRβ gene and TCRα gene rearrangements in all clones (Table 5: Inframe of the TCRα chain region (Table 5). In frame) gene rearrangement, Table 6: In frame gene rearrangement of the TCRβ chain region). There was no combination of NKT cell types in which TRAV24 and TRBV11 were specifically used in all of these reconstructions, confirming that they were peripheral blood T cell-derived iPS cells. In Tables 5 and 6, "iPS Clone" indicates an established T cell-derived iPS cell line, "Realrangement" indicates a combination of segments used for reconstruction, and "Secues of junctional region" indicates a binding region. The base sequence is shown.

(Example 3)
Further, as in Example 2, the rearrangement 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 these DNA rearrangements are known to be involved in the development of normal T lymphocytes in the thymus. There is. 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 trace and is characterized. Therefore, by examining these traces, it is possible to retroactively confirm whether or not the iPS cells obtained in Example 1 are derived from mature peripheral T lymphocytes of a healthy donor.

First, TCRG or TCRB reconstruction 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., Diamond Mol Pathol, 1995). See Volume 4, pp. 108-112, van Dongen, JJ, Leukemia, 2003, Volume 17, pp. 2257-2317). As a result, TCRG or TCRB rearrangements were 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). ..
In addition, the TCRβ chain forms a heterodimer together with the TCRα chain, and most peripheral blood T cells express TCRαβ (Davis, MM et al., Nature, 1998, 334, 395). See page 402). For TCRG and TCRB, the TCRA locus is complex. The TCRA locus is a region spanning more than 1000 kb, including not only the TCRD locus, but also 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 using these for the analysis of multiple structures, the number of combinations of a huge number of primers was narrowed down to the extent that they could be put into practical use. 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, by this method, TCRA rearrangement could be identified in TCRB reconstituted iPS cells (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 sites consisting of three types of complementarity determining regions (CDRs 1, 2, and 3). In these three regions, CDR3 is the most dispersible because it spans the V (D) J binding region where various random nucleotides (N-nucleotides or P-nucleotides) are inserted. Therefore, we identified the sequences encoding individual TCR strands in the CDR3 region of the established iPS cell genome. As a result, it became clear that the functional rearrangement (Rearranged (Producive), in-frame binding and no stop codon) in the original T cells was conserved, and in all T-iPS cells. It was revealed that there was only one functional TCRA and TCRB reconstruction on one allele. The other allele was found to have a complete or non-functional reconstruction (intact or Realranged (Unproducive)), including DJβ reconstruction (see Tables 7 and 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 β receptor and a complex. Indicates whether or not the antigen is recognized (functional: Protein) or not (non-functional: Unproducive), and "Sequence of junctional receptor" 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 identified (see Godfrey, DI et al., Semin Immunol, Vol. 22, pp. 61-67). Therefore, it was clarified that iPS cells were produced from one peripheral blood T cell without changing the TCR-producing ability engraved in the CDR3 sequence.

(Example 4)
< ^{Reprogramming from CD4 +} lymphocytes or CD8 ⁺ lymphocytes to iPS cells>
Peripheral T lymphocytes are primarily ^{derived 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 viral gene transfer (Berger, C. et al., Blood, 2003, Vol. 101, 476- See page 484, 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, reprogrammed to iPS cells, and the efficiency of reprogramming to iPS cells differs depending on the derived T cells. I checked. The induction conditions in each cell are shown below.

<Induction conditions for ^{CD4 + T cells>}
CD3-positive CD56-negative CD4-positive T cell fractions (hereinafter referred to as CD4-positive T cells) of peripheral blood mononuclear cells obtained by blood collection were fractionated by flow cytometry, and anti-CD3 antibody and anti-CD28 antibody (bead binding, solidification) were obtained. , Regardless of addition to the medium). About 20 ng / ml of human IL-2 was added to the medium, and Yamanaka factor gene transfer was performed multiple times on a 24-well plate coated with retronectin 48 to 96 hours after stimulation. The Yamanaka factors used at that time were OCT4, SOX2, KLF4, and C-MYC, or OCT4, SOX2, and KLF4. About 2 days after the last gene transfer, 1 to ⁵ x 105 gene-introduced T cells were placed on the irradiated MEF on a 6 cm dish. When the CD4 positive T cells were not used by being separated by the flow cyto on the first day, the CD4 positive T cells were separated by the flow cyto and placed on the MEF at this timing. For 4 days from the next day of transfer, replace half of the medium with 0.5 μM valproic acid-containing ES cell medium, and then replace the 0.5 μM valproic acid-containing ES cell medium every day or every other day for culturing. Continued. From about 10 days after the transfer, paving stone-like cell collection was observed, and completed iPS cell colonies were observed within a few days.

