JP7277255B2 - Method for evaluating specificity of regulatory T cells in vitro - Google Patents

Description

The present invention relates to a method for evaluating regulatory T cell specificity in vitro.

Various control mechanisms such as cytokines and cell surface molecules exist in vivo to maintain the homeostasis of lymphocytes. Among the lymphocyte fractions, several lymphocyte fractions with immunosuppressive effects have been reported. Among them, regulatory T cells (hereinafter sometimes abbreviated as Tregs) were discovered as suppressive lymphocytes having CD4+CD25+ as surface markers (Non-Patent Document 1). Subsequently, Foxp3 was revealed to be the master transcription factor of Treg regulatory T cells (Non-Patent Document 2), and various pathological conditions such as autoimmune diseases, infectious diseases, transplant immunity, and malignant tumors (Non-Patent Document 8). is known to suppress the immune response in the onset of

Regulatory T cells express T cell receptors (TCRs) on their cell surfaces and are activated and exhibit suppressive effects only in response to specific antigens. That is, regulatory T cells are considered to be a technique that can suppress immune responses to target antigens, and there have been reports of applications such as transplantation medicine using antigen specificity one after another (Non-Patent Documents 5, 6, 7). Therefore, regulatory T cells are often described as having an antigen-specific suppressive effect. However, when focusing on suppressed cells, when regulatory T cells exert their suppressive action, do they non-specifically suppress surrounding suppressed cells, or do they respond to the antigen? It is still unclear whether it specifically suppresses only cells. Although there is a report that it is non-specific (Non-Patent Document 4), there is still room for examination of its evaluation method. Strictly speaking, therefore, regulatory T cells are expressed as ``antigen-specific suppressive power is activated, but as a result, it is not yet clear whether the suppressed cells are antigen-specifically suppressed''. It is an understanding of the current situation.

In other words, when considering the antigen specificity of regulatory T cells, the first thing to consider is whether or not the conditions under which regulatory T cells themselves are activated and exert their suppressive power are antigen-specific (activation specificity). (I will call it here), and whether only the intended specific suppressed cells are suppressed when the suppressed cells are suppressed after the regulatory T cells are activated. Or whether the suppressed cells existing in the vicinity of the regulatory T cells are universally suppressed non-specifically (referred to here as the specificity of the suppressed cells (II)). can think. There are several reports that the former viewpoint is already specific, and it is publicly known (Patent Document 1).

Numerous in vitro methods for quantitatively evaluating the immunosuppressive power of regulatory T cells are known, one of which is a method of co-cultivating regulatory T cells and suppressed cells. In this technique, the intensity of proliferation of co-cultured suppressed cells is measured. By measuring the extent to which this proliferation is attenuated, the suppressive power of regulatory T cells is evaluated. The intensity of proliferation can be measured by staining the cells with a dye before culturing and measuring the attenuation of the dye by flow cytometry, or by measuring the amount of radioisotope 3H-thymidine uptake (Non-Patent Document 5). , 6 and 7).

By using this technique, it has been found that only regulatory T cells stimulated with a specific antigen exert a suppressive effect and suppress suppressed cells. That is, it is known knowledge obtained from these analysis methods that regulatory T cells have activation specificity (I). However, these techniques cannot reveal the specificity (II) of the suppressed cells. That is, the conventional method for evaluating the suppressive power of regulatory T cells is a method of evaluating only the strength of the suppressive power of regulatory T cells, and it is possible to determine whether all the cells to be suppressed are uniformly suppressed. It is impossible to distinguish between only one being suppressed and the rest not being suppressed.

The question of whether only the intended specific suppressed cells are suppressed is actually a very important concern in immunosuppressive therapy. Most of the immunosuppressive agents currently used clinically act non-specifically on lymphocyte clones in vivo, and thus simultaneously suppress clones that are essential for survival. Therefore, it poses a serious problem of increasing the risk of infection and carcinogenesis. If there is an immunosuppressive therapy that can suppress only antigen-specific suppressed cells, it may overcome the problems of conventional immunosuppressants. Regulatory T cells have been suggested to have such potential, but no method has been established to evaluate their antigen specificity before in vivo administration, and regulatory T cells cannot be safely applied. Therefore, development of a method for evaluating the antigen specificity of regulatory T cells in vitro is earnestly desired.

JP 2004-208548 A

Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor α-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. Sakaguchi, S., Sakaguchi, N., Asano, M. et al. J. Immunol., 155, 1151-1164, 1995 Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. Nat. Immunol., 4, 330-336 ,2003 CD4+CD25+Foxp3+ T cells and CD4+CD25-Foxp3+ T cells in aged mice. Nishioka, T., Shimizu, J, Iida, R., Yamazaki, S., and Sakaguchi, S. J. Immunol. 176:6586-6593, 2006. Suppressor Effector Function of CD4+CD25+ Immunoregulatory T Cells IsAntigen Nonspecificm, Angela M. Thornton, J. Immunol. 2000;164;183-190 Dendritic cells expand antigenspecific Foxp3+CD25+CD4+ regulatory T cells including suppressors of alloreactivity, Sayuri Yamazaki, Immunological Reviews 2006,Vol. 212: 314-329 Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice, Uri Sela, The Rockefeller University Press, November 14, 2011 Effective expansion of alloantigen-specific Foxp3 CD25+CD4+regulatory T cells by dendritic cells during the mixed leukocyte reaction Sayuri Yamazaki, 2758-2763 PNAS February 21, 2006 vol.103 no.8 Regulatory T cells in cancer immunotherapy. Nishikawa H, Sakaguchi S. Curr Opin Immunol. 2014 Apr;27:1-7

The present invention was made in view of such problems, and an object of the present invention is to provide a method for accurately evaluating the suppressive specificity of regulatory T cells using an in vitro system.

A method for evaluating regulatory T cell specificity in vitro according to the present invention comprises a preparation step of preparing a first regulatory T cell, a first suppressed cell, and a second suppressed cell; an antigen presenting step of presenting one antigen to the first regulatory T cell to activate the first regulatory T cell with the first antigen; and the first regulatory T cell; co-cultivating the first suppressed cell and the second suppressed cell, wherein cell division of the second suppressed cell is not reduced, but cell division of the first suppressed cell is and determining that, if reduced, the first regulatory T cells are specific only for the first suppressed cells.

INDUSTRIAL APPLICABILITY According to the present invention, the antigen specificity of the suppressive action of regulatory T cells can be accurately evaluated using an in vitro system.

