JP2003033175A - Peripheral blood dendritic cell subset inducting selective immune response suppression - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peripheral blood dendritic cell subset selectively suppressing T-cell immune responses in autoimmune diseases, allergic diseases, and rejection symptoms after transplantation, and to provide an immunosuppressant which can selectively suppress immune responses causing the diseases. SOLUTION: This immunosuppressant comprises a peripheral blood dendritic cell subset which can induce and IL-10 producing ability in T-cells, when contacted with the T-cells having the immune response with a target antigen. The immunosuppressant is prepared by stimulating a peripheral blood dendritic cell subset originated from human peripheral blood and having a surface marker of CD4<+> HLA-DR<+> on the cell surface such as 2 type dendritic cell precursor originated from human peripheral blood or a plasma dendritic cell having a surface marker of CD4<+> HLA-DR<+> CD11c<-> CD123<+> , or the concentrated fraction of the peripheral blood dendritic cell subset, with the target antigen for selectively suppressing the immune response in vitro, thus applying the differentiation- suppressing treatment to the mature dendritic cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a subset of peripheral blood dendritic cells capable of selectively suppressing the immune response of T cells in autoimmune diseases, allergic diseases, and rejection after transplantation. The present invention relates to a composition having an immunosuppressive action.

[0002]

BACKGROUND OF THE INVENTION Dendritic cells (DC) are known as antigen-presenting cells (APCs) that are essential for the initiation of an immune response by priming naïve or dormant T cells (An).
nu. Rev. Immunol. 9, 271-296, 1991, Blood Rev. 13,
51-64, 1999). The ability of DC to present antigenic peptides and initiate a T cell-dependent immune response is the ability of macrophages and B
The ability of other types of professional APC including cells is far superior. Due to its nature, various strategies using DC have been adopted in cancer immunotherapy to enhance tumor immunity, and tumor vaccines using DC from experiments using animal models are anti-tumor CTLs. (Cytotoxic T lymphocyte)
Reported to induce a response and consequently reject the tumor (J.
Exp. Med. 186, 1183-1187, 1997, J. Exp. Med. 186,
1213-1221, 1997). Furthermore, tumor vaccines using DC are actually used in clinical practice (Nat. Med. 4,
328-332, 1998, J. Exp. Med. 190, 1669-1678, 1999).

DCs are a population of cells that are morphologically, phenotypically and functionally different from other cells. DCs are divided into “mature DCs” and “immature DCs” by their functional ability to stimulate T cells, and “Th1-skewing DC: DC1” by their ability to differentiate naive T cells into effector cells that secrete different types of cytokines. And “Th2-skewing
“DC: DC2” and “myeloid DC” and “lymphoid D” due to differences in the lineage and cell surface markers.
C ”(Blood Rev.13, 51-6
4, 1999, Stem Cells, 15, 409-419, 1997, Immunolog
y, 82, 487-493, 1994, Science, 283, 1183-1186, 199
9, J. Immunol. 165, 566-572, 2000, Blood, 95, 2484
-2490, 2000).

DC precursors (pDCs) are present in peripheral blood and are known to become functionally mature DCs by adding appropriate cytokines and / or growth factors in vitro (Immunology, 82, 487-493, 1994, J. Ex
p. Med. 178, 1067-1078, 1993, Proc. Natl. Acad. Sc
i. USA, 87, 7698-7702, 1990, J. Exp. Med. 179, 110
9-, 1118 1994, J. Exp. Med. 180, 83-93, 1994). Human DC precursors in peripheral blood have a morphology resembling medium-sized lymphocytes lacking dendrites, T cells, B cells, monocytes and NK
There are no markers of other cell types including cells, CD4 and M
Identified as a positive subset for both HC class II molecules (J. Exp. Med. 178, 1067-1078, 199
3). Human pDCs in the bloodstream are composed of at least two phenotypes and functionally distinct subsets, including pDC1 (type 1 dendritic cell precursor) and pDC2 (type 2 dendritic cell precursor) (J. Immunol. 161, 740-748, 1998, J.
Immunol.163, 1409-1419, 1999, J. Immunol.163, 3
250-3259, 1999, Eur. J. Immunol. 29, 2769-2778, 19
99). pDC1 has been identified as a myeloid antigen positive pDC such as CD11c and CD33, while pDC2 is
Apparently plasma cell-like, lacking myeloid antigens, pre-T
It is known to express CR (T cell receptor) α chain and CD123 (IL-3 receptor α chain) (J. Immu
nol. 165, 566-572, 2000, Blood, 95, 2484-2490, 200
0, J. Immunol. 163, 1409-1419, 1999).