<Induction conditions for ^{CD8 + T cells>}
CD3-positive CD56-negative CD8-positive T cell fractions (hereinafter referred to as CD8-positive T cells) of peripheral blood mononuclear cells obtained by blood collection were fractionated by flow cytometry, and anti-CD3 antibody and anti-CD28 antibody (bead binding, solidification) were obtained. , Regardless of addition to the medium). Human IL-2 was added to the medium at 20 ng / ml, IL-7 at 10 ng / ml, and IL-15 at 10 ng / ml, and 48 to 96 hours after stimulation, multiple times on a 24-well plate coated with retronectin. Yamanaka factor gene transfer. The Yamanaka factors used at that time were 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 x 105 gene-introduced T cells were placed on the irradiated MEF on a 6 cm dish. When CD8-positive T cells were not used after being separated by flow cytos on the first day, CD8-positive T cells were collected by flow cytos at this timing and placed on the MEF. For 4 days from the next day of transfer, replace half of the medium with 0.5 μM valproic acid-containing ES cell medium, and then replace the 0.5 μM valproic acid-containing ES cell medium every day or every other day for culturing. Continued. A paving stone-like cell cluster was observed from about 2 weeks after the transfer, and completed iPS cell colonies were observed within 1 to 2 weeks.

As a result of examining under the above induction conditions, in this example, ^{the success rate of induction from CD4 +} T lymphocytes to T-iPS cells under the condition that NANOG is not introduced is the condition under which the reprogramming factor NANOG is further introduced. It was twice the success rate of induction from CD8 ^{+ T lymphocytes to T-iPS cells in.} In this example, N from ^{CD4 + T lymphocytes}
T-iPS cells are obtained by 7 or 8 trials without introducing ANOG.

In addition, although not shown in the figure, ^{when inducing CD8 +} T lymphocytes to T-iPS cells, M
_{Hypoxic culture under 5% O 2 in the} presence of 10 μM ROCK inhibitor after transfer onto EF, 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 increase slightly.

Based on the above results, further CD4 ⁺ T lymphocytes were added to CD45RA and CD62L /
Based on the expression of CCR7, it is divided into a subset of naive (CD45RA + CD62L +), central memory (CD45RA-CD62L +), effector memory (CD45RA-CD62L-), and terminal effector T cells (CD45RA + CD62L-), and induced into iPS cells (reprogramming). (See Salusto, F. et al., Nature, 1999, Vol. 401, pp. 708-712, see FIG. 14). As a result, the average reprogramming efficiency in 3 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 effectors. It was 0.0008% in T cells, and it was revealed that T-iPS cells were established relatively better than central memory T cells (see FIG. 15). The high efficiency of reprogramming 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>
Transplantation of CD34-positive cells obtained from human ES cells into SCID-hu mice induces differentiation of T cell lineages in the transplanted human embryonic thymus (Galic, Z et al., PNAS, 2006, Vol. 103, 31). No. 1,1742-7). In vivo, ES cells are induced by OP9-DL1 cells into CD3-positive and TCR-positive cells, mainly CD4 / CD8 double positive (see Timmermans et al., JI, 2009, Vol. 182, pp. 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 mutations 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 and conflicts. It was expected to have a monoclonal TCR depending on the allele exclusion state. However, whether T cells can be induced from T-iPS cells, whether the induced T cells are functional, and the rearranged state of TCR are the same as those of the T cells that are the source of T-iPS cells. Since it is unknown whether or not, these points were investigated as follows.