Adoptive transfer of OTII regulatory T cells with OTII naive T cells, SM naive T cells, OVA peptide-pulsed antigen-presenting cells, and SM peptide antigen-pulsed antigen-presenting mixtures resulted in a response to OTII-naive CD4 T cells only. The immune response was suppressed and no suppression was observed for SM naive T cells. On the other hand, when adoptive transfer of SM regulatory T cells to OTII naive T cells, mixed cells of SM naive T cells, and the above-mentioned antigen-presenting cells, the immune response to only SM naive CD4 T cells is suppressed, and OTII naive T cells are suppressed. was not found (a). When OTII-regulatory T cells were co-cultured with OTII naive T cells, SM naive T cells, and the above antigen-presenting cells, immune responses to only OTII-naive CD4 T cells were suppressed, and SM naive T cells were suppressed. (b). On the other hand, when SM-regulatory T cells were co-cultured with OTII naive T cells, and mixed cells of SM naive T cells and the above-mentioned antigen-presenting cells were co-cultured, OTII-regulatory T cells generated an immune response against only SM-naive CD4 T cells. and no suppression of OTII naive T cells (c). Adoptive transfer of OTII regulatory T cells with a mixture of OTII naive T cells, SM naive T cells, and antigen-presenting cells co-pulsed with OVA and SM peptides suppresses the immune response to OTII-naive CD4 T cells alone, suppressing SM No suppression was observed on naive T cells. On the other hand, when adoptive transfer of SM regulatory T cells to OTII naive T cells, mixed cells of SM naive T cells, and the above-mentioned antigen-presenting cells, the immune response to only SM naive CD4 T cells is suppressed, and OTII naive T cells are suppressed. was not found (a). When OTII-regulatory T cells were co-cultured with OTII naive T cells, SM naive T cells, and the above antigen-presenting cells, immune responses to only OTII-naive CD4 T cells were suppressed, and SM naive T cells were suppressed. (b). On the other hand, when SM-regulatory T cells are co-cultured with OTII naive T cells, SM naive T cells mixed with the above-mentioned antigen-presenting cells, SM-regulatory T cells generate an immune response against SM-naive CD4 T cells alone. and no suppression of OTII naive T cells (c). (a): Exogenous peptide antigen and cell surface-expressed alloantigen are presented by separate antigen-presenting cells, and co-adoptively transferred with H-2d-specific regulatory T cells. FIG. 4 shows that SM-naive CD4 T cells recognizing antigen show good cell division, whereas C57BL/6 naive CD4 cells recognizing alloantigen show very little cell division. (b): Exogenous peptide antigen and cell surface-expressed protein alloantigen are presented by separate antigen-presenting cells, and H-2d-specific regulatory T cells are cultured in vitro. Despite this, SM-naive CD4 T cells showed good cell division, but cell division of C57BL/6 naive CD4 cells that recognize alloantigens was suppressed by the addition of regulatory T cells. When the exogenous peptide antigen OVA and the cell surface expressed protein alloantigen H-2 ^bm12 were presented by the same antigen-presenting cells and co-adoptively transferred with OTII-regulatory T cells, C57BL/6 naive CD4 T cells CFSE decay histogram (a) and the number of dividing cells (b), showing that the cell division observed in OTII-naive CD4 T cells was suppressed, although the OTII-naive CD4 T cells showed good cell division. When OVA and alloantigen H-2 ^bm12 were presented by the same antigen-presenting cells, and co-cultured with OTII-regulatory T cells and C57BL/6 naive CD4T, OTII-naive CD4T cells, C57BL/6 naive CD4T Although the cells showed good cell division, the cell division observed in OTII-naive CD4 T cells was suppressed (c). When the anti-H-2 bm12 -regulatory T cells were presented with two types of cell surface-expressed alloantigens and co-cultured with the anti-H-2 ^bm12 -regulatory T cells, the anti-H-2 ^d- responsive suppressed cells showed favorable cell division, Growth of H-2 ^bm12- responsive suppressed cells was remarkably suppressed. On the other hand, when co-cultured with H-2 ^d -regulatory T cells, anti-H-2 ^bm12- responsive suppressed cells show good cell division, but only anti-H-2 ^d -responsive suppressed cells proliferate. FIG. 10 shows that suppression was observed. FIG. 4 shows that coexistence of non-specific factors reduces the antigen specificity of OTII-regulatory T cells. Similar to Fig. 4(b), exogenous peptide antigen OVA and cell surface expressed protein alloantigen H-2 ^bm12 were presented by the same antigen-presenting cells, and OTII-regulatory T cells and C57BL/6 When co-cultured with naive CD4T and OTII-naive CD4T cells, cell division of OTII-naive CD4T cells was suppressed, whereas C57BL/6 naive CD4T cells showed favorable cell division (a left). However, coexistence of regulatory T cells suppresses not only OTII-naive CD4T cells but also C57BL/6 naive CD4T cells in a dose-dependent manner. Antigen specificity of suppression by cells was reduced. Adoptive transfer of thymic-derived OTII regulatory T cells with OTII-naive T cells, SM-naive T cells, and a mixture of antigen-presenting cells co-pulsed with OVA and SM peptides suppressed the immune response to OTII-naive CD4 T cells alone. , but no suppression of SM naive T cells was observed. On the other hand, when adoptive transfer of thymus-derived SM regulatory T cells to OTII naive T cells and mixed cells of SM naive T cells and the antigen-presenting cells described above suppresses the immune response to only SM naive CD4 T cells, OTII naive T cells (a). When a mixture of thymic-derived OTII-regulatory T cells, OTII-naive T cells, and SM naive T cells was co-cultured with the above-mentioned antigen-presenting cells, immune responses to only OTII-naive CD4 T cells were suppressed. No suppression on T cells was observed (b). On the other hand, when thymus-derived SM-regulatory T cells were co-cultured with OTII naive T cells, SM naive T cells mixed with the above antigen-presenting cells, thymus-derived SM-regulatory T cells enhanced SM-naive CD4 T cells. Suppression of the immune response to OTII naive T cells was observed but not to OTII naive T cells (c)

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. The embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is as follows. The scope of the present invention is not limited to the embodiments, and other embodiments in which the configurations of the following embodiments are appropriately replaced by those skilled in the art are also included in the scope of the present invention.

The in vitro regulatory T cell specificity evaluation method according to the present embodiment includes:
A preparation step of preparing a first regulatory T cell, a first suppressed cell, a second suppressed cell, and optionally a third or more suppressed cells; an antigen-presenting step of presenting to a first regulatory T cell and activating the first regulatory T cell with a first antigen; a first regulatory T cell; a first suppressed cell; Co-cultivating two suppressed cells and, optionally, a third or more suppressed cells, and cell division of the suppressed cells of the second and, optionally, the third or more suppressed cells determining that the first regulatory T cell is specific only for the first suppressed cell if cell division of the first suppressed cell is reduced but not reduced and have

Preferably, in the antigen-presenting step, the first antigen and the second antigen are presented to the first regulatory T cell, and the first antigen activates the first regulatory T cell.

Also, prepare a first regulatory T cell, a second regulatory T cell, a first suppressed cell, a second suppressed cell, and, if necessary, a third or more suppressed cells a preparing step, presenting the first antigen and the second antigen to the first regulatory T cell to activate the first regulatory T cell with the first antigen; an antigen-presenting step of presenting a second antigen to the second regulatory T cell to activate the second regulatory T cell with the second antigen; a first regulatory T cell; co-cultivating the suppressed cells and the second suppressed cells, and optionally the third or more suppressed cells, wherein the cell division of the second suppressed cells is not reduced, but the first suppressed cell determining that the first regulatory T cell is specific only for the first suppressed cell when cell division of the suppressed cell is reduced, the second regulatory T cell and the first co-cultivating the suppressed cell and the second suppressed cell, and optionally the third or more suppressed cells, wherein the cell division of the first suppressed cell is not reduced, but the second suppressed cell determining that the second regulatory T cell is specific only for the second suppressed cell if cell division of the suppressed cell is reduced.

In addition, by using the above principle, the cells to be suppressed may be a population of cells having innumerable types of specificity, and the difference in the cell population and the difference in the degree of proliferation before and after co-culture can be measured. Thus, methods of identifying suppressed cell populations can also be used. Similarly, by using the principle described above, regulatory T cells can also be used for the purpose of screening target regulatory T cells or suppressed cells by co-cultivating multiple types of cells at the same time.

The expression "antigen specific" in the immune system goes back to the discovery of T cell receptors on T cells, or the specific reaction between antigens and antibodies expressed and produced by B cells. Taking the T-cell receptor as an example, each T-cell receptor molecule expressed by each cell has a different molecular structure, and when viewed as a whole organism, it has diversity. As a result, each cell has the characteristic of reacting only with a specific antigen molecule. In other words, the expression "the reaction is specific" means that one T cell receptor of interest reacts to an extremely limited target (molecules such as antigens), and does not show binding to other molecules and does not react. It means that there is. Plural pairs of animals expressing antigen molecules and T cell receptors responsive to the antigen are known to those skilled in the art and are widely applied to biological experiments. For example, using antigen A and A' cells that respond to it, under two conditions: with A' cells & without antigen A, with A' cells & with antigen A, if the condition is confirmed in the latter, then asthma is an antigen. It is concluded that it is triggered by a specific immune response. Alternatively, it is concluded that asthma is induced by an antigen-specific immune response even when pathological conditions are confirmed under the two conditions of no A' cells and antigen A, and A' cells and antigen A present. ing.

It should be noted that when the concept of antigen specificity is applied to the study of immunosuppression, the situation is slightly different from the above. That is, the response of A' cells (suppressed cells A) responding to antigen A is suppressed by the addition of regulatory T cells A, and the response of B' cells (suppressed cells B) responding to antigen B is suppressed by regulatory T cells. Although it is known that addition of cell A does not suppress T cells (Non-Patent Document 6), whether or not it can be concluded that regulatory T cells A exerted antigen-specific suppressing ability will be discussed below.

In a conventional experimental environment, A' cells (suppressed cells A), antigen-presenting cells that presented antigen A, and regulatory T cells A were mixed and cultured, and the immune response of A' cells (suppressed cells A) to antigen A was confirmed. to determine whether regulatory T cell A can suppress On the other hand, B' cells (inhibited cells B), antigen-presenting cells that presented antigen B, and regulatory T cells A were mixed and cultured to allow regulatory T cells A to immunize B' cells (inhibited cells B) against antigen B. Determine if responses can be suppressed. It has been concluded that regulatory T cells act in an antigen-specific manner, since the former exerts suppressive power and the latter does not exert suppressive power in these two sets of experiments. However, when the response of A' cells (inhibited cells A) by regulatory T cells A decreases, regulatory T cells A coexist with antigen A and are continuously stimulated by antigens and activated. On the other hand, when regulatory T cell A responds to B' cells (inhibited cell B), regulatory T cell A coexists with antigen B and stimulates regulatory T cell A. Antigen A is absent. Therefore, regulatory T cell A was compared under two conditions that are not the same: an environment with stimulating antigens (the former) and an environment without stimulating antigens (the latter). Instead, it is as if the inhibitory effect was exerted only in the former, but not in the latter. That is, on the former side, regulatory T cells A are in an awake state after receiving antigenic stimulation A, but on the latter side they are in a so-called dormant state without receiving antigenic stimulation A. It does not stand up to the criticism that it could not exert its inhibitory action on B and the B' cells (inhibited cells B) that respond to it.

Based on the above, it can be said that regulatory T cells themselves can exhibit suppressive power when activated by their specific antigens, that is, (activation specificity (I)) is specific. However, after regulatory T cells are activated, when suppressing suppressed cells, whether only the intended specific suppressed cells are suppressed, or whether suppressed cells existing in the vicinity of regulatory T cells are suppressed. However, the specificity of the suppressed cells (II) has not been elucidated.

What the present inventors want to clarify is true antigen specificity, that is, specificity of suppressed cells (II). In both experiments, regulatory T cells A were specifically stimulated with antigen A to prepare for wakefulness, and then A' cells (inhibited cells A) stimulated by antigen A, antigen Comparing the responses of B' cells (inhibited cells B) stimulated by B, B' cells (inhibited cells B) stimulated by antigen B do not exert inhibitory power, but are stimulated by antigen A. Regulatory T cell A exerted its suppressive power specifically only on A' cells (suppressed cells A) only after suppressing only A' cells (suppressed cells A). and express. This is the true antigen specificity or suppressed cell specificity (II) proposed by the present inventors.

