KR20190032198A - Method of Measuring Immunogenicity - Google Patents

Abstract

A method for identifying, measuring, or predicting immunogenicity in a recipient of an implant is provided. The method comprises the steps of: contacting an implant with a composition for antigen stimulation comprising an immune system derived from a recipient (allogeneic antigen stimulation step); and measuring the number of CD8 T cells in the obtained product, and/or the presence or number of memory CD8 T cells or memory CD4 T cells. An immune response such as rejection reaction is predicted in advance, thereby enabling more effective treatment.

Description

Method of Measuring Immunogenicity [0002]

Contacting the graft with an antigen-stimulating composition comprising an immune system derived from a recipient (antigen stimulating step); And measuring the number of CD8 T cells in the resulting reaction and / or the presence or number of memory CD8 T cells or memory CD4 T cells. And how to predict.

Human mesenchymal stem cells are used in about 70% of clinical trials using stem cells, and their applications are also very broad, including heart, neurological, orthopedic, digestive and immune system diseases. In the early stage of clinical trials using mesenchymal stem cells, most of them were autologous cells, but the percentage of using homologous cells, which can be mass-produced gradually, is increasing (see the following figure).

In particular, mesenchymal stem cells do not have MHC (major histocompatibility complex) class II and CD40, CD80, and CD86, which play a major role in the recognition of allogeneic antigens, and MHC Class I expression It is known that not only the immunogenicity is relatively low but also the therapeutic effect using clinically immunoregulatory action can be expected.

On the other hand, it is known that when allogeneic mesenchymal stem cells are used in clinical use, allogeneic antigens derived from donors induce immune responses to recipients and cause safety problems. When the immune response to the allogeneic antigens occurs, the administered cells may not be able to grow and may be eliminated, resulting in a decrease in the therapeutic effect. In particular, the surface antigen is increased by inflammatory cytokines (IFN-gamma etc.) There is also a possibility that the risk of reaction induction increases. Therefore, in order to utilize allogeneic mesenchymal stem cells in an actual treatment, predicting the possibility of inducing immunogenicity prior to administration to the human body is important for securing safety.

Immunogenicity assays for allogeneic antigen have been used in both in vitro and in vivo assays. Among these, the in vitro evaluation method has limitations in that it does not reflect the overall immune response in the same conditions as in vivo , but it can be used to evaluate the immune response by using human cells as in vivo The study has the advantage of revealing the detailed mechanism of action of immune cells that are difficult to understand. Efforts have been made to establish an in vivo test using humanized animal models, etc., but there is still a limit to reflect the human immune system. Therefore, in order to evaluate the immune response by allogeneic antigens to human mesenchymal stem cells, it is recommended to perform evaluation in in vitro conditions first, and to comprehensively evaluate the result with animal experimental results based on the results.

In vitro assays for allogeneic immune responses include MLR (Mixed Lymphocyte Reaction), Carboxyfluorescein diacetate succinimidyl ester (CFSE) assay, specific immunocytes differentiated by fluorescence-activated cell sorting (FACS) ), And enzyme-linked immunospot (ELISPOT) assay using enzyme immunoassay. In general, the MLR assay used in in vitro immunogenicity assays is to assess the proliferation of T cells after reacting responder T cells with allogeneic stimulator lymphocyte cells. The MLR assay can assess the proliferation of T cells by inserting 3H-thymidine into the T cells or using the CFSE label, but it is impossible to analyze the proliferation and differentiation of the T cells. Given that T cells differ in function depending on subpopulation, and even if total T cell proliferation occurs at the same level, the interpretation of the results may vary if the number of proliferating cells and the ratio of cell population between the subpopulation groups are significantly different. The presence or absence of T cell proliferation obtained from this test method has limitations in interpreting clinical significance. On the other hand, when FACS is used, it is possible to perform selective analysis by functional subgroups using markers capable of distinguishing memory T cells, and when ELISPOT is used, specific cytokine-releasing T cells (Th1, Th2, Th17) can be selectively analyzed.

Until now, there has been no evaluation method to obtain definite results in the single immunity test for the evaluation of stem cell immunity. Efforts are being made to find evaluation conditions that can be analyzed in the environment close to the immune system, Development of new techniques for evaluating the immunogenicity of stem cells is needed.

The present invention relates to a method for the treatment or prevention of CD8 T cells derived from a recipient (or allogeneic) at the time of administration (infusion or transplantation) of a transplant (for example, a homologous or autologous cell on a transplant recipient basis) CD4 T cells, CD3 T cells, T cell-depleted PBMC (Td-PBMC), and non-immune cells (recipient reference allogeneic or autologous cells, meaning stem cells for immunogenicity evaluation) And then immunostaining the graft with the antigen to thereby measure the immunogenicity of the graft in the recipient.

The invention relates to the use of recipient derived CD8 T cells, CD4 T cells, and / or CD3 T cells as markers for identification or measurement of immunogenicity of said foreign cells in recipient.

One example provides a composition for antigen stimulation, comprising an immune system derived from a recipient. The composition for allogeneic antigen stimulation may be used for identifying or predicting the immunogenicity of the graft (recipient-based foreign cells and / or autologous cells) in the recipient. The composition for antigen- (Or predicting) whether an immune rejection has occurred in the recipient of the graft, that is, whether the graft has an immunogenicity to the recipient at the time of administration (injection or transplant). In one example, the composition may further comprise a cytokine, such as an inflammatory cytokine (interleukins (ILs), interferons (IFNs), tumor necrosis factor (TNFs), etc.)).

The composition for antigen stimulation may include an immune system derived from a recipient, for example, a T cell isolated from a recipient, wherein the peripheral blood mononuclear cells derived from the recipient and the non-immunized cells (e.g., stem cells) And at least one selected from the group consisting of < RTI ID = 0.0 > In one example, the T cells, peripheral blood mononuclear cells, and non-immune cells may be isolated from recipients.

The T cell may be a CD8 T cell, a CD4 T cell, and / or a CD3 T cell, and more preferably a CD8 T cell. The T cells may be isolated from the blood of the recipient, for example, peripheral blood mononuclear cells.

Another example provides a composition for identifying or measuring immunogenicity, which comprises at least one member selected from the group consisting of a T cell detection agent and a memory T cell detection agent. The T cell may be a CD8 T cell, a CD4 T cell, and / or a CD3 T cell, and more preferably a CD8 T cell. The memory T cell may be an effector memory T (TEM) differentiated from the T cell, a central memory T (TCM), or a combination thereof. For example, the memory T cell may be a memory CD8 T cell and / or a memory CD4 T cell. In one example, the memory cell may be a Th1, Th2, and / or Th17 cell subtype that produces IFN-gamma, and / or IL-17A, but is not limited thereto.

The T cell detection agent may be an antibody that specifically binds to a protein (for example, cytokine or the like) specifically present in each T cell (for example, cell surface) and / or a memory T cell differentiated therefrom, (small molecular chemical), platemer, and the like, but is not limited thereto. The T cell detection agent may be labeled with a conventional labeling substance (for example, a fluorescent substance, a luminescent substance, a radioactive substance, or the like).

Other examples include the number of T cells in the reactants and / or the presence or absence of memory T cells differentiated from the T cells to identify, measure, or predict immunogenicity upon recipient administration (implantation or transplantation) of the implant Or the number of the cells.

In another example,

(1) contacting the graft with a composition for antigen stimulation comprising the recipient ' s immune system (antigen stimulation step); And

(2) measuring the number of T cells in the reactant of step (1), and / or the presence or number of memory T cells differentiated from the T cells

Measuring or predicting the immunogenicity of the recipient in the recipient, or providing information to confirm, measure or predict the immunogenicity of the recipient in the recipient. The method is for confirming (or predicting) whether the implant has immunogenicity to the recipient upon administration to the recipient, that is, whether the recipient causes the recipient's immune rejection response. The method can also be used to confirm or predict whether the implant is suitable for administration to the recipient.

The immune system may include T cells isolated from recipient, and may further include at least one selected from the group consisting of peripheral blood mononuclear cells and non-immune cells. In one example, the T cells, peripheral blood mononuclear cells, and non-immune cells may be isolated from recipients. The immune system may further comprise a cytokine to provide an environment similar to the immunostimulatory state of the recipient (e. G., Inflammatory state, etc.). The T cell may be a CD8 T cell, a CD4 T cell, and / or a CD3 T cell, and more preferably a CD8 T cell. The T cells may be isolated from recipient or recipient and allogeneic, preferably recipient, blood, such as peripheral blood mononuclear cells.

(I) comparing the number of T cells in the reactant of step (1), such as the number of cells of CD8 T cells, with the immune system derived from the recipient prior to, or not reacting with, the graft (Ii) the presence of a memory T cell differentiated from the T cell, for example, a memory CD8 T cell and / or a memory CD4 T cell, in the reactant of step (1) (Iii) in both cases (i) and (ii), the graft has an immunogenicity to the recipient at the time of transplantation to the recipient (I. E., Causing the recipient ' s immune rejection response). (I-1) the number of T cells in the reactant of step (1), for example, the number of cells of CD8 T cells, the immune system originating from the recipient not reacting with the transplant or reacting with the transplant (Ii-1) when the reactant of step (1) does not have a memory T cell differentiated from the T cell, such as a memory CD8 T cell and / or a memory CD4 T cell, (Iii) when all of the above (i-1) and (ii-1) are compared with the immune system derived from the recipient before or after the immune response, Water is not immunogenic to the recipient at the transplantation (i.e., does not induce recipient's immune rejection response), or that the transplant is suitable for transplantation into the recipient Or predicted.