First, from the established T-iPS cells, one allele of both the TCRα chain and the TCRβ chain undergoes in vitro reconstitution, and the contralateral allele undergoes out frame reconstitution in the α chain, β chain. In, a clone stopped by DJ reconstruction was selected (TkT3V1-7), and the 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) are ES-sac (Takayama et al., Blood, 2008). Year, Vol. 111, No. 11, pp. 5298-5306) was formed to induce CD45-positive blood cells inside by day 14 (see (a) in FIG. 16). Blood cells were transferred to OP9-DL1 on days 12 to 14, medium exchange was repeated every 3 days to continue culturing, and CD3 positive cells were collected by FACS on days 21 and 40 to collect T cell-related genes. Expression was evaluated. Although the CD3 positive cells on the 40th day were mainly CD4 / 8 negative cells, some CD8 single positive cells were observed (see (b) in FIG. 16).

In addition, after the sack was transferred onto the OP9-DL1 cell layer, floating cells emerged from the sack were collected and analyzed weekly using flow cytometry (see (a) in FIG. 17). As a result, in the first week, most floating cells weakly expressed CD45, and in some cells CD34, CD7, and CD1a were expressed, while cells expressing the CD3-TCRαβ complex and CD5. There was no. In the second week, CD45 in most cells
Expression was upregulated, and the CD3-TCRαβ complex was also beginning to be expressed in some cells. By the third week, CD34 cells had completely disappeared, and CD3-TCRαβ strongly positive cells were the cell group with the largest number of floating cells. Incidentally, in CD3 + cells TCRarufabeta ^- cells was not (see (b) of FIG. 17). Also, even after the third week, most redifferentiated CD3 ⁺ TCRαβ ⁺ cells were in the co-negative (DN) stage, but in some cell groups, only CD4 positive cells (8. 5 ± 8.2%) and CD8-positive cells (15.5 ± 15.9%) were also detected (see FIGS. 18 and 19).

In general, it is known that even at the thymocyte stage, TCR-expressing T lineage cells can respond to TCR stimulation and can also produce cytokines (Fisher, M. et al., J Immunol, 1991, Vol. 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 the floating cells with 30 ng / ml OKT-3 and 600 IU / ml hIL-2 for 5 days, the CD3 ⁺ cell group increased, and the CD3-expressing cells were activated by morphological criteria. It was found (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 calcium ionophore (ionomycin), 12.5% of T lineage cells produced IFN-γ, 1.0% of T lineage cells produced IL-2, and 6.2%. It was revealed that the T lineage cells of T lineage produced both of them (see FIG. 21). ^{Therefore, it was clarified that CD3 +} TCRαβ ⁺ cells derived from T-iPS cells contribute to the induction to T lineage cells both morphologically and functionally.

In addition, as described above, since the TCR gene generally does not undergo high-frequency somatic mutations (somatic hyper-variable mutations), the redifferentiated T lineage cells derived from T-iPS cells frequently become the source of T-iPS cells. It is expected that TCRαβ encoded in the genome of the cell has been expressed. However, since it is unclear whether the rearranged state of the TCR is the same as that of the T cell that was the source of the T-iPS cell, this point was investigated as follows.

As a result, when the TCR expression of the induced CD3 cells was analyzed, only one type of TCRβ 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). Although the TCRα chain showed diversity, most of it was derived from the reconstituted α chain (see FIG. 23). It was revealed that the reprogrammed T cells express TCR clonally when they are re-induced into T cells.

^{In addition, CD3 high} TCRαβ ^high T lineage cells redifferentiated from 4 T-iPS cells derived from two donors were analyzed. That is, a cDNA library of these cells was constructed by SMART-mediated reverse transcription reaction (see Du, G., et al., J Immunol Methods, 2006, 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 T-iPS cell before differentiation (see FIGS. 13, 23, and 25). On the other hand, most transcripts were derived from the functional chain, and there were also transcripts derived from the non-functional chain (see Table 8).
.. However, no transcripts different from the sequences from the functional or non-functional rearrangements were identified (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, that is, a β receptor and a complex. Indicates whether or not the antigen is recognized (functional: Protein) or not (non-functional: Unproducive), and "Sequence of junctional receptor" indicates the base sequence of the binding region. Further, in Table 9, "Productivity" refers to whether or not the reconstituted T cell receptor has a function, that is, whether or not it forms a complex with the β receptor and recognizes an antigen (functional: Productive) or not (functionality: Productivity). Non-functional: Unproducive), "No. of Sequencing Samples" indicates the number of samples analyzed for each TCR gene, and "Sequence receptor with T-iPSC's genome" in the analyzed samples before redifferentiation. The number and proportion (Indental) consistent with the genome of the T-iPS cell, or the number and proportion different from the genome (Different) are shown.