Therefore, the present inventor added antigen A and regulatory T cell A to the coexistence of A' cells (suppressed cells A) and B' cells (suppressed cells B), and compared their effects. Suggest a method.

Preferably, in a state in which A' cells (inhibited cell A) and B' cells (inhibited cell B) coexist, antigen A and antigen B and regulatory T cell A are added and the effects are compared. Suggest a method.

Next, if it is possible to create regulatory T cells B, create a coexistence state of A' cells (suppressed cells A) and B' cells (suppressed cells B), then antigen A and antigen Adding B and regulatory T cell B and comparing their effects yields results that further improve the reliability of the experiment.

The specificity confirmed by the present invention is of vital interest in vivo. This is because, if regulatory T cells were administered for therapeutic purposes, it would have been expected that the regulatory T cells would suppress the disease-causing lymphocytes, but in an unexpected biological environment, This is because it is necessary not to suppress the action of important lymphocytes. After all, the superiority of regulatory T cells to conventional immunosuppressive therapy is not certain if the suppressed cells in the vicinity of the suppressive regulatory T cells are nonspecifically suppressed. That is, in order for regulatory T cells to finally specifically suppress suppressed cells, not only activation specificity (I) but also suppressed cell specificity (II) is antigen-specific at the same time. There is a need.

INDUSTRIAL APPLICABILITY According to the present invention, regulatory T cells with clarified specificity (II) for cells to be suppressed can be generated, and it can be demonstrated in vitro that only cells intended to be suppressed can be suppressed. This provides tremendous value in the medical field of immunosuppressive therapy. That is, it is possible to evaluate the safety and efficacy of therapeutic cells in vitro prior to actual therapeutic administration.

In addition, in the analysis of factors involved in immune response, in vivo analysis involves the death of experimental animals and a corresponding burden in the process of collecting assay products from living organisms. You can only get point information. However, in vitro analysis allows samples to be collected and analyzed at any timing during the culture process, and it is easy to increase analysis conditions. Furthermore, addition or removal of constituent cells, other cells or reagents can be easily performed. As described above, the in vitro analysis method increases the degree of freedom of analysis compared to in vivo analysis, and brings about great efficiency in the development of new drugs and the analysis of biological components.

By using the present invention, it is possible to detect a minute inhibitory ability that could not be evaluated by conventional analysis methods.

The principle of the present invention can also be used as a method for discovering minute inhibitory effects that have hitherto gone undetected.
That is, in conventional known test methods, when evaluating the suppressive ability of target regulatory T cells, a method of introducing polyclonal suppressed cells into an in vitro assay is widely known (Non-Patent Documents 2 and 3). , 5, 6, 7).

In this approach, the inhibitory capacity is
G: Number of proliferating suppressed cells in the presence of regulatory T cells
H: Number of proliferating suppressed cells in the absence of regulatory T cells
In this case, it is calculated by the formula of inhibitory capacity = 1 - (G/H). This calculation method has been premised on the premise that all suppressed cells are equally suppressed. However, what the present inventors have clarified is that in such an examination method, when the injected cells to be suppressed are a polyclonal population, all of the injected cells to be suppressed are subject to the inhibitory action. The point is that it is not a cell, but only a suppressed cell that recognizes the same antigen as the regulatory T cell injected into the assay. Another point of view is that among the injected suppressed cells, a cell population that does not recognize the same antigen as the regulatory T cells injected into the assay is not suppressed in the first place.

Therefore, in the calculation method in the conventional method, a calculation method was created in which the behavior of proliferating cells of all the suppressed cells put into the in vitro assay system was analyzed and the ratio was calculated under conditions with/without regulatory T cells. In that case, cells that should not be suppressed in the first place are included in the calculation. If such cells that should not be suppressed account for a large number of the injected suppressed cells, the suppressive ability of the original regulatory T cells will be underestimated, and the suppressive ability that should be detected will be detected. There is a possibility that a situation where it is difficult to be

According to the technique of the present invention, in the above-described evaluation method, the cells to be evaluated are limited to the suppressed cells that recognize the same antigen as the regulatory T cells used in the assay, among the suppressed cells. are proposing to Therefore, we propose to limit the target of analysis to those cells in advance, and then to calculate the suppressive ability by analyzing the behavior of only those limited suppressed cells under conditions with/without regulatory T cells. do.

This method makes it possible to detect inhibitory activity with high sensitivity even in situations that were previously underestimated and difficult to detect.

Calculation method of inhibitory ability in the present invention
J: Number of proliferating suppressed cells that recognize the same antigen as the utilized regulatory T cells Conditions for presence of regulatory T cells
K: The number of proliferating suppressed cells that do not recognize the same antigen as the utilized regulatory T cells Conditions for presence of regulatory T cells
L: Number of proliferating suppressed cells recognizing the same antigen as the utilized regulatory T cells Condition without regulatory T cells
M: The number of proliferating suppressed cells that do not recognize the same antigen as the regulatory T cells used.
G = J + K
H = L + M, and
Inhibitory capacity in the present invention = 1 - (J/L)
is calculated as

For example, in the following cases where the number of proliferating suppressed cells that recognize the same antigen as the utilized regulatory T cells is only a fraction of the total number of suppressed cells:
i.e.
G = J + K = 1 + 95 = 96
H = L + M = 5 + 95 = 100
Assuming that
According to the conventional method,
Inhibitory capacity (conventional method) = 1 - (G / H) = 1 - ( 96 / 100) = 0.04 = 4 %
is underestimated, and it is judged that there is not much suppressive power,
By using the calculation method according to the invention,
Inhibitory capacity (method) = 1 - (J / L) = 1 - (1 / 5) = 0.8 = 80 %
It is calculated that it has a strong inhibitory ability, and it becomes possible to clearly show the inhibitory ability.

In other words, in the conventional calculation method, the non-responsiveness of the suppressed cells, which do not recognize the same antigen as the regulatory T cells used, masks the original suppressive action of the regulatory T cells. There is a risk that they may underestimate the inhibitory capacity.

Thus, the principle of the present invention can also be used as a method for discovering minute inhibitory effects that have hitherto been undetected.

This suggests that there may be cases in which successful results in the development of regulatory T cells to date have been erroneously concluded as unsuccessful. The calculation method of the present invention finds the significance of re-evaluating and re-examining the methods that have been rejected up to this point.

the claimed first regulatory cell, the second regulatory cell,
The present invention is described mathematically using the expressions first suppressed cell and second suppressed cell.

P = number of proliferating first suppressed cells: (conditions without any regulatory T cells)
Q = number of proliferating cells of second suppressed cells: (conditions without any regulatory T cells)
R = number of proliferating cells of the first suppressed cell: (in the presence of the first regulatory cell)
S = number of proliferating cells of the second suppressed cell: (in the presence of the first regulatory cell)
T = number of proliferating cells of the first suppressed cell: (in the presence of the second regulatory cell)
U = number of proliferating cells of second suppressed cells: (in the presence of second regulatory cells)
In the conventional method, the suppressing ability is
Suppressive ability of the first regulatory cell (conventional method) = 1 - ((R+S)/(P+Q))
Suppressive capacity of secondary regulatory cells (conventional method) = 1 - ((T+U)/(P+Q))
The inhibitory ability according to the present invention is
Suppressive capacity of the first regulatory cell (this method) = 1 - ((R)/(P))
Suppressive capacity of secondary regulatory cells (this method) = 1 - ((U)/(Q))
is described as

Therefore, if P is significantly less than Q,
for example,
P = 100
Q = 1000
R = 5
S = 990
T = 95
U = 200
if was
The suppression ability by the conventional method is
Without distinguishing P and Q, find the sum of P and Q,
without distinguishing between R and S, find the sum of R and S,
Since T and U are not distinguished and the sum of T and U is calculated before calculating the inhibitory capacity,
Suppressive capacity of first regulatory cell (conventional method) = 1 - ((R + S) / (P + Q))
= 1 - ((5 + 990) / (100 + 1000))
= 1 - (995 / 1100)
= 0.09545
= 9.545%
Suppressive capacity of secondary regulatory cells (conventional method) = 1 - ((T + U) / (P + Q))
= 1 - ((95 + 200) / (100 + 1000))
= 1 - (295 / 1100)
= 0.731
= 73.2%
is calculated as
The suppressive capacity according to the present invention is
Identify P and Q, first find the individual values of P and Q,
Identify R and S, first find the individual values of R and S,
Since T and U are identified and the individual values of T and U are obtained first, then the inhibitory capacity is calculated,
Suppressive capacity of the first regulatory cell (this method) = 1 - ((R) / (P))
= 1 - (5/100)
= 0.95
= 95%
Suppressive capacity of secondary regulatory cells (this method) = 1 - ((U) / (Q))
= 1 - (200/1000)
= 0.80
= 80%
is calculated as

In particular, it can be seen that the inhibitory ability of the first regulatory cell was underestimated at 9.545% by the conventional method. The superiority of the present invention in such situations is high, and it is possible to clearly detect 95% of the suppressive ability of minute regulatory T cells in situations where conventional methods are difficult to detect.