Thus, the method further comprises, after the measuring step (2)

(3) determining the number of T cells, the number of memory T cells, and / or the generation of memory T cells, measured in step (2), from the recipient not reacting with the transplant or reacting with the transplant Comparing with the number of T cells measured in the immune system, the number of memory T cells, and / or the generation of memory T cells

. ≪ / RTI >

The method may be performed in vitro (ex vivo or ex vivo).

The step of contacting the graft of step (1) with a recipient or a recipient with an immune system derived from the same species may be carried out by subjecting the graft to antigen stimulation to determine whether the graft- Confirmation or prediction. The above step (1) can be carried out by culturing the graft together with an immune system derived from a recipient. Such culturing can be carried out under conventional culture conditions and can be carried out in a non-heterogeneous culture medium which does not contain a heterogeneous component (for example, donor and / or recipient and heterologous serum, etc.). The non-heterogeneous culture medium may further comprise autologous serum derived from a recipient. In step (1), the transplant is added to the immune system of the same species as the recipient or recipient once or more (for example, once, twice, three times, or four times) until measurement of the T cell and / .

The transplant may be cultured in a non-xenogenic medium containing xeno-free medium or autologous serum (recipient-derived serum).

The method may further include the step of culturing the graft in a non-xenogenic medium containing xeno-free medium or autologous serum (donor-derived serum) prior to the step (1). The xeno-free medium means a medium containing no heterologous antigen. The heterologous antigen can be any protein derived from a different species than the stem cell-derived species (for example, bovine serum albumin (BSA) Albumin derived from different species or albumin-containing serum or blood such as FBS (Fetal Bovine Serum), and the like). At this time, in order to simulate the immunostimulatory state, cytokine may be added to the medium and cultured. The cytokine may be at least one inflammatory cytokine selected from the group consisting of interleukins (ILs), interferons (IFNs), tumor necrosis factor (TNFs), transforming growth factors (TGFs)

Measuring the number of T cells in the reactant of step (1) of step (2), and / or the presence or number of memory T cells differentiated from said T cells, Or by a T cell quantification or qualitative analysis method using a conventional detection substance (for example, an antibody specific to the cell surface protein, an extramammer, a small molecule compound, etc.) for a substance specifically secreted from the cells .

In one embodiment, the measuring step may be performed in one or more of the following ways, but is not limited thereto:

1. Antigen-specific T cell proliferation and production of memory cells: Cellular analysis (for example, fluorescence activated cell sorting (FACS)) using fluorescent labeling (eg, CFSE (Carboxyfluorescein succinimidyl ester)

2. Analysis of toxic substances such as inflammatory cytokines or granzyme B secreted from antigen-specific T cells: analyzed using FACS and / or ELISPOT (Enzyme-Linked ImmunoSpot);

3. Analysis of survival rate of MSCs due to cytotoxic immune responses of antigen-specific T cells: using conventional cell counting methods (e.g., CCK-8 (Cell Counting Kit-8));

4. Analysis of mortality of MSCs due to cytotoxic immune responses of antigen-specific T cells: conventional apoptosis and / or necrosis analysis.

The method may further comprise, after the comparing step (3)

(a) comparing (i) the number of T cells in the reactant of step (1), such as the number of CD8 T cell cells, the immune system derived from the recipient with or without reacting with the graft, (Ii) the presence of a memory T cell differentiated from the T cell, such as a memory CD8 T cell and / or a memory CD4 T cell, in the reactant of step (1) (Iii) when both of (i) and (ii) above, the implant is not administered to the recipient or the recipient of the transplant is treated with the recipient The method comprising the steps of: or

(b) (i) comparing the number of T cells in the reactants of step (1), for example, the number of CD8 T cell cells, with an immune system derived from a recipient not reacted with the graft or with the graft (Ii-1) the reaction product of step (1) does not contain a memory T cell differentiated from the T cell, such as a memory CD8 T cell and / or a memory CD4 T cell, (Iii) when both (i-1) and (ii-1) are compared with the immune system derived from the recipient prior to or not reacting with water, Determining to administer to the recipient

. ≪ / RTI >

Hereinafter, the present invention will be described in more detail.

The above implants are used for various purposes such as treatment, molding, and the like. Means a stem cell for administration (e.g., transplantation) in vivo to a mammal such as a human, and may be for use in autotransplantation, allograft, or xenotransplantation. In one embodiment, the transplant may be an autologous stem cell derived from a recipient or a homologous derived stem cell derived from the same species as the recipient. The transplant may be cultured in a non-xenogenic medium containing xeno-free medium or autologous serum (recipient-derived serum). The non-heterogeneous medium may be any one or more of a transplantable material derived from a transplant-derived subject and a recipient (for example, a human) and a heterologous individual (for example, a non-human animal) if the transplant-derived subject and recipient are homologous May refer to a medium that does not contain components (e. G., Serum).

The stem cells include all embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPS cells), and progenitor cells. For example, the stem cells may be at least one selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells, and pre-developmental cells. The stem cell may be a stem cell derived from the same species and / or a stem cell derived from an autosome. Embryonic stem cells are stem cells derived from embryonic stem cells that are capable of differentiating into all tissue types. Induced pluripotent stem cells (iPS cells), also referred to as degenerating stem cells, induce pluripotent stem cells to regenerate pluripotent stem cells by introducing a cell differentiation-related gene into the differentiated somatic cells, Means cells that have been removed. Progenitor cells have the ability to differentiate into specific types of cells similar to stem cells, but they are more specific and targeted than stem cells, and unlike stem cells, the number of divisions is finite. The pre-developmental cells may be pre-developmental cells derived from the mesenchyme, but are not limited thereto. In this specification, pregenerating cells are included in the category of stem cells, and unless otherwise stated, 'stem cells' are interpreted as including the entire developmental cells.

Adult stem cells are stem cells extracted from umbilical cord blood (umbilical cord blood) or adult bone marrow, blood, nerve, etc., which means primitive cells just before being differentiated into cells of specific organs. The adult stem cells may be at least one selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and the like. The adult stem cells may be mammals, such as human adult stem cells. Adult stem cells are difficult to proliferate and tend to differentiate easily. Instead of using various kinds of adult stem cells, they can be used for various long-term regeneration needed in actual medicine. In addition, they can differentiate according to the characteristics of each organ And can be advantageously applied to the treatment of incurable diseases / incurable diseases.

In one example, the adult stem cells may be mesenchymal stem cells, such as human mesenchymal stem cells. Mesenchymal stem cells (MSCs), also called mesenchymal stromal cells (MSCs), are known as osteoblasts, chondrocytes, myocytes, adipocytes, And a multipotent stromal cell capable of differentiating into various types of cells. The mesenchymal stem cells are non-marrow stem cells such as placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, dental pulp, May be selected from pluripotent cells derived from non-marrow tissues.

In this specification, graft recipients and recipients are used in the same sense.

The recipient means the individual to be implanted (transplanted), and may be selected from mammals including humans. The recipient may be the same, homologous, or heterogeneous entity as the individual from which the transplant is derived, for example, it may be a homozygous entity or the same entity.

As used herein, an antigen stimulus may mean an immunostimulation by an immunogenic (immune-stimulating) substance derived from a recipient carried out on an implant, and the composition for antigen stimulation may be a substance derived from a recipient May refer to a composition comprising an immunogenic material.

The composition for antigen stimulation or the immune system comprises at least one member selected from the group consisting of T cells, peripheral blood mononuclear cells, and non-immunocytes (for example, recipient and allogeneic or autologous stem cells) isolated from recipient , And may include, for example, all of T cells isolated from recipient, peripheral blood mononuclear cells, and non-immunized cells.

The T cell may be a CD8 T cell, a CD4 T cell, and / or a CD3 T cell, and more preferably a CD8 T cell. The T cells may be isolated from the blood of the recipient, for example, peripheral blood mononuclear cells. The separation of the T cells may be performed by any conventional means, such as, but not limited to, using an antibody specific for each T cell or a bead to which the antibody is attached. The T cell may be one with a detectable label attached thereto. The label may be at least one selected from the group consisting of all substances capable of being detected (or verified) by a conventional method, for example, a fluorescent substance, a luminescent substance, a radioactive substance, and the like.

The peripheral blood mononuclear cells may be separated from blood of recipient. The peripheral blood mononuclear cells may be T cells, such as CD8 T cells, CD4 T cells, or CD3 T cells). Thus, when T cells are removed from peripheral blood mononuclear cells, B cells serve as antigen presenting cells. In one embodiment, the peripheral blood mononuclear cell may be one in which proliferation is suppressed by irradiation, but the present invention is not limited thereto.

The non-immune cells may be selected from all cells except immune cells isolated from recipient, such as stem cells, and the like. The stem cells are as described above. In one example, the non-immune cells may be the same kind of stem cells as the transplanted stem cells. The non-immune cells can be used intact (in the form of cells) (which can be used for acute rejection) or as a cell lysate form (usable for chronic rejection).

In this specification, CFSE marker cells are exemplarily applied to FACS to analyze T cell proliferations after antigen stimulation, or T cell subtypes that secrete specific cytokines using ELISPOT, We suggest an immunogenicity assay using the allogeneic-antigen stimulation test designed to maximize the benefits of the assay. The assay proposed herein should be understood as one of the methods designed to assess the immunogenicity of homozygous antigens at high sensitivity at the current scientific level.