Furthermore, the induction from other pluripotent cells to CD34-positive 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)
Although CD3-positive cells were induced in all of PSC), 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. Found (see Figures 16, 17, 26, and 27).

(Example 6)
<Induction of iPS cells established from human T cells to functional T cells 2>
Using T-iPSC clones (6 clones) derived from multiple donor CD4T cells or CD8T cells, an attempt was made to induce a T cell population having a monoclonal TCR. ^{That is, irradiation OP9 in which 3 × 10 5} T-iPS cells were spread on a 10 cm dish on the induction start date.
The cells were sprinkled on cells or 10T1 / 2 cells and co-cultured using a differentiation medium for about 14 days. Blood cells that entered the sac-like structure appeared during co-culture from the 12th to the 14th day. At that time, blood cells appeared without the combined use of cytokines, but it was revealed that the addition of VEGF improved the recovery of CD34-positive hematopoietic stem cells (hereinafter referred to as 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 tendencies were also reflected in the recovery of the final product, T lineage cells.

^{In addition, on any of the 12th to 14th days, 3 × 10 5} irradiated OP9 / DL1 cells, OP9 / DL4 cells, or 10T1 / 2 / DL4 cells in which blood cell cells containing those CD34 cells were spread on a 10 cm dish. 20% FBS in the presence of sowing, IL-7 and Flt3L
It was co-cultured in the containing α-MEM medium. SCF was not used because it strongly promotes differentiation into NK cells. As mentioned above, some of the floating cells in the second week of co-culture become CD3-positive TCRαβ-positive T lineage cells in the third week, but these cells are double negative in the expression of CD4 and CD8 ( It included DN), double positive (DP), and single positive (SP), and was not a uniform population (see FIG. 17). Therefore, as a result of further detailed examination, it was found that the composition 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 and CD56-negative T cells themselves was small, and most of them were DN. At IL-7 5-10 ng / ml, a CD3-positive CD56-positive NKT-like cell population appeared, again mostly DN. On the other hand, at IL-7 1 ng / ml, CD3-positive and CD56-negative T lineage cells accounted for the majority, and differentiation from DN to DP, and to CD4SP and CD8SP was confirmed. Moreover, when these SP cells were analyzed with peripheral blood T cell differentiation markers, about 80% of CD8SP ^{were CD45RA + CD62L +} naive T cells, and most of CD4SP ^{were CD45RA- CD62L +} central memory T cells. That is, it was suggested that T cells that had advanced to the effector memory stage could be rejuvenated into naive T cells (in the case of CD8SP) and central memory T cells (in the case of CD4) via T-iPS cells.

As described above, the method for producing human T lymphocytes of the present invention is particularly specific for the target antigen, and thus is specific for the target antigen and is suitable for the desired human tissue. It excels in producing sex antigen (HLA) -restricted CD4 single-positive cells or CD8 single-positive cells. 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: 1-109
<223> Nucleotide sequence of artificially synthesized primer

Claims (5)

A method for producing a population of human CD4 single positive T cells.
A step of inducing iPS cell colonies from human T cells having antigen specificity and expressing a CD4 molecule {however, a p53 inhibitor is not used in the induction} .
A step of culturing the iPS cell colony in a medium suitable for culturing iPS cells, and
The iPS cells are differentiated into human CD4 single positive T cells selected from the group consisting of CD45RA + CD62L + naive T cells and CD45RA-CD62L + central memory T cells, and the differentiation is confirmed to confirm the differentiation, and the human CD4 single containing the differentiated cells. A method comprising the step of obtaining a population of positive T cells. The method of claim 1, wherein the human T cells induced by iPS cells are isolated CD4 + CD45RA + CD62L + naive T cells. The method of claim 1, wherein the human T cells induced by iPS cells are isolated CD4 + CD45RA-CD62L-effector memory T cells. The method of claim 1, wherein the human T cells induced by iPS cells are isolated CD4 + CD45RA + CD62L-terminal effector T cells. Claims 1 to 4, wherein the human T cells induced by iPS cells are T cells activated with an anti-CD3 antibody and an anti-CD28 antibody in the presence of interleukin-2 (IL-2). The method according to any one item.

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