In the preparation step, the first regulatory T cells, the second regulatory T cells, the first suppressed cells, and the second suppressed cells are not particularly limited in kind as long as they are animal cells. Non-human animal cells such as human, mouse, rat, rabbit, etc. can be mentioned.

Animal cells may be derived from healthy humans or animals, or humans or animals suffering from a given disease, or animals that have undergone genetic manipulation, gene deletion, or forced gene expression manipulation, or given disease model animals. may

As used herein, the term "antigen" refers to peptides, proteins, viruses, nucleic acid-related agents, prions, liposomes, or animal, plant tissues, alloantigens, or microbial cells capable of inducing a T cell response. Preparations or similar agents.

An antigen may be a protein obtainable by spontaneous genetic mutation, recombinant DNA technology, purification from a different tissue or cell source than other cells. Such proteins are not limited to native proteins, but also include modified proteins or chimeric constructs obtained, for example, by altering selected amino acid sequences or by fusing portions of different proteins. On the other hand, when the antigen of interest is expressed in cells and has the ability to stimulate T cells, it can be used as cells, cell aggregates, or tissue fragments without being extracted from cells. The regulatory T cells used herein may be added at the same time, staggered 0.5 days before, 1 day, 4 days, 7 or more days before, or 0.5 days after, 1 It may be added after days, after 4 days, after 7 days or more.

Foxp3-positive regulatory T cells are preferably used as the regulatory T cells used herein. can also be used.

Specifically, regulatory T cells include IL-10-producing T cells, TGFbeta-producing cells, LAG3 (CD223) expressing cells, CD39 expressing cells, CD54 expressing cells, CD73 expressing cells, CD103 expressing cells, CD134 expressing cells, CD154-expressing cells, CXCR4-expressing cells, CXCR5-expressing cells, CXCR6-expressing cells, GITR-expressing cells, GRP83-expressing cells, GranzymeB-expressing cells, Perforin-expressing cells, Nrp-1-expressing cells, IL-9-expressing cells, IL-35-expressing cells, PD-1-expressing cells, PD-L1-expressing cells, BTLA-expressing cells, CTLA-4-highly expressing T cells, more preferably Foxp3-positive regulatory T cells.

Regulatory T cells used herein may be regulatory T cells composed of monoclonal cells, or regulatory T cells composed of polyclonal cells.

The regulatory cells used herein desirably express a receptor that recognizes an antigen, and one of the receptors that recognizes a specific antigen is the T cell receptor (TCR). , an antigen of interest, a surface-expressed receptor capable of recognizing a molecule, a molecule with an artificially altered antigen-recognition site, a cell expressing an antigen-recognition receptor, an extracellular domain with an artificially altered antigen-recognition site Cells expressing a receptor whose intracellular domain has a chimeric structure of a T-cell receptor, B-cell receptors recognizing target antigens, and cells expressing immunoglobulins may also be used. Alternatively, even in cells whose suppressive action is not clear by themselves, cells that coexist with other lymphocytes to produce suppressive action in cooperation with other lymphocytes and have receptors for specific antigens can also be used.

Regulatory T cells used herein can also be cells that have regulated nuclear transcription factor expression to exert a suppressive effect, even if they do not endogenously express Foxp3.

The method of antigen presentation in the antigen presentation step may be a method using an antibody capable of stimulating T cell receptors, or a method using MHC-expressing cells to present antigens to MHC molecules. It may be presented using major histocompatibility antigen (MHC) class I or II expressed by suppressed cells. A preferred method is to present the antigen to antigen-presenting cells. Alternatively, it may be a method of recognizing and stimulating antigen recognition receptors (TCR, etc.) without the intervention of MHC molecules.

When antigen-presenting cells are used as the antigen-presenting method in the present invention, the antigen-presenting cells can be collected from a different individual or from the same individual as the suppressed cells (effector cells).

When antigen-presenting cells are used in the present invention, the cells used are preferably cells expressing MHC molecules when antigen recognition is performed by T cell receptors. Although dendritic cells expressing MHC class I or MHC class II are preferably used, other cells expressing MHCII such as B cells, Langerhans cells, macrophages, monocytic cells, Kupffer cells ) can also be used. Furthermore, the cells are not limited to the above as long as they express MHC molecules.
However, when MHC-independent antigen recognition is used, TCR (T cell receptor) can be recognized and stimulated without MHC molecules. In this case, any cell expressing the antigen of interest can be used. It is possible.

When antigen-presenting cells are used in the present invention, any of immature dendritic cells, mature dendritic cells, other antigen-presenting cells, artificial antigen-presenting cells, and mixtures thereof may be used. In addition, artificial antigen-presenting cells are antigen-presenting cells that are artificially produced, for example, to express at least major histocompatibility antigen (MHC) class II and co-stimulatory molecules (e.g., CD80, CD86, etc.). Examples include genetically engineered cells. Additionally (MHC) class I may be expressed. Furthermore, the artificial antigen-presenting cells may be cell lines derived from affected organs of autoimmune diseases or tumors and modified to express MHC class I, class II, and co-stimulatory molecules as described above. .

For antigen presentation in the present invention, one or more antigens can be used. When multiple antigens are used, they can be presented by separate antigen-presenting cells, or multiple antigens can be presented by the same antigen-presenting cell. This includes the case of presentation using artificially produced cells without using antigen-presenting cells. Also, the antigen may be added at the same time as the start of the assay, or may be added before or after with a time lag.

Antigens or multiple antigens that can be used in the present invention may be exogenous peptide antigens presented in an MHC-restricted manner, or cell surface-expressed proteins such as alloantigens (alloMHC molecules) that are allorecognized by T cells. good. They may be a combination of exogenous antigens, a combination of exogenous antigens and alloantigens, or a combination of alloantigens.

Cells used as suppressed cells (effector cells) in the present invention are immunocompetent cells generally called leukocytes. Granulocytes, monocytes, lymphocytes, innate immune system cells, adaptive immune system cells are included, preferably lymphocytes. Lymphocytes expressing a T cell receptor (TCR) are preferred. CD4 lymphocytes are preferred, but CD8-positive lymphocytes and NKT cells, for example, can also be used.

Cells used as cells to be inhibited (effector cells) in the present invention may be cells in an unactivated state or cells after activation. Cells after addition of a test substance for screening purposes can also be used. Alternatively, the test article can be applied during or after the assay.

Cells used as suppressed cells (effector cells) in the present invention can be collected from the same individual as regulatory T cells or from a different individual. Cells used as cells to be suppressed (effector cells) in the present invention can be collected from the same individual as the antigen-presenting cell or from a different individual.

Cells used as cells to be suppressed (effector cells) in the present invention can be a cell population with one specificity or multiple specificities at the same time.

Suppressed cells (effector cells), as well as antigen-presenting cells, can be isolated using cell surface antigens such as CD4 or CD8, CD11c, CD19, T cell receptor (TCR), TCRVbeta chain specific antibody, NK1.1, CD90, Using CD56, CD16, CD56, CD25, CD44, CD45, MHCII, MHCI, and CD4 as markers, cell sorting or MACS magnetic separation can be used for isolation. Alternatively, separation can also be performed using FSC and SSC measurements of a flow cytometer without using surface markers.

Elements that can be used herein include, as cell components, regulatory T cells, suppressed cells (effector cells), antigen-presenting cells, cell lysates of interest for screening purposes, lysed tissue fragments, , organs, tissue sections, disrupted tissue sections, cell populations, cell populations separated by specific surface markers, animal, plant, microbial cells, tissues, or similar agents of interest. In addition, the culture media contains immunosuppressants, immunomodulators, cytokines, antibodies, growth factors, hormones, culture supernatant extracts, intracellular humoral components, cell-derived organic substances, and liposomes that are of interest for screening purposes. , nucleic acid components, prions, or similar agents.

During the culturing process of the in vitro assay, it can coexist with these cell components or liquid components from the beginning of the culturing process, from 1 day, from 2 days, or from 3 days. At the start of the reaction, the sample can be centrifuged and the old supernatant portion can be washed and removed.

In another aspect of the invention, the antigen is crude or partially purified tissue or cells obtained by various biochemical procedures known to those skilled in the art (e.g., fixation, lysis, subcellular fractionation, density gradient separation). is a preparation of Alternatively, synthetic peptides chemically synthesized based on peptide sequence information are used. Alternatively, molecules expressed in cells are used as antigens.

A specificity-adjusting substance capable of altering the antigen specificity of regulatory T cells obtained by the screening method for a substance promoting or suppressing the spontaneous onset of autoimmune disease according to one embodiment of the present invention is an in vivo antigen-specific It is useful for elucidating the mechanism of immunosuppression, and substances that increase specificity may be used to reduce side effects caused by unintended immunosuppression of administered regulatory T cells and to enhance their intended effects. That is, it can be used for the purpose of destroying tolerance and exterminating pathogenic microorganisms in pathological conditions in which the tolerance mechanism involving non-specific inhibitory action favors the survival of pathogenic microorganisms in the host. Similarly, it can be used to break tolerance in other infectious diseases and malignancies involving non-specific suppression. Post-transplantation immunosuppressive therapy, when the above-mentioned substances expected to prevent or improve autoimmune diseases are used as pharmaceuticals, the usual pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, Various formulation ingredients such as pH buffers, disintegrants, solubilizers, solubilizers and isotonic agents can be added. In the prophylactic or therapeutic method using these pharmaceuticals, an appropriate dose according to the patient's sex, body weight and symptoms can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms such as powders, granules, capsules, syrups and suspensions, or in dosage forms such as solutions, emulsions and suspensions. It can be administered parenterally in the form of an injection, and can also be administered intranasally in the form of a spray.