The allogeneic-antigen stimulation evaluation method is an example of a test method designed to evaluate the immunogenicity that can be induced through direct and indirect stimulation of mesenchymal stem cells, , Which is the most sensitive and clinically significant in vitro test method. The evaluation method proposed in the present invention is one of the methods designed to evaluate the immunogenicity of the homozygous antigens with high sensitivity at the present scientific level. Therefore, in the field of research and development, the kind of cells used, clinical administration conditions, And appropriate test design should be made. One example of the present invention can be used to evaluate immunogenicity to a researcher or a licensor who develops a therapeutic agent using allogeneic mesenchymal stem cells.

Hereinafter, the application of the present invention to human mesenchymal stem cell transplantation will be described in more detail.

When human allogeneic mesenchymal stem cells are immunogenic, they induce an adaptive immune response when administered to recipients, resulting in transplant rejection, in which the transplanted cells are removed in vivo . This rejection is mainly due to the cell mediated immunity mediated by the recipient CD4 T and CD8 T cells. Cell-mediated immune responses are induced by stimulation of the recipient's T lymphocytes with the direct antigen presentation or indirect antigen presentation stimulation of the mesenchymal stem cells of the treated mesenchymal stem cells (see " ≪ / RTI > < RTI ID = 0.0 >

[T-cell stimulation pattern of recipients with allogeneic antigens]

T cell activation leads to 1) T cell division, 2) T cell differentiation (differentiation of CD4 T cells into Th1, Th2, and Th17), 3) memory T cell generation, A variety of subsequent immune responses are produced through the generation of hypersensitivity (DTH), cytotoxic T cell response, and allogenic antibodies, resulting in graft removal. Thus, the cell-mediated immune response induced by the antigen-mediated mesenchymal stem cell transfection is dependent on the degree of fragmentation of the recipient T cell, the differentiation of CD4 T cells (Th1, Th2, Th17), the activity of antigen-specific CD4 T and CD8 T cells , Memory CD4 T cells (central and effector memory T cells), and the presence or absence of memory CD8 T cells (see figure below "T cell differentiation pattern following antigen stimulation"):

Therefore, induction of allogeneic antigen stimulation by direct stimulation and indirect stimulation, and then FACS and ELISPOT were used to determine antigen-specific CD4 T and CD8 T cell proliferation and differentiation and memory immune cell generation, antigen-specific CD4 T cell And methods of analyzing inflammatory cytokine production by CD8 T cells can be used to assess immunogenicity. In addition, the cytotoxic immune response to stem cells by the activity of antigen-specific T cells can be evaluated by analyzing cell viability and cell death rate of stem cells.

Immunostimulation of each test condition Exam conditions Donor antigen Recipient immune system Antigen presenting cells (APC) Acceptor T cells Direct Antigen Stimulation Group Human mesenchymal stem cells T cell removal PBMC CFSE labeled CD4 T and CD8 T cells Indirect antigen stimulation group Fragmented human mesenchymal stem cells T cell removal PBMC CFSE labeled CD4 T and CD8 T cells Control group Untreated T cell removal PBMC CFSE labeled CD4 T and CD8 T cells Negative control group Untreated Untreated CFSE labeled CD4 T and CD8 T cells

In the case of direct stimulation, 1) donor mesenchymal stem cells, 2) T-cell-depleted PBMC (PBMC, Peripheral Blood Mononuclear Cell) from which T cells have been removed by irradiation, 3) (CD3 T, CD4 T, CD8 T) of the recipient recipient. In the test group according to the indirect stimulation, 1) crushed donor mesenchymal stem cells, 2) peripheral blood mononuclear cells (T cell-depleted PBMC) of the recipient in which T cells were removed by irradiation, 3) (CD3 T, CD4 T, and CD8 T), and the cells are disrupted by liquid nitrogen or ultrasonic waves to antigenize the cells. The antigen-presenting cell (APC) B cells in recipient T cell-depleted PBMCs (Table 1).

Table 2 summarizes the principles and evaluation items of immunogenicity assessment using FACS and ELISPOT:

Overview of Mesenchymal Stem Cell Immunogenicity Assay Evaluation method Analysis principle Evaluation items Interpret results FACS T cell selection assay using cell surface markers (TCM, T cell Central Memory), active memory T cell (TEM, T cell Effector Memory) In the test group with allogeneic dendritic stimulation, the increase of memory T-cell subpopulation group induction of antigen-specific immune response Cell proliferation assay using CFSE label CFSE low cell line (evidence for antigen-specific T cell proliferation) ELISPOT Specific cytokine detection by T cell subset Th1, Th2, and Th17 cell groups Increased production of cell subtypes differentiated from activated CD4 T cells compared to the control group in the test group with allogeneic dendritic stimulation confirmed antigen-specific immune response induction

The following considerations should be taken into account when assessing the immunogenicity of allogeneic MSCs using the allogeneic antigen stimulation assay described in this specification: 1) The culture medium used for the allogeneic antigen stimulation test for mesenchymal stem cells Reported that FBS (Fetal Bovine Serum) was added to FBS, which is a heterologous protein, may affect the immunogenicity test. Therefore, in the culturing process of this test method for the evaluation of immunogenicity by allogeneic antigen stimulation, in addition to the mesenchymal stem cell to be evaluated, serum derived from the same recipient is used to exclude an additional factor that may cause an immune response . In addition, the use of a culture medium in a xeno-free condition should be considered to eliminate the possibility of immunogenicity.

2) Among clinical indications to which mesenchymal stem cells are applied, especially in the case of immune system disease, inflammation of the patient may be frequently exacerbated. In this state, the blood cytokine concentration may be elevated, and the increased cytokine may further increase the allogeneic immune response. Therefore, it is necessary to consider the clinical condition of the recipient, and it can be considered to increase the degree of prediction of the immune response result by adding a representative cytokine to the experimental conditions.

The technique provided by the present invention enables the effective treatment by predicting whether or not an immune response such as a rejection reaction is induced in various treatments using stem cells by confirming the immunogenicity of the stem cells.

FIGS. 1A-1C show the effects of inflammatory cytokines on the expression of HLA and co-stimulatory molecules on the surface of adipose derived mesenchymal stem cells (ADSC).
Figures 2a and 2b are the results showing the immunosuppressive effect of human ADSCs on the proliferation of artificially stimulated mouse T cells,
Figures 3a and 3b show the results of a comparison of antigen recognition pathways for the evaluation of immunogenicity of ADSCs through allogeneic antigen stimulation.
Figures 4a-4d show the results of generation of antigen-specific CD8 T cells following allogeneic antigen challenge.
Figures 5a-5d show the results of antigen-specific memory CD4 T and memory CD8 T cell generation during allogeneic antigen stimulation.
Figures 6a-6d show the production of IFN-gamma and IL-17A by CFSE-low-CD4 T or -CD8 T cells following allogeneic antigen stimulation.
Figures 7a and 7b show that the HLA-blocking antibody inhibits the generation of antigen-specific memory CD4 T and antigen-specific memory-CD8 T cells after allogeneic antigen challenge.
Figures 8a and 8b show the cytotoxicity of CD8 T cells to ADSC in terms of cell viability.
FIGS. 9A and 9B show the cytotoxicity of CD8 T cells to ADSC by the cell death rate. FIG.
Figures 9c and 9d are graphs showing that cytotoxicity of CD8 T cells to ADSCs is inhibited by the HLA Blocking Ab complex.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the scope of the present invention. The embodiments described below may be modified without departing from the gist of the invention.

Example 1. Allogeneic Antigen Stimulation

1.1. Reagents and equipment

end. reagent

StemPro®Accutase: Life Technologies, A11105-01

Fetal Bovine Serum: Gibco, Mesenchymal Cell Qualified 12664-025,

Certified 16000044

Penycillin-streptomycin: Gibco, 15140-122

Glutamax: Gibco, 35050-079

ACK Lysis Buffer: Gibco, A10492-01

Blood collection tube: BD Vacutainer 367955 (Silicone coated),

367874 (Heparin coated)

CFSE: Sigma, 21888

Ficoll: GE healthcare, 17-1440-02

4% paraformaldehyde (in PBS): Santa Cruz, 30525-89-4

Human CD3 T cell isolation kit - Negative isolation (Pan T cell isolation kit)

Miltenyi, 130-096-535

Human CD3 T cell isolation kit-Positive isolation:

Miltenyi, 130-050-101

Human CD4 T cell isolation kit: Miltenyi, 130-096-533

Human CD8 T cell isolation kit: Miltenyi, 130-096-495

LS separation column: Miltenyi, 130-042-401

Xenofree-media: CellGro, 24803-0500 (Human MSC medium)

Cellstart: Life Technologies, A10142-01

ELISPOT

Human IL-4 kit (Mabtech, 3410-2A), Human IL-17A kit (Mabtech, 3520-2A), Human IFN-gamma kit (BD Biosciences, 551849)

ELISPOT plate: Millipore, S2EM004M99

BCIP / NBT substrate: Sigma (B5655)

I. equipment

Flow cytometry (Canto II)

Gamma irradiator (10 Gy)

Macs separator, Miltenyi

Centrifugal separator

microscope

ELISPOT Reader: Autoimmun Diagnostika GmbH

1.2. Allogeneic Antigen Stimulation Method

The allogeneic antigen challenge test is performed through several steps. This example is a pretreatment step that must be performed before evaluating the allogeneic immune response of human mesenchymal stem cells using FACS, ELISPOT, and cytotoxic immune response.

(1) Separation of serum from human blood

In case of cell culture for allogeneic antigen stimulation in which the allogeneic type antigen of the test mesenchymal stem cell (donor) is reacted with the human immune system (recipient's immune system) Serum was used. To obtain the autologous serum, blood was collected from the recipient (5 healthy male and female, 20 to 40 years old, no disease state), and the blood was coagulated and the blood was coagulated by placing it in a silicone coated- After centrifugation, serum was collected.

(2) Separation of PBMC (Peripheral blood mononuclear cells) from blood

For the purpose of isolating PBMCs, use heparin coated-tubes to prevent blood clotting when collecting blood from recipients (five healthy men and women aged 20 to 40 years, no disease status). The process of separating PBMC from blood by centrifugation after Ficoll treatment is shown schematically in the figure below:

[Schematic diagram of PBMC separation using Ficoll]

The specific process is as follows:

1) Blood was collected from the recipient in an amount of 15 to 20 ml, and diluted 3-fold with PBS.

2) 3 ml of Ficoll was put into a 15 ml tube, 7 ml of diluted blood was slowly poured, and the tube was centrifuged at 400 × g and 20 ° C. for 30 minutes.

3) Carefully remove the supernatant and transfer the mononuclear cell layer with PBMC to a new 15 ml tube. Fill with PBS, centrifuge at 400 × g and 20 ° C for 10 min, remove the supernatant, PBMC were washed.

4) PBMC obtained by removing the supernatant was resuspended with 1 ml of PBS and stored at 4 ° C until the experiment.

(3) Isolation of T cells from PBMC

Two separate fractions were separated from the prepared recipient PBMCs by 1) T cells and 2) T cell-depleted PBMCs, respectively.

Negative selection is the most effective method for isolating T cells from PBMC cells, since beads used to capture T cells with beads (positive selection) can affect the allogeneic antigen-stimulating immune response T cells were isolated.

Since T-cell separation-PBMC separation is aimed to remove T cells, T cells (CD3 T, CD4 T and CD8 T cells) are captured and removed using beads as a positive selection method, and the remaining cell fraction T cell depletion-PBMC). The purpose of this procedure is to allow B cells in the T cell removal-PBMC to act as antigen-presenting cells.

end. Negative selection: Isolation of T cells

human CD4 T cell isolation kit (Miltenyi, 130-096-535), Human CD4 T cell isolation kit (Miltenyi, 130-096-533) and Human CD8 T cell isolation kit (Miltenyi, 130-096-495) CD3 T cells, CD4 T cells, and CD8 T cells were separately isolated from the PBMC prepared in Example 1.2 (2) above.

More specifically, 10 μl of a Pan-T cell biotin-antibody cocktail was added to PBS to adjust the volume to 40 μl per ⁷ PBMCs, mixed with light, and stored in ice for 10 minutes. 10 ⁷ PBMC was added to PBS 30㎕ per dog, and here the Pan T cell cocktail 20㎕ microbead (according to the test object using a Human CD4 T cell cocktail 20㎕ microbead, or Human CD8 T cell cocktail microbead 20㎕ each) was added to After mixing, they were mixed and stored in ice for 10 minutes. To this, 1 ml of PBS was added to float the cells.

After attaching a Macs LS column to a magnetic separator, the column was washed with 3 ml of cold PBS, and 1 ml of the cell suspension was added to the LS column. An additional 3 ml of PBS was added to the column. CD3 T cells (or CD4 T cells, CD8 T cells) passing through the column were centrifuged at 400 xg and 4 ° C for 5 minutes. 1 ml of a non-heterogeneous culture medium (containing 5% (v / v) recipient serum) was added to isolated CD3 T cells (or CD4 T cells, CD8 T cells) and stored at 4 ° C until the experiment.

* T cells are separated into CD3 T, CD4 T, or CD8 T cells, respectively, and used according to the purpose of the experiment.

I. Positive selection: T cell removal PBMC  (T cell-depleted PBMC)

T cell removal PBMC (T cell-depleted PBMC) is isolated by removing T cells from PBMC. At this time, T cells are removed by positive separation method.

In the case of the positive separation method, the following method can be used. Example 1.2. (2) In order to remove T cells from the PBMC prepared from, human CD3 positive T isolation kit (Miltenyi, 130-050-101) were .15 ml cornical 10 ⁷ PBMC per tube using the above prepared PBS was added to a volume of 80 ml, 20 ml of CD3 T cell microbead was added, and the mixture was mixed and kept on ice for 15 minutes. To this, 1 ml of PBS was added to float the cells.

Macros LS column was attached to a magnetic separator and washed with 3 ml of cold PBS. 1 ml of the cell suspension was added to the thus prepared LS column. 3 ml of PBS was added to the column. The reaction products that passed through the column were centrifuged at 400 xg and 4 ° C for 5 minutes to separate T cell depleted-PBMC cells.

1 ml of a non-heterogeneous culture medium (containing 5% human serum) was added to the separated T cell depleted-PBMC cells and stored at 4 ° C until the experiment.

(4) T cell removal-PBMC (T-cell depleted PBMC) was irradiated with radiation

The prepared T cell depleted-PBMC was irradiated with 1,000 rad radiation to lose the ability to proliferate B cells functioning as antigen-presenting cells. When the dose is more than 2,000 rad, the function of the B cell as an antigen presenting cell is markedly decreased. Therefore, in this embodiment, the dose of radiation is set with reference to this fact.

Example 1.2 (3) above b. T cells removed from prepared recipients-PBMC were irradiated with 1,000 rad. Removal of irradiated T cells-1 ml of a heterogeneous culture medium (containing 5% fetal calf serum) was added to PBMC and stored at 4 ° C until the experiment. Removal of T cells to be used for repeated stimulation-PBMCs were stored in liquid nitrogen with 10% DMSO (a non-heterogeneous culture medium containing 10% fetal bovine serum).

(5) CFSE labeling of CD4 T, CD8 T, or CD3 T cells

T cells were labeled with CFSE (carboxyfluorescein succinimidyl ester) in order to evaluate whether the recipient's T-cell division was due to antigen stimulation of mesenchymal stem cells. CFSE expresses fluorescence by forming strong covalent bonds with living primary amines. The detection principle is that fluorescence sensitivity decreases as CFSE content decreases as cell proliferation proceeds. In the following examples, antigen-specific T cell proliferation was evaluated by the presence or absence of CFSE-low T and CFSE-low memory T cells during FACS analysis.

First, 10 ml of PBS was added to the prepared recipient CD3 T (or CD4 T, or CD8 T) cells, and the cells were washed by centrifugation at 400 xg for 5 minutes at 4 ° C. CD3 T (or CD4 T, or CD8 T) cells were suspended in 1 ml of PBS. 10 ml of 100 uM CFSE was added to the suspension of CD3 T cells on a clean bench, immediately mixed with 1 ml of PBS, and the final concentration of CFSE was adjusted to 0.5 uM. Tube was kept at room temperature for 5 minutes with light blocked. The Xeno-free media was then filled and washed by centrifugation at 400xg and 4 ° C for 5 minutes. This washing process was performed twice using xeno-free media or PBS. 1 ml of xeno-free media was added to CFSE-labeled CD3 T (or CD4 T, or CD8 T) cells and kept on ice until the experiment.

Example 2. Evaluation of specific memory T cell group using FACS

Allogeneic antigenic stimulation of human mesenchymal stem cells (adipose-derived stem cells (ADSCs) from human normal abdomen or breast adipose tissue was performed by two methods, indirect stimulation and direct stimulation. Both methods were cultured for 21 days, and the culture medium was changed every 7 days. For indirect stimulation, shredded stem cells were added to 0d, 7d, and 14d. For direct stimulation, stem cells were added to 0d (or added to 7d) Lt; / RTI > In this example, the allogeneic antigen challenge test was designed as shown in the following figure (test method for allogeneic antigen stimulation).

In the above picture, allogeneic-antigen stimulation of mesenchymal stem cells (AAS) induces homologous antigen-induced stimulation of donor's immune cells (T cells and Td-PBMC) with donor allogeneic mesenchymal stem cells Quot; In addition, allogeneic mesenchymal stem cells can be further added on day 7. Allogeneic antigen stimulation is performed by changing the culture medium every 7 days after the reaction and replacing only half of the medium volume with fresh medium. The cytokine treatment is an additional test condition for adding cytokines in the administration conditions of allogeneic mesenchymal stem cells or reflecting conditions in which the patient's inflammatory condition is exaggerated.

Existing memory CD4 and CD8 T cells were tested in mice using in vivo It has been difficult to apply the present invention to studies for evaluating the immunogenicity of human mesenchymal stem cells in addition to studies on human memory T cells because mouse is a xenogenic model for human cells. Thus, in this Example, a test method for evaluating the production of human memory CD4 T and CD8 T cells by antigen of human mesenchymal stem cells using in vitro allogeneic antigen stimulation test method is presented.

To eliminate the immunoreactivity factor in the culture medium such as FBS, the recipient (5 healthy men and women aged 20 to 40, no disease state; the following results are representative values of 5 outcomes or 3 or more Allogeneic antigen stimulation experiments were performed in a cell culture medium (Cellgro) for xeno-free mesenchymal stem cells containing 5% (v / v) autologous serum. The stimulation conditions of allogeneic antigens are as follows.

Indirect allogeneic-antigen stimulation

: Tremor of recipient T-cells removed - PBMCs (Td-PBMC) + CFSE-CD3 T, CFSE-CD4 T, or CFSE-CD8 T cells + recipient of shaved allogeneic mesenchymal stem cells Stem cells) treatment;

- Direct allogeneic-antigen stimulation

: Treatment of allogeneic mesenchymal stem cells (adipose tissue-derived mesenchymal stem cells) of CFSE-CD3 T, CFSE-CD4 T, or CFSE-CD8 T cells + recipient of recipient T cell removal-PBMC + recipient.