The regulatory T cells used in the assay are cultured for several hours, one day, two days or more, preferably three days or more, depending on the purpose. The culture period is 30 days or less, more preferably 14 days or less, 7 days or less.

A suitable medium for practicing the present invention is any culture medium suitable for the growth, survival and differentiation of immunocompetent cells. Typically, it consists of a basal medium containing nutrients (carbon sources, amino acids), pH buffers and salts, which may be supplemented with serum of human or other origin and/or growth factors and/or antibiotics. . Typically, the basal medium can be RPMI 1640, DMEM medium, all of which are commercially available standard media.

Identification of transfected cells used for in vitro assays includes autofluorescent dyes (GFP, YFP, RFP, luciferase, etc.), transgenic markers (hCD2, etc.), Thy1.1, CD90.1 markers, Thy1.2, CD90.2 markers, CD45.1 marker, CD45.2 marker, etc., may be used as long as they can be identified on a cell-by-cell basis, but are not limited to these methods. Furthermore, the combination of these markers can increase the identifiable cell types by combining multiple markers, eg GFP+ Thy1.1, GFP- Thy1.1, YFP+ GFP+, YFP+ RFP+ CD45.1+. Alternatively, cells to be suppressed are prepared in an unidentified state, an in vitro assay is performed, and after the assay, the antigen recognition receptor is labeled with the antigen of interest to identify the cells having the receptor for the antigen of interest. can also A specific example is a commercially available reagent in which an antigen-MHC complex is bound to a fluorescent dye, but the method is not limited to this method.

Cell division evaluation methods used in the assay include the BrdU method (bromodeoxyuridine method), thymidine (3H-thymidine) uptake method, cytochrome staining method, and the like, but are not limited to any of these methods. As the cell dye staining method, for example, Carboxyfluorescein diacetate succinimidyl ester (CFSE), PKH26 (Sigma Aldrich), CellVue (Sigma Aldrich), Cell Trace Violet (ThermoFisher Scientific) and the like are sold by various companies.

(Example 1) Antigen specificity analysis assay for immunosuppression using two types of peptide antigens [mouse]
C57BL/6 and Balb/c mice were purchased from CLEA Japan (Tokyo, Japan). RAG-2-deficient OTII TCR transgenic mice (hereinafter referred to as OTII-mice) and LCMV-specific mice (hereinafter referred to as SM-mice) were bred in an animal facility. All mice were used at 6-8 weeks of age and maintained under specific pathogen-free conditions in accordance with institutional animal care guidelines. Mouse B6 CD45.1 (Jax mice Stock No: 002014) and B6.PL-Thy1(a)/CyJ (Jax406) were purchased from Oriental Yeast Co., Ltd. OTII-mouse and SM-mouse were mated with RAG2-deficient mice, Jax002014 and Jax406 to prepare CD45.1 and Thy1.1 positive mice in each strain.

[Antibodies, reagents and media]
The following reagents were purchased from BD PharMingen (San Diego, Calif.). CD3ε(145-2C11), CD28(37.51), CD16/32, APC-Cy7 Mouse Anti-Rat CD90/Mouse CD90.1(Clone OX-7), PerCP Mouse Anti-Rat CD90/Mouse CD90.1(Clone OX-7) -7), Qdot605-CD4(RM4-5), PE-TCR Vb8.3(1B3.3)
The following were purchased from Biolegend. CD45.2-BV785 Brilliant Violet 785(TM) anti-mouse CD45.2 Antibody Clone 104, CD45.1 (A20)-PECy7,PerCP anti-mouse CD25 Antibody (Clone PC61), APC anti-mouse TCR Vβ5.1, 5.2 Antibody (clone MR9-4), Brilliant Violet 785(TM) anti-mouse CD45.2 Antibody Clone 104,
The following reagents were purchased from Lonza. RPMI 1640
The following reagents were purchased from Peprotech. hIL-2 Recombinant Human IL-2,
Recombinant Human TGF-β1
CD90.1(+) SM-naive CD4 T cells and CD45.1(+) OTII-naive CD4 T cells were used as suppressed cells.

[Generation and purification of regulatory T cells]
Spleens were removed from C57BL/6 CD45.1(+) CD45.2(+) OTII RAGKO mice and C57BL/6 CD45.1(+)CD45.2(+) SM RAGKO mice. The splenic tissue was crushed using a cell strainer to separate floating cells into a single cell state. The number of cells was calculated, the required amount was taken, and CD4 T cells were isolated using cell sorting or MACS magnetic separation using CD4 as a marker. The isolated CD4 T cells were cultured for 5 days in a CD3e/CD28-coated incubator together with TGFbeta and IL2 with the addition of TFGβ and IL-2 to generate Foxp3-positive regulatory T cells. In addition to these, ATRA (all-trans-Retinoic Acid) was added to the culture medium. The cultured cells were stained with Cell Trace Violet (ThermoFisher Scientific), a dye for cell division detection.

[Isolation of suppressed cells (effector cells)]
As suppressed cells (effector cells), spleens were taken from CD45.1(+) OTII- mice and CD90.1(+) SM- mice, and floating cells were separated using a cell strainer. A portion of the floating cells was isolated by treating the required amount of CD4 positive T cells with a CD4 marker and using MACS magnetic separation. After that, they were stained with a dye for cell division detection (Cell Trace Violet (ThermoFisher Scientific)).

[Isolation of antigen-presenting cells]
Spleens were collected from wild-type C57BL/6 mice, and floating cells were separated using a cell strainer. Some of the floating cells were isolated as antigen-presenting cells by treating CD11c-positive T cells with MACS magnetic separation using the CD11c marker.

As antigens, I-Ab-restricted OVA (323-339) and I-Ab-restricted Lymphocytic Choriomeningitis Virus-derived peptide: LCMV GP (61-80) were used. Each peptide was cultured in separate tubes with dendritic cells isolated from CD45.2(+)-derived C57BL/6 cells and presented to MHC. After antigen presentation, centrifugation at 300 g for 5 minutes was repeated four times, and the supernatant was discarded to wash the cells.

Regulatory T cells were generated from CD45.1(+) CD45.2(+) SM-naive CD4 T cells, CD45.1(+) CD45.2(+) OTII-naive CD4 T cells and are designated were defined as SM-regulatory T cells and OTII-regulatory T cells. We analyzed the expression levels of Foxp3(+) in these SM-regulatory T cells and OTII-regulatory T cells, and confirmed that both were over 95%.

After the above cell components were mixed in a combination of without and with regulatory T cells, the cells were transvenously transferred into the tail vein of wild-type C57BL/6 mice using a 29G syringe. After 3 days, splenocytes were collected, and the splenocytes were crushed using a cell strainer to separate floating cells. Cell numbers were calculated and 2×10 ⁶ splenocytes per animal were analyzed using a flow cytometer. As a result, SM-naive CD4T cells, which are suppressed cells, and OTII-naive CD4T cells, both of which were adoptively transferred, showed good cell division, but were co-transferred with OTII-regulatory T cells. SM-naive CD4 T cells show good cell division, whereas OTII-naive CD4 T cells show very little cell division, and OTII-regulatory T cells provide an immune response to OTII-naive CD4 T cells. suppression was observed. Since almost no suppression of SM-naive CD4 T cells was observed, it was shown that immunosuppression by OTII-regulatory T cells acts specifically only on OTII-naive CD4 T cells in vivo. .

Next, when co-adoptively transferred with SM-regulatory T cells, OTII-naive CD4 T cells show good cell division, whereas SM-naive CD4 T cells show very little cell division, and SM-regulatory Sexual T cells suppressed the immune response to SM-naive CD4 T cells. Almost no suppression of OTII-naive CD4 T cells was observed, suggesting that immunosuppression by SM-regulatory T cells acts specifically on SM-naive CD4 T cells in vivo. (Fig. 1a).

In vitro culture of the cell components was performed. RPMI 1640, 500 mL was used as the basal medium, and a medium adjusted to the following composition was prepared and used.
50ml FBS (frozen at -20℃)
stock FBS are already HI (heat inactivated 56℃,30min)
5ml L-glutamine stock (200mM, 100x) (frozen at -20℃)
5ml NEAA stock (100x) in refrigerator
5ml sodium pyruvate stock (100mM, 100x) at refrigerator
5ml HEPES stock (1M, 100x) at room temperature
5ml streptomycine/penicillin stock (100x) at -20°C
0.5ml 2-mercaptoethanol (1000x working stock;
37ul 2ME in 10ml of PBS in refrigerator (4°C))
500ml RPMI-1640 with phenol red in cold room
Sterilize by filtration (0.2um)
As a result, as observed in vivo, both SM-naive CD4 T cells and OTII-naive CD4 T cells, which are suppressed cells, were cultured alone, and showed good cell division.