Allogeneic antigen stimulation was induced by the recipient's immune system (T cell depletion-PBMC + CFSE-CD4 T, -CD8 T, or -CD3 T) and recipient allogeneic mesenchymal stem cells (adipose tissue-derived mesenchymal stem cells) Followed by culturing for 21 days. This reaction was carried out every 7 days while replacing 50% of the culture medium.

Previously, all of the cells were harvested after 7 days, and then the medium was added. However, this method affects the T cell damage and the irritation reaction due to the use of Trypsin-EDTA, I did. In addition, there is a risk of cell loss during centrifugation washing and replating after the collection of cells in the conventional method. Thus, in this example, the allogeneic antigens stimulation experiment was conducted by replacing the medium every 7 days. When replacing the medium, the plate was carefully treated so that immersed immune cells were not removed, and the medium was removed and a new medium was added. Allogeneic mesenchymal stem cells were used for disruption of the allogeneic stem cells for indirect antigen stimulation and added on the first day, 7th day and 14th day of the reaction.

For direct antigen stimulation, allogeneic mesenchymal stem cells can be added on the first or seventh day of the reaction, and the specific addition timing and addition amount can be appropriately adjusted according to the purpose of the experiment. The immunogenicity of allogeneic mesenchymal stem cells can be evaluated by this allogeneic antigen stimulation reaction.

Analysis of antigen-specific immune responses of T cells using FACS was performed 21 days after AAS administration. If necessary, all of the cells in culture can be collected on days 7 and 14 after AAS, and FACS analysis and ELISPOT analysis can be performed.

Using FACS analysis after allogeneic antigen stimulation, antigen-specific activated T cells among CFSE-labeled CD4 T cells and CD8 T cells can be analyzed. And we can analyze the ratio (ratio,%) and cell number of CD4 and CD8 T cells between the central memory T cell group (TCM, T cell Central Memory) and the active memory T cell group (TEM, T cell Effector Memory) In addition, the number of CFSE-low TCM and TEM cells can be analyzed to identify antigen-specific newly generated subpopulation memory cells. Since allograft memory cells play a central role in rejection of grafts and rejection of secondary grafts at allograft, it is important to identify whether memory cells are generated in recipients.

In the FACS analysis, CFSE-labeled T cells (negative control) were cultured alone without addition of other immune systems or mesenchymal stem cells to determine the selection criteria for CFSE-low T cells. For comparison, T cell depletion-PBMC + CFSE-labeled T cell groups were used as experimental control groups for mesenchymal stem cell addition group. All T cells used in the experiment were labeled with CFSE.

To examine the immunogenicity of the mesenchymal stem cells pretreated with the inflammatory cytokines, the mesenchymal stem cells were pretreated with cytokines 4 days before the allogeneic antigen stimulation and the plate was treated the day before ; Cellstart, Life Technologies). On the next day (day of allogeneic antigen stimulation; day 0), the recipient's immune system was added to perform the allogeneic antigen stimulation experiment.

Allogeneic mesenchymal stem cells for indirect antigen stimulation were added to the experimental group on the day of the experiment. The composition of the medium for the reaction and the specific experimental method are as follows.

Analysis process

(1) Indirect allogeneic-antigen stimulation

1) removal of irradiated T cells from recipient-PBMC (1x10 ⁵ cells; prepared in Example 1.2. (4)) and CFSE-labeled CD4 T or CD8 T, CD3 T (2x10 ⁵ cells; 5). Cells were plated in 12-well plates. PBMCs were dosed with DMSO (10%) for repeated stimulation and stored in liquid nitrogen. As experimental controls, CD4 T + irradiated T cell depleted-PBMC (control) test groups with CD4 T cell alone (negative control) and no antigen treatment conditions were prepared.

2) Allogeneic mesenchymal stem cells (1 × 10 ³ cells) were pulverized by repeating freezing-thawing 5 times or more in liquid nitrogen, and then added to the prepared 12-well plate to induce an allogeneic allogeneic antigen stimulation. Crushed mesenchymal stem cells were stored at -70 ° C for use in repeated stimulation.

3) The cultures of 2) above contained 5% of autologous serum in xeno-free media and human recombinant interleukin-2 (IL-2) (eBioscience) . And the final culture medium was adjusted to 1 ml / well so that the content was 1 ng / ml (this is the basic medium composition for allogeneic antigen stimulation experiments).

IL-2 is added in vitro for maintenance of T cells and appropriate responses to antigens, which do not induce T cell activity (see Negative Controls of Experimental Results). T cells have differentiated IL-2R (CD25, CD122, CD132) that responds to IL-2 according to their subtypes.

4) After 7 days of incubation, 50% of the supernatant was carefully exchanged while careful not to remove the submerged cells. To this was added donor-irradiated T cell depleted-PBMC (3x10 ⁵ cells; prepared in Example 1.2. (4)). T cell depletion-PBMC were added at 7 day intervals.

5) The culture was replaced at 7 days intervals as in step 4) and cultured until day 21.

6) CFSE-labeled CD4 / CD8 T cells were analyzed by FACS for the presence or absence of immunogenicity on surface antigen of human allogeneic mesenchymal stem cells using the allogeneic dendritic antigen stimulation method as described above. CD4 / CD8 T cell proliferation and generation of CD4 / CD8 memory T cells (TCM and TEM cells). (Or anti-CD45RA) and anti-CD62L (or anti-CCR7) mAbs, which are memory T cell markers, are used for FACS analysis of human memory T cells using human anti-CD3, anti-CD4 and anti- (Each antibody was purchased from BioLegend) (TCM: CD4 (or CD8) + CD45RO + CD62L +, TEM: CD4 (or CD8) + CD45RO + CD62L-). Thus, the experimental control group required for the analysis of repeated allogeneic-antigen stimulation immune responses was T cells + T cells removed-PBMC (irradiated).

(2) Direct allogeneic-antigen stimulation

1) Allogeneic Antigen Stimulation Experiments Allogeneic mesenchymal stem cells were plated at 1 × 10 ³ cells / well in a 12-well plate one day prior to the next day to prepare a direct allogeneic antigen stimulation experiment.

2) dimorphic antigen stimulation experiment day grantor of CD3 on T (2x10 ⁵ cells. Example 1.2 5 preparation) and removing the irradiated radiation-T cells in -PBMC (1x10 ⁵ cells; Example 1.2 (4) T cell removal-PBMC) was dispensed into a 12-well plate.

3) The culture medium of 2) was prepared to a final culture volume of 1 ml / well in a non-heterogeneous culture medium containing 5% of autologous serum and 1 ng / ml of human recombinant IL-2.

4) The cells were cultured for 7 days and the medium was changed. At the time of medium change, 50% of the supernatant was removed from the supernatant, and the supernatant was carefully removed without any shaking to prevent the floating cells on the bottom from disappearing. The non-heterogeneous culture medium of the composition described in 3) was filled to 1 ml / well.

5) When replacing the culture medium at 7-day intervals, the irradiated T cells removed in liquid nitrogen-PBMC were further thawed and washed (same number of cells as in day 0, 1x10 ⁵ cells). The addition of these irradiated T cell depleted-PBMCs was performed at 7 day intervals.

The presence or absence of immunogenicity on the surface antigen of human allogeneic mesenchymal stem cells using the direct allogeneic antigen stimulation method was measured by FACS on the 21st day using CFSE labeled T cells and T cell proliferation and memory T cells (TCM and TEM cells ) Were compared with the control group. In order to analyze human memory-T cells by FACS, anti-CD45RO (or anti-CD45RA), anti-CD62L (or anti-CCR7) mAbs BioLegend) (TCM: CD4 (or CD8) + CD45RO + CD62L +, TEM: CD4 (or CD8) + CD45RO + CD62L-). Experimental controls used CFSE-labeled T cells + T cell depletion-PBMC (radiation).

Example 3. Evaluation of cytotoxic immune response of T cells to allogeneic mesenchymal stem cells

Cytotoxic immune response was evaluated by performing allogeneic antigen stimulation by direct stimulation. Direct stimulation methods may be more effective in immunogenicity analysis than indirect stimulation methods. Allogeneic antigen stimulation was continued for 14-24 days (cell survival analysis and cell death analysis), and T cell depletion-PBMC was added every 7 days with culture medium exchange. In order to remove the immune response inducers such as FBS in the allogeneic antigen stimulation, autologous serum of the recipient was used for xeno-free mesenchymal stem cells containing 5% (v / v) Allogeneic antigen stimulation experiments were performed under the condition of CellGenix. The stimulation conditions and methods of allogeneic antigens for the evaluation of cytotoxic immune responses are as follows.

Cellular composition for direct allogeneic-antigen stimulation was as follows: T cell removal-PBMC + T cells (CD3 T, CD4 T, or CD8 T) + allogeneic mesenchymal stem cells 2. (2)).

1) Allogeneic Antigen Stimulation Experiment Allogeneic mesenchymal stem cells (1 × 10 ³ cells / well) were plated on a 12-well plate one day prior to the experiment.

2) Removal of T cells (irradiated T cells) with T (CD3 T, CD4 T, or CD8 T) (2x10 ⁵ cells; prepared in Example 1.2. -PBMC (1x10 ⁵ cells; preparation in Example 1.2.4); T cell removal-PBMC) was dispensed into a 12-well plate. As a control for the cell death rate test, only the allogeneic mesenchymal stem cells were cultured without the immune cells (CD3 T and T cell removal-PBMC) (ie, comparison of ADSCs versus immune cells + ADSCs). As a control for cell viability, mesenchymal stem cells (XF-ADSC) were used (ie, ADSCs compared to non-heterologous ADSCs (XF-ADSCs) versus heterologous antigens).

3) The culture medium of 2) was incubated at 1 ml / well in a non-heterogeneous culture medium containing 4 ~ 5% (v / v) of autologous serum and 1 ng / ml of human recombinant IL- .

4) The cells were cultured for 7 days and the medium was changed. At the time of medium change, 50% of the supernatant was removed from the supernatant, and the supernatant was carefully removed without any shaking to prevent the floating cells on the bottom from disappearing. The non-heterogeneous culture medium of the composition described in 3) was filled to 1 ml / well.

5) When replacing the culture medium at 7-day intervals, the irradiated T cells removed in liquid nitrogen-PBMCs were further thawed and washed (same number as in day 0, 1x10 ⁵ cells).

6) The cytotoxic immune response of T cells using direct allogeneic antigen stimulation to mesenchymal stem cells was evaluated by microscopic observation of mesenchymal stem cell morphology between 14 and 24 days after the reaction, And cell viability and cell death rate were examined. At the time of analysis, morphological damage of mesenchymal stem cells due to allogeneic antigen stimulation was observed with a microscope (compared with the control group), and time analysis of the optimal analysis time may be necessary.

Allogeneic mesenchymal stem cells can be administered repeatedly with a single dose at the time of allogeneic antigen challenge, depending on the purpose of the experiment. In the case of repeated administration, allogeneic mesenchymal stem cells can be administered in the same manner as in day 0 on day 7 in the above-described experimental method.

(1) Cell survival analysis

1) After the allogeneic antigens stimulation, the mesenchymal stem cells were observed under the microscope for 14-24 days.

2) If damaged mesenchymal stem cells were observed, the 12-well plate was shaken gently to remove T cells and T cells, and the supernatant was removed by suspending PBMCs. To remove floating cells, they were carefully washed once or twice with 400 μl of PBS (warmed in a 37 ° C water bath). At this time, be careful not to let the attached mesenchymal stem cells fall.

3) To the 12-well plate from which the floating cells had been removed, 0.3 ml of DMEM culture medium was added, 30 μl of water-soluble tetrazolium salt, WST-8 was added, and the mixture was reacted for 3 to 4 hours in a CO ₂ incubator.

4) Each sample in a 12-well plate was transferred to a 96-well plate and dispensed.

5) The cell viability of mesenchymal stem cells was evaluated by analyzing the absorbance at 450 nm using an ELISA reader.

(2) Analysis of cell death rate

1) It was observed microscopically whether mesenchymal stem cell injury was induced between 14 and 24 days after the allogeneic antigen stimulation.

2) T cell and PBMC cells were suspended by gently shaking the 12-well plate which was subjected to the allogeneic antigen stimulation reaction to remove the supernatant. To remove floating cells, they were carefully washed 1-2 times with 400 μl PBS (warmed in a 37 ° C water bath).

3) The cells attached to the plate were reacted with Accutase in a 37 ℃ incubator for 5 minutes, and the separated cells were collected in a FACS analysis tube.

Accutase has relatively low cell damage and can be used as an alternative to trypsin-EDEA. In addition, the analysis time may be different according to the experiment, and it is recommended to measure by time difference such as 20 days and 22 days to select an effective analysis point.

4) The recovered cells were washed once with 3 ml of PBS, added with anti-CD73, -CD90, -CD45 antibody (eBioscience) conjugated with fluorescent materials, mixed well and reacted on ice for 15 minutes. Each antibody can be diluted to 1: 100 ~ 1: 500. Optimal appropriate concentration needs to be determined by the information of each reagent company and the experimenter.

Anti-CD73 and anti-CD90 antibodies were used to exclude immune cells to fractionate mesenchymal stem cells in FACS analysis. Anti-CD45 (lymphocyte common expression markers) were excluded.

5) After adding 2 ml of PBS, the mixture was centrifuged at 400 xg and 4 ° C for 5 minutes.

6) Annexin V binding buffer (eBioscience) (10X buffer diluted 1X with distilled water) was further washed once with 0.5 ~ 1 ml.

7) The cells were suspended (from 1 to 5 x 10 ⁶ cells / ml) with 100 to 200 μl of Annexin V binding buffer.

8) Annexin V (5 μl) per 100 μl of cell suspension was added and allowed to react for 15 minutes under constant temperature (light blocking) conditions.

9) Annexin V binding buffer (2 ml) was added and centrifuged at 400 xg for 5 minutes at 4 ° C.

10) After removal of supernatant, 200 ㎕ of Annexin V binding buffer was added to suspend the cells.

11) 5 ㎕ of Propidium Iodide (PI) was added and reacted on ice for 15 minutes and analyzed by FACS within 1 ~ 2 hours. Cells were stored at 4 [deg.] C in which light was blocked until analysis.

12) In the analysis of cell death of mesenchymal stem cells using FACS, the CD73 and CD90 regions, which are markers of mesenchymal stem cells, were selected and analyzed to select only the mesenchymal stem cells. The CD45 region (lymphocyte common expression marker ) Were excluded.

Example 4. Evaluation of specific cytokine-releasing T-cell subpopulations using ELISPOT

In this Example, ELISPOT was used to evaluate the subtype differentiation of activated T cells upon mesenchymal stem cell stimulation on mesenchymal stem cells according to secretory cytokines, not surface antigens. It is possible to confirm the differentiation of Th1 and Th17 cell subpopulations, which produce IFN-gamma and IL-17A, among activated T cells activated by allogeneic antigen stimulation.

As shown in Fig. 3A, the allogeneic antigen stimulation was applied, and all the cells were collected on the 14th day, followed by ELISPOT reaction, and analyzed on the 17th day after 3 days. In FIG. 3A, AAS means addition of mesenchymal stem cells for allogeneic antigen stimulation (AAS, allogeneic antigen stimulation), performed at day 0, added at day 7, day 14, and / or day 21 Can be performed). " Treating cytokines" Is an additional test condition for adding cytokines in the administration conditions of allogeneic mesenchymal stem cells or reflecting conditions in which the patient's inflammatory condition is enhanced (Fig. 3A).

The ELISPOT used in this example was a commercially available kit (BD Biosciences, Mabtech) . Human-anti-IFNgamma was assayed using a kit provided by BD Biosciences Inc. and human anti-IL-17 using a kit provided by Mabtech, referring to respective manufacturers' manuals.

Analysis process

1) Allogeneic antigens were stimulated by indirect and direct routes according to 1) to 5) of 1) to 4) or 2.) of Example 2. (1) The ELISPOT test was performed on the following procedure.

2) On the day before the ELISPOT experiment (on the 13th day), 100 μl of 30% ethanol was added to a 96-well plate for ELISPOT, allowed to stand for 30 seconds, and discarded. After washing three times with PBS, PBS was added one more time to prevent the plate from drying and then removed. Capture-antibody (diluted with PBS according to manufacturer's instructions) was added to each well of the wells and incubated overnight at 4 ° C. This procedure was performed one day before the ELISPOT experiment (day 13)

3) On the 14th day, the capture-antibody attached to the plate was removed from the ELISPOT plate and washed three times with PBS.

4) In the case of 1) allogeneic antigen-stimulated group, all of them were collected using acutase and centrifuged to remove supernatant. To this, 1 ml of the new culture was added and the cells were suspended and T cell depleted-PBMC were thawed. 100 μl of triplicate or quadruplicate was added to the ELISPOT 96-well filter plate (PVDF) prepared in 3) and cultured in an incubator for 3 days. At this time, 10% of the cells harvested after allogeneic antigen stimulation were subjected to flow cytometry and left for analysis of antigen-specific T cells (CFSE-low T cells). CFSE-low T cells were analyzed by flow cytometry after anti-CD3, anti-CD4 and anti-CD8 antibodies.

5) On the ELISPOT plate, allogeneic antigen stimulation was performed for 3 days (day 17), and the culture solution was removed and washed twice with 200 μl of distilled water. And then washed three times with PBS (Washing buffer I) containing 0.05% (v / v) Tween-20.

6) Detection antibody (biotin conjugated-Ab) was diluted in PBS according to the manufacturer's instructions, added to the plate in an amount of 100 μl, and reacted at room temperature for 2 hours.

7) Washing with Washing buffer I (PBS containing 0.05% Tween-20) three times.

8) Streptavidin-ALP (Alkaline phosphatase, Mabtech) was diluted 1,000-fold with PBS, and 100 μl of each was added to the plate and incubated for 1 hour at constant temperature.

9) Washing buffer I was washed 4 times, and PBS was washed 2 times.

10) 100 μl of BCIP / NBT substrate (Mabtech or Sigma) solution was added to each well. Spot formation was observed for 5 to 60 minutes, preventing overdevelop. This creates a high background which makes data analysis difficult.

11) The plate was washed twice with distilled water to stop the reaction, and the plate was dried at constant temperature.

12) The spot was analyzed with an ELISPOT reader (Autoimmun Diagnostika GmbH).