When co-cultured with OTII-regulatory T cells, SM-naive CD4 T cells showed good cell division, whereas OTII-naive CD4 T cells showed very little cell division, and OTII-regulatory T cells , suppressed the immune response to OTII-naive CD4 T cells. Almost no suppression of SM-naive CD4 T cells was observed, indicating that immunosuppression by OTII-regulatory T cells acted specifically only on OTII-naive CD4 T cells in vitro ( Figure 1b).

Next, when co-cultured with SM-regulatory T cells, OTII-naive CD4 T cells show good cell division, whereas SM-naive CD4 T cells show very little cell division, indicating that SM-regulatory T cells are not susceptible to SM-regulatory T cells. Suppression of the immune response to SM-naive CD4 T cells was observed by T cells. Little suppression of OTII-naive CD4 T cells was observed, indicating that immunosuppression by SM-regulatory T cells acts specifically against SM-naive CD4 T cells in vitro. (Fig. 1c).

From the above results, it was shown that the in vitro culture system implemented according to the present invention yields the same results as the immunosuppressive specificity analysis shown in vivo.

At the same time, cultures were also performed with regulatory T cells mixed in 30%, 60%, and 100% of the suppressed cells. - Percentage proliferation of SM-naive CD4 T cells with regulatory T cells and OTII-percentage of proliferation of naive CD4T cells with SM-regulatory T cells) is well-expanded and reduced in division No findings were observed, and specific suppression was maintained, indicating that stable specificity analysis performance was exhibited.

(Example 2) Antigen-specific immunosuppressive assay using two types of peptide antigens In Example 1, the two types of peptide antigens were presented by separate antigen-presenting cells. Different peptide antigens were presented by the same antigen-presenting cells.

CD90.1(+) SM-naive CD4 T cells and CD45.1(+) OTII-naive CD4 T cells were used as suppressed cells.

In the same manner as in Example 1, regulatory T cells were generated and purified, suppressed cells (effector cells) were isolated, and antigen-presenting cells were isolated.

As antigens, I-Ab-restricted OVA (323-339) and I-Ab-restricted Lymphocytic Choriomeningitis Virus-derived peptide: LCMV GP (61-80) were used. Both peptides were co-cultured in the same vessel with dendritic cells isolated from CD45.2(+)-derived C57BL/6 cells and presented to MHC. After antigen presentation, centrifugation at 300 g for 5 minutes was repeated four times, and the supernatant was discarded to wash the cells.

Regulatory T cells were generated from CD45.1(+) CD45.2(+) SM-naive CD4 T cells, CD45.1(+) CD45.2(+) OTII-naive CD4 T cells. After the above-described cell components were mixed in a combination of without and with regulatory T cells, the cells were transvenously transferred into the tail vein of wild-type C57BL/6 mice using a 29G syringe.

After 3 days, splenocytes were collected, and the splenocytes were crushed using a cell strainer to separate floating cells. Cell numbers were calculated and 2×10 ⁶ splenocytes per animal were analyzed using a flow cytometer.

As a result, SM-naive CD4T cells, which are suppressed cells, and OTII-naive CD4T cells, both of which were adoptively transferred, showed good cell division, but were co-transferred with OTII-regulatory T cells. In this case, SM-naive CD4 T cells show good cell division, but OTII-naive CD4 T cells show very little cell division, and OTII-regulatory T cells enhance the immune response to OTII-naive CD4 T cells. Suppression was observed. Since almost no suppression of SM-naive CD4 T cells was observed, it was shown that immunosuppression by OTII-regulatory T cells acts specifically only on OTII-naive CD4 T cells in vivo. .

Next, when co-adoptively transferred with SM-regulatory T cells, OTII-naive CD4 T cells show good cell division, whereas SM-naive CD4 T cells show very little cell division, and SM-regulatory Sexual T cells suppressed the immune response to SM-naive CD4 T cells. Almost no suppression of OTII-naive CD4 T cells was observed, suggesting that immunosuppression by SM-regulatory T cells acts specifically on SM-naive CD4 T cells in vivo. (Fig. 2a).

In vitro culture of the cell components was performed. The basal medium was the same as in Example 1. As a result, as observed in vivo, both SM-naive CD4 T cells and OTII-naive CD4 T cells, which are suppressed cells, were cultured alone, and showed good cell division. When co-cultured with OTII-regulatory T cells, SM-naive CD4 T cells showed good cell division, whereas OTII-naive CD4 T cells showed very little cell division, and OTII-regulatory T cells , suppressed the immune response to OTII-naive CD4 T cells. Almost no suppression of SM-naive CD4 T cells was observed, indicating that immunosuppression by OTII-regulatory T cells acted specifically only on OTII-naive CD4 T cells in vitro ( Figure 2b).

Next, when co-cultured with SM-regulatory T cells, OTII-naive CD4 T cells show good cell division, whereas SM-naive CD4 T cells show very little cell division, indicating that SM-regulatory T cells are not susceptible to SM-regulatory T cells. Suppression of the immune response to SM-naive CD4 T cells was observed by T cells. Little suppression of OTII-naive CD4 T cells was observed, indicating that immunosuppression by SM-regulatory T cells acted specifically against SM-naive CD4 T cells in vitro. (Figure 2c).

From the above results, antigen specificity of immunosuppression is demonstrated in vivo by using the in vitro culture system implemented by the present invention, even when multiple antigens are presented to the same antigen-presenting cells. It was shown to give the same results as the homogeneous immunosuppressive specificity analysis. As in Example 1, cultures were also performed at the same time by increasing the mixing ratio of regulatory T cells to 30%, 60%, and 100% of the suppressed cells. The suppressed side (in the case of OTII-regulatory T cells, the proportion of proliferation of SM-naive CD4 T cells, in the case of SM-regulatory T cells, the proportion of proliferation of OTII-naive CD4 T cells) proliferated well. was observed, no findings of decreased division were observed, and specific suppression was maintained, indicating that stable specificity analysis performance was exhibited.

(Example 3) When two types of antigens are combinations of heterogeneous substances In Examples 1 and 2, antigen specificity was analyzed in the coexistence state of two types of exogenous antigen peptides. That is, in Examples 1 and 2, both of the two types of antigens are peptide antigens, and both are presented in an MHC-restricted manner. In Example 3, the case where one is an exogenous antigen peptide and the other is a cell surface-expressed protein was analyzed. In the case of typical alloantigens that have antigenicity as cell surface-expressed proteins, for example, H-2d MHC class II molecules were taken up to examine whether immunosuppression was antigen-specific.

H-2d-specific regulatory T cells were generated by co-culturing CD90.1(+) C57BL/6 naive CD4 T cells with H-2d-positive expressing cells. Unlike Examples 1 and 2, the C57BL/6 naive CD4 T cells are a polyclonal cell population with TCR gene diversity, so the regulatory T cells prepared here are a polyclonal cell population. In the same manner as in Examples 1 and 2, cells to be suppressed (effector cells) and antigen-presenting cells were isolated. In Example 3, CD90.1(+) C57BL/6 naive CD4 T cells and CD45.1(+) SM-naive CD4 T cells were used as cells to be suppressed.

As antigens, MHC H-2d expressed on H-2d-positive expressing cells and an I-Ab-restricted Lymphocytic Choriomeningitis Virus-derived peptide: LCMV GP(61-80) were used. Peptides were co-cultured with dendritic cells isolated from CD45.2(+)-derived C57BL/6 cells and presented to MHC. After antigen presentation, centrifugation at 300 g for 5 minutes was repeated four times, and the supernatant was discarded to wash the cells.

After the above cell components were mixed in a combination of without and with regulatory T cells, the cells were transferred to the plantar of wild-type C57BL/6 mice using a 29G syringe. Three days later, popliteal lymph nodes were collected, and the lymph node tissue was crushed using a cell strainer to separate floating cells. Cell numbers were calculated and 2×10 ⁶ splenocytes per animal were analyzed using a flow cytometer.

As a result, when only C57BL/6 naive CD4T cells and SM-naive CD4T cells, which are suppressed cells, were adoptively transferred, both of them showed good cell division. When adoptively transferred, SM-naive CD4 T cells show good cell division, whereas C57BL/6 naive CD4 cells show very little cell division, and H-2d-specific regulatory T cells promote C57BL/ 6 Suppression of immune response to naive CD4 cells was observed. Since almost no suppression of SM-naive CD4 T cells was observed, immunosuppression by H-2d-specific regulatory T cells acts specifically only on C57BL/6 naive CD4 cells in vivo. It has been shown. (Figure 3a).

In vitro culture of the cell components was performed. The basal medium is the same as in Example 1. As a result, as observed in vivo, both C57BL/6 naive CD4 cells and SM-naive CD4 T cells, which are suppressed cells, showed good cell division when cultured alone. When co-cultured with H-2d-specific regulatory T cells, SM-naive CD4 T cells show good cell division, whereas cell division observed in C57BL/6 naive CD4 cells is regulatory T cell dose-dependent. H-2d-specific regulatory T cells suppressed the immune response to C57BL/6 naive CD4 cells. Since almost no suppression of SM-naive CD4 T cells was observed with increasing doses of regulatory T cells, immunosuppression by H-2d-specific regulatory T cells was only observed in C57BL/6 naive CD4 cells in vitro. (Fig. 3b).

Based on the above results, it is possible to construct an in vitro assay method that can show the same results as in vivo for the antigen specificity of immunosuppression when one is a foreign antigen peptide and the other is a cell surface expressed protein. showing.