13) Cytokine spots secreted from antigen-specific T cells (CFSE-low T) were analyzed (eg, spot number / 1x10 ² CFSE-low T cells)

Example  5. Local origin Of mesenchymal stem cells (ADSC)  On the surface HLA  And the effects of inflammatory cytokines in the expression of co-stimulatory molecules

In order to evaluate the correlation between allogeneic surface antigens and immunogenicity in human ADSCs, the changes in HLA expression on the surface of ADSCs stimulated with various inflammatory cytokines were measured and compared with untreated ADSCs, The effect of inflammatory cytokines on the expression of co-stimulatory molecules was investigated. ADSCs were isolated from normal human adipose tissue and cultured in complete DMEM. In this example, the ADSC was used for less than 8 times. Specifically, for the ADSC surface marker analysis, ADSCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (MSC-qualified, Life Technologies), 1% penicillin-streptomycin (Life Technologies). For surface marker analysis, ADSC positive markers were analyzed using anti-human CD13, anti-human CD44, anti-human CD73, anti-human CD90 and anti-human CD105 (eBioscience). The markers associated with the immunogenicity of ADSCs include anti-human CD80, anti-human CD86 (eBioscience), anti-HLA-ABC, anti-HLA- DR, anti- HLA- DR / DP / DQ (BioLegend) human natural killer group 2D ligand (NKG2DL) (anti-human MIC-A, anti-human MIC-B, anti-human ULBP1, and anti-human ULBP2 / 5/6; R & D Systems) was used. Surface markers were analyzed with Facs.

The obtained results are shown in Figs. 1A, 1B, and 1C.

(10 ng/ml, 14-8012-62) 또는 이들의 조합(eBioscience). 각각의 실험은 적어도 세 번씩 수행하였다.Figure 1A shows the results obtained by staining ADSCs with monoclonal antibodies against CD44, CD105, CD13, CD90 and CD73, respectively. Figure 1b shows that the ADSCs and non-ADSCs (regions that do not exhibit ADSC positive markers do not express any markers associated with immunogenicity induction as a result, meaning that they do not affect immunogenicity studies) CD86, HLA-DR, HLA-ABC, IL-17, and IL-17 without treatment or treatment with the IFN-gamma, TFN-alpha, IFN-gamma + IL- MIC-A, MIC-B, ULBP-1 and ULBP-2/5/6, respectively. Figures 1b and 1c show that ADSCs were stained with mAbs for CD80 and CD86 (A) or as mAbs for HLA-DR and HLA-ABC (B), respectively, for analysis of HLA surface antigen of ADSC cells, and NKG2DL (Generic name of the following markers recognized by NK cells or CD8 T cells), the ADSCs were each stained with mAbs for MIC-A, MIC-B (C), ULBP1 and ULBP2 / 5/6 . ADSCs were either untreated or treated with the following various cytokines: interferon gamma (IFN- ?, 50 ng / ml, 34-8319-85), tumor necrosis factor alpha (TNF-a; 10 ng / ml, 34-8329- (50 ng / ml, 34), IL-12 (10 ng / ml, 34-8129-85) -8069-85), IL-23 (10 ng / ml, 14-8239-63), IL-1

(10 ng / ml, 14-8012-62) or a combination thereof (eBioscience). Each experiment was performed at least three times.

As shown in Figures 1b and 1c, in various inflammatory environments, ADSCs do not express CD80 and CD86. However, HLA-ABC expression on the surface of ADSCs was increased in IFN-gamma, IL-1beta, and TNF-alpha treated ADSCs as compared to cytokine-untreated (see FIGS. In particular, in ADSCs stimulated with cytokine combination (IFN-gamma + IL-17A / F + IL-23), the increase in HLA-ABC expression was more markedly induced than in ADSCs treated with IFN-gamma. HLA-DR was weakly expressed after cytokine combination or stimulation with IFN-gamma. ADSCs do not express the NKG2DL family of MIC-A, MIC-B, ULBP-1 and ULBP-2/5/6 in both non-inflammatory (cytokine treated) and inflammatory conditions (cytokine treated) . IL-1beta induced an increase in HLA-ABC expression on the surface of ADSC, but did not induce an increase in NKG2DL and co-stimulatory molecules (Fig. 1c).

These results show that ADSC-stimulated ADSCs effectively increase HLA-ABC expression compared to IFN-gamma-treated ADSCs and weakly induce HLA-DR expression compared to that induced by untreated ADSCs. But. Expression of CD80, CD86 and NKG2DL was not induced.

Example  6. Human response to the proliferation of artificially stimulated mouse T cells ADSC  Immunosuppressive effect

To better understand the properties of ADSC, we examined the development of immunogenicity and its ability to regulate the immune response. For this experiment, mouse T cells were stimulated with anti-mouse CD3 and anti-mouse CD28 antibody (BioLegend) and human ADSC was added on the day of stimulation or the following day.

Specifically, mouse CD3 T cells were stimulated with anti-CD3 and soluble anti-CD28 antibodies coated on the plate in a 24-well plate. On the day of stimulation, CD3 T cells (1 × 10 ⁵ cells / well) were suspended with ADSC (7 × ^{10 4} cells / well) (see FIG. In addition, CD3 T cells were suspended at 1 × 10 ⁵ cells / well, stimulated, and the following day ADSC cells were added at 7 × ^{10 4} cells / well (FIG. CFSE-low CD4 T cells and CD8 T cells were analyzed by flow cytometry on the fourth day after stimulation (*, p <0.05; **, p <0.01). The results are shown in FIGS.

As shown in FIGS. 2A and 2B, ADSCs added on the day of stimulation inhibited the proliferation of CD8 T and CD4 T cells. In addition, ADSC effectively inhibited the proliferation of T cells even when added the next day after T cell stimulation. These results demonstrate that human ADSCs can exert immunosuppressive effects on the proliferation of stimulated T cells.

Example  7. Allogeneic antigen stimulation  through ADSC  Comparison of antigen recognition pathways for immunogenicity assessment

We investigated whether HLA-ABC expression on homologous ADSC surface induces immunogenicity after allogeneic antigen stimulation. In this example, immunogenicity assays of ADSCs were investigated using human immune cells and homologous ADSCs. The ADSCs used for homologous ADSC immunogenicity tests used mesenchymal stem cells (referred to as XF-ADSCs) cultured in xenofree-media immediately after isolation.

FIG. 3A schematically shows a three week experimental plan (see Example 4).

ADSCs were pretreated or untreated with various inflammatory cytokines in a heterogeneous antigen-free medium four days prior to allogeneic antigen challenge. CFSE-labeled CD3 T cells and T-cell-depleted peripheral blood mononuclear cells (T cell depletion-PBMCs) were incubated with homologous ADSCs for allogeneic antigens stimulation. For direct pathway analysis, ADSCs were suspended in 12-well plates the day before allogeneic antigen stimulation and then added for sensitization 7 days after stimulation. For indirect pathway analysis, the shattered ADSCs were added on the first day of the stimulation response and further on days 7 and 14. For ELISPOT analysis, whole cells were harvested after 2 weeks. Cells were suspended in quadruplicate on a 96-well filter plate (PVDF plate) and cultured for another 3 days. Ten percent of the cells harvested after allogeneic antigen challenge were used for flow cytometry for CFSE-low T cell counts.

To analyze cytokine spots secreted from antigen-specific T cells, the number of spots was calculated by dividing by the number of CFSE-low T cells. Cells to which ADSC was not added were compared and analyzed using a control (T cell + T cell removal-PBMC); (***, p <0.001; XF-ADSC, ADSC cultured in a heterogeneous medium; AAS, allogeneic antigen challenge).

The results obtained are shown in Fig. 3B. As shown in FIG. 3B, ADSC significantly induced IFN-gamma and IL-17A release by CFSE-low CD3 T cells after allogeneic antigen stimulation ex vivo. Antigen recognition by direct pathway was more effective in evaluating immunogenicity compared to indirect pathway. These results suggest that ADSC induces immunogenicity after allogeneic antigen stimulation mainly through direct pathway.

Example 8. Antigen-specific CD8 T cell generation test after stimulation of allogeneic antigen

In order to clarify the generation of antigen-specific T cells, the number of CFSE-low-CD4 T or -CD8 T cells in total CD3 T cells was analyzed, and the ADSCs were subjected to CFSE-low CD8 T cells after allogeneic antigen stimulation , But not CFSE-low CD4 T cells.

ADSC was added on day 7 after stimulation. After 3 weeks, all cells were harvested after allogeneic antigens stimulation through the direct pathway, and T cells were analyzed by flow cytometry and the results are shown in FIGS. 4a to 4d.

4A shows a plot for flow cytometry analysis, and the dot plot in FIG. 4B shows the distribution of CD4 T and CD8 T cells on day 21 after allogeneic antigen stimulation compared to the control group, and the histogram in FIG. Low CD4 T and CFSE-low CD8 T cells in a population of CD8 T cells, and FIG. 4d shows a graph of the number of CFSE-low CD4 or CFSE-low CD8 T cells corresponding to FIG. , p <0.05; ADSCs cultured in XF-ADSC, non-heterogeneous media, not significant In FIGS. 4b and 4d, the control (control) means the ADSC-free group and Untreated means the cytokine-untreated group, respectively. As shown in FIGS. 4C and 4D, the number of CFSE-low CD8 T cells was significantly increased compared to the control group, but no significant increase in CFSE-low CD4 T cells was observed. These results show that ADSC significantly induces the production of CFSE-low CD8 T cells, that is, antigen-specific CD8 T cells.

Example 9. Allogeneic Antigen Stimulation  Of antigen-specific memory CD4 T and memory CD8 T cells

Memory T cells are essential for defending pathogens. However, inhibition of alloreactive memory T cells is necessary for successful allografts.