Considering Example 3 from the viewpoint of the clonality of regulatory T cells, not only regulatory T cells consisting of monoclonal cell populations as in Examples 1 and 2, but also polyclonal lymphocyte populations of T cell receptors It is also shown that the in vitro assay system implemented in the present invention stably possesses specificity analysis ability even when the constructed regulatory T cells are used.

(Example 4)
In Example 3, when one is a foreign antigen peptide and the other is a cell surface-expressed protein, the case where each antigen is presented to separate cells was verified. and T cell-responsive cell surface-expressed proteins (alloantigens) were presented to the same cells.

Splenocytes were prepared from CD45.1(+) CD45.2(+) OTII mice, and after ACK lysis treatment, some of the cells that passed through a cell strainer were isolated as naive CD4T cells using MACS magnetic separation. . The isolated CD4 T cells were added with TFGβ and IL-2 and cultured for 5 days in a CD3e/CD28 coated incubator to generate Foxp3-positive regulatory T cells. In addition to these, ATRA (all-trans-Retinoic Acid) was added to the culture medium. The cultured cells were stained with Cell Trace Violet (ThermoFisher Scientific), a dye for cell division detection.

We isolated suppressed cells (effector cells) and isolated antigen-presenting cells. In Example 4, CD90.1(+) C57BL/6 naive CD4 T cells and CD45.1(+) OTII-naive CD4 T cells were used as cells to be suppressed.

As antigen, I-Ab restricted OVA(323-339) peptide was used. As an alloantigen, H-2bm12xb mouse-derived dendritic cells were used, and OVA peptide was mixed with dendritic cells and presented to MHC. After antigen presentation, centrifugation at 300 g for 5 minutes was repeated four times, and the supernatant was discarded to wash the cells.

The above cell components were admixed in a combination of no and with regulatory T cells and then adoptively transferred intravenously into the tail vein of wild-type C57BL/6 mice using a 29G syringe. After 3 days, the spleen was collected, and the tissue was disrupted using a cell strainer to separate floating cells. Cell numbers were calculated and 2×10 ⁶ splenocytes per animal were analyzed using a flow cytometer. As a result, when only C57BL/6 naive CD4T cells and OTII-naive CD4T cells, which are suppressed cells, were adoptively transferred, they both showed good cell division, but they were co-adoptively transferred with OTII-regulatory T cells. In contrast, C57BL/6 naive CD4 T cells exhibit good cell division, but OTII-naive CD4 T cells exhibit very little cell division, and OTII-regulatory T cells provide an immune response to OTII-naive CD4 T cells. suppression was observed. Almost no suppression of C57BL/6 naive CD4 T cells was observed, suggesting that immunosuppression by OTII-regulatory T cells acts specifically on OTII-naive CD4 T cells in vivo. rice field. (Figures 4a, 4b).

In vitro culture of the cell components was performed. The basal medium is the same as in Example 1. As a result, as observed in vivo, both C57BL/6 naive CD4T cells and OTII-naive CD4T cells, which are suppressed cells, were cultured alone, and showed good cell division. When co-cultured with OTII-regulatory T cells, C57BL/6 naive CD4 T cells showed good cell division, whereas OTII-naive CD4 T cells showed a decrease in cell division in a regulatory T cell dose-dependent manner. Over time, OTII-regulatory T cells were found to suppress the immune response to OTII-naive CD4 cells. Immunosuppression by OTII-regulatory T cells was also specific to OTII- naive CD4 cells in vitro, as little suppression of C57BL/6 naive CD4 T cells was observed with increasing doses of regulatory T cells. (Fig. 4c).

From the above results, the antigen specificity of immunosuppression is similar to that in vivo when one is a foreign antigen peptide and the other is a cell surface-expressed protein, and even when the number of presenting cells is one. This indicates that a viable in vitro assay procedure can be constructed.

(Example 5) Antigen-specific immunosuppressive assay using two types of cell surface-expressed proteins Although the antigen-specific immunosuppressive action of regulatory T cells in a coexisting environment was verified, in Example 5, verification was performed in the case where both types were cell surface-expressed proteins (alloantigens).

Splenocytes were prepared from CD45.1(+) CD90.1(+) C57BL/6 mice, and after lysing with ACK, some of the cells that passed through the cell strainer were isolated as naive CD4T cells using MACS magnetic separation. released. The isolated CD4 T cells were co-cultured with dendritic cells from H-2bm12 mice and dendritic cells from H-2d mice to generate H-2bm12-specific regulatory T cells and H-2d-specific regulatory T cells. generated sexual T cells.

Inhibited cells (effector cells) and antigen-presenting cells were isolated. In Example 5, naive CD4T cells collected from CD45.2(+)CD45.1(+) H-2b/d mice and anti-H-2d responsive suppressed cells were used as anti-H-2bm12 responsive suppressed cells. Naive CD4T cells collected from CD45.2(+) CD90.1(+) H-2b/bm12 mice were used as cells.

As antigens, H-2bm12 mice and dendritic cells collected from H-2d mice were mixed and used. The above cell components were cultured in vitro. The basal medium is the same as in Example 1.

As a result, anti-H-2bm12-responsive suppressed cells and anti-H-2d-responsive suppressed cells were found to proliferate well in the absence of regulatory T cells. When co-cultured with H-2bm12-regulatory T cells, the anti-H-2d responsive suppressed cells exhibited favorable cell division, but the anti-H-2bm12 responsive suppressed cells exhibited marked suppression of proliferation. , H-2bm12-regulatory T cells were shown to have an antigen-specific immunosuppressive effect only on H-2bm12-naive T cells. It was found to suppress regulatory T cells in a dose-dependent manner (Fig. 5a).

When co-cultured with H-2d-regulatory T cells, the anti-H-2bm12-responsive suppressed cells exhibited favorable cell division, but only the anti-H-2d-responsive suppressed cells exhibited growth suppression. H-2d-regulatory T cells were shown to have antigen-specific immunosuppressive effects only on H-2d-regulatory T cells. Moreover, the suppression was found to be dose-dependent on regulatory T cells (Fig. 5b).

The above results demonstrate that the in vitro assay of the present invention can exhibit an antigen-specific immunosuppressive reaction even in an environment where cell surface-expressed proteins (alloantigens) coexist.

(Example 6) Screening for antigen-specificity modulators Screening for antigen-specificity modulators can be performed using the present invention.

Splenocytes were prepared from CD45.1(+) CD45.2(+) OTII mice, and after ACK lysis treatment, some of the cells that passed through a cell strainer were isolated as naive CD4T cells using MACS magnetic separation. . The isolated CD4 T cells were added with TFGβ and IL-2 and cultured for 5 days in a CD3e/CD28 coated incubator to generate Foxp3-positive regulatory T cells. The cultured cells were stained with Cell Trace Violet (ThermoFisher Scientific), a dye for cell division detection.

We isolated suppressed cells (effector cells) and isolated antigen-presenting cells. In Example 4, CD90.1(+) C57BL/6 naive CD4 T cells and CD45.1(+) OTII-naive CD4 T cells were used as cells to be suppressed.

As antigen, I-Ab restricted OVA(323-339) peptide was used. As an alloantigen, H-2bm12xb mouse-derived dendritic cells were used, and OVA peptide was mixed with dendritic cells and presented to MHC. After antigen presentation, centrifugation at 300 g for 5 minutes was repeated four times, and the supernatant was discarded to wash the cells.

The above cell components were admixed in a combination of no and with regulatory T cells and then adoptively transferred intravenously into the tail vein of wild-type C57BL/6 mice using a 29G syringe. After 3 days, the spleen was collected, and the tissue was disrupted using a cell strainer to separate floating cells. Cell numbers were calculated and 2×10 ⁶ splenocytes per animal were analyzed using a flow cytometer. As a result, when only C57BL/6 naive CD4T cells and OTII-naive CD4T cells, which are suppressed cells, were adoptively transferred, they both showed good cell division, but they were co-adoptively transferred with OTII-regulatory T cells. In contrast, C57BL/6 naive CD4 T cells exhibit good cell division, but OTII-naive CD4 T cells exhibit very little cell division, and OTII-regulatory T cells provide an immune response to OTII-naive CD4 T cells. suppression was observed. Almost no suppression of C57BL/6 naive CD4 T cells was observed, suggesting that immunosuppression by OTII-regulatory T cells acts specifically on OTII-naive CD4 T cells in vivo. rice field. (Figures 4a, 4b).

In vitro culture of the cell components was performed. The basal medium is the same as in Example 1. As a result, as observed in vivo, both C57BL/6 naive CD4 T cells and OTII-naive CD4 T cells, which are suppressed cells, were cultured alone, and showed good cell division. When co-cultured with OTII-regulatory T cells, C57BL/6 naive CD4 T cells showed favorable cell division, but the cell division seen in OTII-naive CD4 T cells was reduced in the presence of regulatory T cells. , OTII-regulatory T cells suppressed the immune response to OTII-naive CD4 cells. Almost no suppression of C57BL/6 naive CD4 T cells was observed, suggesting that immunosuppression by OTII-regulatory T cells acts specifically only on OTII-naive CD4 cells in vitro. (Fig. 6).

Next, an in vitro assay was performed in the presence of 50 units or 100 units of non-specific factor. No significant change was observed under the 50 unit condition. However, under 100 unit conditions, co-culture with OTII-regulatory T cells significantly reduced cell division in C57BL/6 naive CD4 T cells as well.

Under the condition of 100 units, when C57BL/6 naive CD4T cells and OTII-naive CD4T cells, which are the suppressed cells, were cultured alone, good cell division was observed. When co-cultured with OTII-regulatory T cells, the cell division seen in OTII-naive CD4 T cells was reduced, and C57BL/6 naive CD4 T cells also showed a significant reduction in cell division. This indicates that coexistence of non-specific factors causes suppression by OTII-regulatory T cells not only on OTII-naive CD4 cells but also on C57BL/6 naive CD4 T cells. -Suggests that it reduces the antigen specificity of regulatory T cells.

Based on the above results, an example of screening for antigen-specific regulatory factors using the present invention was presented.

(Example 7) Immunosuppressive antigen specificity analysis assay using thymus-derived regulatory T cells [Generation and purification of regulatory T cells]
Spleens were removed from C57BL/6 CD45.1(+) CD45.2(+) OTII RAG-WT mice and C57BL/6 CD45.1(+) CD45.2(+) SM RAG-WT mice. The splenic tissue was crushed using a cell strainer to separate floating cells into a single cell state. Cell numbers were calculated, the required amount was removed, CD25 labeled and CD25+ cells were isolated using MACS magnetic separation. From the obtained cells, CD4T+Foxp3+ cells were isolated using a cell sorting method using CD4 and Foxp3GFP as markers. The isolated CD4T+CD25+Foxp3+ cells were added with IL-2 and cultured for 5 days in a CD3e/CD28 coated incubator to generate Foxp3-positive regulatory T cells. CD4T+Foxp3+ cells were isolated from the cultured lymphocytes by cell sorting using CD4 and Foxp3GFP as markers. An assay was performed using the thymus-derived regulatory T cells obtained as described above.
In Example 7, two types of peptide antigens were presented by the same antigen-presenting cells and assayed.

CD90.1(+) SM-naive CD4 T cells and CD45.2(+) OTII-naive CD4 T cells were used as suppressed cells. Spleens were removed from C57BL/6 CD90.1(+) SM RAG-KO mice and C57BL/6 CD45.2(+) OTII RAG-KO mice. The splenic tissue was crushed using a cell strainer to separate floating cells into a single cell state. Cell numbers were calculated, the required amount was removed, CD4 was labeled and MACS magnetic separation was used to isolate CD90.1(+) SM-naive CD4 T cells, CD45.2(+) OTII- naive CD4 T cells. . The obtained cells were stained according to a standard method using Cell Trace Violet (ThermoFisher Scientific), a dye for cell division detection. Assays were performed using these cells as suppressed cells.

As antigens, I-Ab-restricted OVA (323-339) and I-Ab-restricted Lymphocytic Choriomeningitis Virus-derived peptide: LCMV GP (61-80) were used. Both peptides were co-cultured in the same vessel with dendritic cells isolated from CD45.1(+)-derived C57BL/6 cells and presented to MHC. After antigen presentation, centrifugation at 300 g for 5 minutes was repeated four times, and soluble peptides in the supernatant were washed to obtain antigen-presented dendritic cells.

OTII and SM regulatory T cells generated by the above treatment were mixed in a combination of without and with regulatory T cells, and then injected intravenously into the tail vein of wild-type C57BL/6 mice using a 29G syringe. cells were transfected.

After 3 days, splenocytes were collected, and the splenocytes were crushed using a cell strainer to separate floating cells. Cell numbers were calculated and 2×10 ⁶ splenocytes per animal were analyzed using a flow cytometer.

As a result, SM-naive CD4T cells, which are suppressed cells, and OTII-naive CD4T cells, both of which were adoptively transferred, showed good cell division, but were co-transferred with OTII-regulatory T cells. In this case, SM-naive CD4 T cells show good cell division, whereas OTII-naive CD4 T cells show very little cell division, and OTII-regulatory T cells enhance the immune response to OTII-naive CD4 T cells. Suppression was observed. We found that OTII-regulatory T cells hardly suppressed SM-naive CD4 T cells, suggesting that immunosuppression by OTII-regulatory T cells specifically acts only on OTII-naive CD4 T cells in vivo. It was shown that

Next, when co-adoptively transferred with SM-regulatory T cells, OTII-naive CD4 T cells show good cell division, whereas SM-naive CD4 T cells show very little cell division, and SM-regulatory Sexual T cells suppressed the immune response to SM-naive CD4 T cells. Almost no suppression of OTII-naive CD4 T cells was observed, suggesting that immunosuppression by SM-regulatory T cells acts specifically on SM-naive CD4 T cells in vivo. (Fig. 7a).

In vitro culture of the cell components was performed. The basal medium was the same as in Example 1. That is, the culture conditions in which the responder cells (effector cells) consisted only of mixed cells of SM-naive CD4T cells and OTII-naive CD4T cells, and the number of these responder cells was 50% or 100% of the number of effector cells. Thus, in vitro assays were performed in culture conditions that included the addition of equal numbers of OTII-regulatory T cells. Furthermore, as a comparison, culture conditions in which responder cells (effector cells) consisted only of a mixture of SM-naive CD4T cells and OTII-naive CD4T cells, and 50% of the number of effector cells in these responder cells, or In vitro assays were performed in culture conditions that consisted of adding 100% or equal numbers of SM-regulatory T cells.

As a result, as observed in vivo, both SM-naive CD4 T cells and OTII-naive CD4 T cells, which are suppressed cells, were cultured alone, and showed good cell division. When co-cultured with OTII-regulatory T cells, SM-naive CD4 T cells showed good cell division, whereas OTII-naive CD4 T cells showed very little cell division, and OTII-regulatory T cells , suppressed the immune response to OTII-naive CD4 T cells. Almost no suppression of SM-naive CD4 T cells was observed, indicating that immunosuppression by OTII-regulatory T cells acted specifically only on OTII-naive CD4 T cells in vitro ( Fig. 7b left).

Next, when co-cultured with SM-regulatory T cells, OTII-naive CD4 T cells show good cell division, whereas SM-naive CD4 T cells show very little cell division, indicating that SM-regulatory T cells are not susceptible to SM-regulatory T cells. Suppression of the immune response to SM-naive CD4 T cells was observed by T cells. Little suppression of OTII-naive CD4 T cells was observed, indicating that immunosuppression by SM-regulatory T cells acted specifically against SM-naive CD4 T cells in vitro. (Fig. 7b right).

From the above results, antigen specificity of immunosuppression is demonstrated in vivo by using the in vitro culture system implemented by the present invention, even when regulatory T cells derived from thymus are used. It was shown that the same results as the specificity analysis of immunosuppression can be obtained. Even if the mixing ratio of regulatory T cells to the suppressed cells is increased to 50% or 100%, the suppressed cells on the side of the antigen system that do not correspond to the corresponding antigen of the regulatory T cells, that is, OTII-regulatory T cells In the case of SM-naive CD4 T cells with SM-regulatory T cells, the rate of proliferation of OTII-naive CD4 T cells was observed to be good, and no evidence of hypomitosis was observed. It was shown that antigen-specific suppression was maintained and stable specificity analysis performance was exhibited.

It can be used for the development of autoimmune disease inhibitors. It can be used for the development of novel cancer therapeutic immunomodulators. It can be used for the development of transplant immune response inhibitors.

Claims (4)

Preparing for providing a first regulatory T cell and at least two suppressed cells selected from a first suppressed cell , a second suppressed cell, and a third or more suppressed cells process and
an antigen presenting step of presenting a first antigen to the first regulatory T cell to activate the first regulatory T cell with the first antigen;
the first regulatory T cell, and at least two suppressed cells selected from the first suppressed cell , the second suppressed cell , and the third or more suppressed cells When the cell division of the second suppressed cell, and the third or more suppressed cells is not reduced, but the cell division of the first suppressed cell is reduced, the first A method for evaluating the specificity of regulatory T cells in vitro, comprising the step of determining that one regulatory T cell is specific only for the first suppressed cell. characterized by comprising an antigen-presenting step of presenting a first antigen and a second antigen to the first regulatory T cells, and activating the first regulatory T cells with the first antigen. The in vitro regulatory T cell specificity evaluation method according to claim 1. a preparatory step of providing a first regulatory T cell, a second regulatory T cell, a first suppressed cell, and a second suppressed cell;
presenting a first antigen and a second antigen to the first regulatory T cell to activate the first regulatory T cell with the first antigen; an antigen presenting step of presenting two antigens to the second regulatory T cell to activate the second regulatory T cell with the second antigen;
co-culturing the first regulatory T cells with the first suppressed cell and the second suppressed cell, wherein cell division of the second suppressed cell is not reduced, but the determining that the first regulatory T cell is specific only for the first suppressed cell if cell division of the first suppressed cell is reduced;
co-culturing the second regulatory T cells with the first suppressed cell and the second suppressed cell, wherein cell division of the first suppressed cell is not reduced, but the determining that the second regulatory T cell is specific only for the second suppressed cell if cell division of the second suppressed cell is reduced;
A method for evaluating the specificity of regulatory T cells in vitro, characterized by comprising: 4. The in vitro regulatory activity according to any one of claims 1 to 3, wherein the regulatory T cells are Foxp3-positive regulatory T cells or IL-10-producing regulatory T cells. A method for evaluating T cell specificity.

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