In this example, changes in the memory T cell population between the experimental and control groups were compared (see Example 2). The marker for human effector memory T cells (TEM) is CD3 + CD4 + CD45RA-CD62L- for CD4 T cells and CD3 + CD8 + CD45RA-CD62L- for CD8 T cells. The marker of a human central memory T cell is CD3 + CD4 + CD45RA-CD62L + in CD4 T cells and CD3 + CD8 + CD45RA-CD62L + in CD8 T cells. In this example, the increase of antigen-specific memory T cells was examined. In this study, we investigated whether CFSE-low memory T cells were induced after 3 weeks of allogeneic dendritic stimulation and that ADSCs were significantly associated with CFSE-low memory-CD4 and CFSE-low memory-CD8 T cells And proliferation was induced.

ADSC was added on the next day after addition on the day of stimulation. After 3 weeks, whole cells were harvested after allogeneic antigens stimulation through the direct pathway, and T cells were analyzed by flow cytometry and the results are shown in FIGS. 5a to 5d.

Figure 5A shows a scheme for flow cytometry analysis. The histogram of FIG. 5b shows CFSE-low memory-CD4 T and CFSE-low memory-CD8 T cells among the CD4 and CD8 memory T cell population compared to the control group at 21 days after allogeneic antigen stimulation. Figure 5c is a graphical representation of the number of CFSE-low memory-CD4 T or CFSE-low memory-CD8 T cells corresponding to 5b. In Figure 5d, ADSCs were stimulated with IFN-gamma or cytokine combination for 4 days, and ADSCs were stained with monoclonal antibody (red line) or isotype antibody against HLA-DR / DP / DQ and then negative control (gray filled histogram) (*, P <0.05; ***, p <0.001; XF-ADSC, ADSCs cultured in a heterogeneous medium, ns, not significant). In FIGS. 5A to 5D, the control (control) means an ADSC-free group, and the Untreated means a cytokine-untreated group, respectively.

As shown in FIGS. 5B and 5C, the number of CFSE-low CD4 TCM cells was significantly increased as compared with the control group. Analysis of antigen-specific CD8 memory T cells for ADSC revealed that the number of CFSE-low CD8-TEM and -TCM cells was significantly increased compared with the control group. These results demonstrate that the use of homologous ADSCs is associated with an important immunogenicity problem in transplant rejection reactions such as the generation of antigen-specific CD4 TCM, CD8 TEM and CD8 TCM cells compared to the control.

Example  10. Allogeneic antigen stimulation  after CFSE Production of IFN-gamma and IL-17A by -low-CD4 T or -CD8 T cells

In this example, it was confirmed that ADSC significantly induced IFN-gamma and IL-17A release by antigen-specific CD4 T and CD8 T cells in response to allogeneic antigen stimulation.

Specifically, ADSC was added on the day of stimulation and then on day 7 further. Two weeks later, allogeneic haplotypes were stimulated through the direct pathway and whole cells were harvested and suspended in quadruplicate on a 96-well filter plate for ELISPOT analysis. Ten percent of the cells harvested after allogeneic antigen stimulation were used for flow cytometry.

The cytokine secreted by CD4 T cells after allogeneic antigens stimulation was analyzed by ELISPOT and shown in FIG. 6A. FIG. 6B is a graph showing the results of dividing the number of spots by the number of CFSE-low CD4 T cells in order to analyze cytokine spots secreted from antigen-specific CD4 T cells corresponding to FIG. 6A. FIG. FIG. 6c shows ELISPOT analysis of cytokines secreted from CD8 T cells after allogeneic antigen stimulation. FIG. 6D is a graph showing the results of dividing the number of spots by the number of CFSE-low CD8 T cells in order to analyze the cytokine spot secreted in the antigen-specific CD8 T cells of FIG. 6C (***, p < 0.05; XF-ADSC, ADSC cultured in non-heterogeneous media). In FIGS. 6A to 6D, the control (control) means an ADSC-free group, and the Untreated means a cytokine-untreated group, respectively.

As shown in FIGS. 6a to 6d, the analysis of CFSE-low-CD4 T and -CD8 T cells showed that ADSC significantly increased IFN-gamma and IL-17A release compared to the control. In addition, ADSCs pretreated with a combination of IFN-gamma, IL-17A / F, and IL-23 significantly increased IFN-gamma and IL-17A release compared to cytokine untreated ADSC. These results show that the increased expression of HLA-ABC (including the production of HLA-DR) on the surface of ADSC further increases the release of inflammatory cytokines. These results suggest that ADSCs should induce significant inflammatory cytokine release by both antigen-specific CD4 T and CD8 T cells as compared with the control group in allogeneic antigen-stimulating reactions, so that more consideration should be given to allogeneic transplantation of ADSCs It suggests.

Example  11. HLA - by blocking antibody Allogeneic antigen challenge  Inhibition test for generation of post-antigen-specific memory CD4 T and antigen-specific memory-CD8 T cells

This example tested that HLA-blocking antibodies inhibit antigen-specific memory-CD4 T and -CD8 T cell generation in response to allogeneic antigen stimulation.

CD4 T and CD8 T cells were incubated for 3 weeks with or without neutralizing antibodies (Abs) during allogeneic antigen challenge. A mixture of anti-human IL-17A antibody and anti-human IFN-gamma antibody was used to neutralize proinflammatory cytokines. A mixture of anti-human IL-17A antibody and anti-human IFN-gamma antibody, anti-HLA-ABC antibody, anti-HLA-DR antibody and anti-HLA-DQ antibody was used to neutralize inflammatory cytokines and block HLA Respectively. The resulting antigen-specific memory CD4 T and antigen-specific memory-CD8 T cell levels are shown in Figure 7a. Figure 7b is a graph showing the number of CFSE-low memory T cells corresponding to Figure 7a (*, p <0.05; ***, p <0.001; XF-ADSC, ADSC cultured in a heterogeneous medium).

As shown in FIGS. 7A and 7B, in the treatment group treated with HLA-blocking antibody during allogeneic antigen stimulation, the CFSE-low memory-CD4 T (anti-ADSC) And -CD8 T cells were significantly inhibited. However, neutralizing antibodies against pro-inflammatory cytokines secreted by alloreactive T cells did not significantly inhibit the production of memory T cells. These results demonstrate that homologous HLA surface antigens on the ADSC are the major cause of immunogenicity associated with the production of antigen-specific memory T cells.

Example 12. Cytotoxic immune response test (cell viability)

On day 14 after stimulation with allogeneic antigens, differences in cytotoxic effects of CD8 T cells on ADSCs under various culture conditions were tested (see Example 3 (1)).

8A schematically shows a test schedule. FIG. 8B is a photograph (left) showing the cell survival of ADSC cultured under various culture conditions on the 14th day after stimulation with allogeneic antigens and a graph showing the quantitative results (right).

As shown in Fig. 8B, -3d XF-ADSC (exchanged into a non-heterogeneous medium in a medium containing FBS 3 days before allogeneic antigen stimulation) and -1d XF-ADSC (a medium containing FBS 1 day before allogeneic antigen stimulation ), Induced cytotoxic effects of CD8 T cells compared to XF (Xenofree) -ADSC cultured in a non-heterogeneous medium from the beginning. However, CD4 T cells did not exhibit cytotoxic action. Thus, cytotoxicity against allogeneic mesenchymal stem cells is shown to be induced by CD8 T cells.

Example 13. Cytotoxic Immune Response Test (Apoptosis + necrosis)

XF-ADSC also exhibits cytotoxicity by recipient CD8 T cells (Figs. 9a and 9b), and this cytotoxicity is inhibited by the HLA Blocking Ab complex (Fig. 9c and 9d). In this example, the homologous HLA antigen of ADSC was found to be responsible for such cytotoxic immune response (see Example 3 (2)).

The results obtained in this embodiment are shown in Figs. 9A to 9D. As shown in FIGS. 9A to 9D, it is confirmed that apoptosis and necrosis are induced by XF-ADSC due to allogeneic immune response, and that it is significantly inhibited by HLA blocking Ab.

Claims (8)

(1) contacting the implant with a composition for stimulating an antigen; And
(2) measuring the number of T cells in the reactant of step (1), or the presence or number of memory T cells differentiated from the T cells
Lt; / RTI &gt;
The implant of step (1) is a stem cell derived from the recipient or recipient and derived from the same species,
The composition for antigen stimulation of step (1) comprises CD8 T cells, CD4 T cells, or all of them separated from the recipient,
Wherein the T cell of step (2) comprises CD8 T cells, CD4 T cells, or both.
And providing information for predicting immunogenicity in the recipient of said implant. 2. The method according to claim 1, wherein said graft is a mesenchymal stem cell or induced pluripotent stem cell derived from a recipient or from a recipient. 3. The method of claim 1, wherein the implant is cultured in a heterogeneous medium The method of claim 1, wherein the composition for antigen stimulation further comprises peripheral blood mononuclear cells, stem cells, or both, isolated from the recipient. 5. The method of claim 4, wherein said peripheral blood mononuclear cells are T cells removed. 2. The method of claim 1, wherein the step (1) of contacting the graft with the composition for antigen stimulation comprises culturing the graft and the composition for antigen stimulation in a non-heterogeneous medium comprising autologous serum of the recipient How, one. 2. The method of claim 1, wherein the measuring step (2) comprises measuring at least one selected from the group consisting of CD8 T cells, memory CD8 T cells, and memory CD4 T cells. A CD8 T cell, a memory CD8 T cell, and a memory CD4 T cell, wherein the composition is capable of detecting at least one selected from the group consisting of CD8 T cells, memory CD8 T cells, and memory CD4 T cells.

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