Activation of T cells requires two signals: TCR occupancy by antigenic peptides presented on MHC molecules and binding of costimulatory molecules on APC and T cell ligands ( Two signal theories:
J. Exp. Med. 184, 1-8, 1996). When there is no costimulation and TCR occupancy occurs, T cells do not proliferate even when optimal restimulation with functional APC and antigen is given (anergy)
(J. Exp. Med. 184, 1-8, 1996). Mature D
C expresses MHC class I and class II molecules as frequently as costimulatory molecules (CD80 and CD86),
High stimulation ability against allogeneic or foreign antigen-specific T cell responses (Blood Rev. 13, 51-64, 1999, Stem C
ells, 15, 409-419, 1997). On the other hand, pDC immediately after isolation from peripheral blood, especially pDC2, is CD80 and CD86.
Is expressed at a very low level even when expressed, and the ability to stimulate T cell responses to foreign antigens is poor (J. Immunol. 165, 566-572, 2000, Blood, 95, 2484-
2490, 2000, J. Exp. Med. 178, 1067-1078, 1993, Pro
c. Natl. Acad. Sci. USA, 87, 7698-7702, 1990, J. E
xp. Med. 179, 1109-1118, 1994, J. Exp. Med. 180, 8
3-93, 1994, J. Immunol. 163, 3250-3259, 1999).

[0006]

Many human diseases are caused by an excessive immune response to a specific antigen. For example, allergic diseases such as atopic dermatitis and hay fever are induced by an excessive immune reaction against specific foreign antigens (ticks, pollen, food, drugs, etc.). In addition, the immune response to self components causes autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. Further, in organ transplantation where the number of practice cases is increasing, rejection of transplanted organs after transplantation is a serious problem. In these pathologies, treatment by suppressing the immune response to the causative antigen is possible, but in cedar pollinosis, immune response to cedar pollen, in idiopathic thrombocytopenic purpura, immune response to platelet membrane protein, rejection In the reaction, it is difficult to selectively and specifically suppress only the immune response to the donor antigen, and at present, immunosuppressive therapy that suppresses all immune functions including normal immune functions is used for autoimmune diseases, allergic diseases, It is used for the treatment of rejection.

[0007] Currently used immunosuppressive therapies for autoimmune diseases, allergic diseases, and rejection reactions include administration of corticosteroids, administration of immunosuppressive agents, and removal of the spleen and thymus involved in immune control. Although known, these immunosuppressive therapies are effective to some extent, and although many patients have improved symptoms, there are not a few cases where the effects are insufficient, and serious side effects are also a major problem. It has become. These problems stem from the fact that conventional immunosuppressive therapy simultaneously suppresses not only immune responses related to diseases but also normal immune functions. If the immune functions are excessively suppressed, infections caused by bacteria, viruses, molds, etc. Will increase the incidence of malignant tumors. Therefore, reducing the immunosuppressive effect decreases the therapeutic effect. For these reasons, the current immunosuppressive therapy is based on a balance between conflicting therapeutic effects and side effects, and it is said that its effectiveness is limited. Therefore, a treatment method that selectively suppresses the immune response that causes the disease without affecting normal immune function is ideal, and its development is strongly desired in the medical field. The subject of the present invention is
Peripheral blood dendritic cell subsets that can selectively suppress the immune response of T cells in autoimmune diseases, allergic diseases, and rejection after transplantation, and selectively suppress the immune responses that cause the disease An object of the present invention is to provide a composition having an immunosuppressive action that can be performed.

[0008]

In connection with the "two signal theory" relating to T cell activation, the present inventors have developed a CD4, a dendritic cell subset present in human peripheral blood.
⁺ Plasmacytoid dendritic cells with surface markers of HLA-DR ⁺ CD11c ^- CD123 ⁺
ell) and type 2 dendritic cell precursor (pDC2). Dendritic cell subsets such as pDC2 that lack the expression of costimulatory molecules essential for T cell activation are activated T
It is hypothesized that binding to cells via an antigen may not induce the necessary costimulatory signals during interaction with T cells, and as a result may induce T cell anergy. To confirm, we first established a method for preparing fractions containing high concentrations of pDC2 from peripheral blood mononuclear cells (PBMC)
A T cell anergy induction experiment was conducted in a model system using such a fraction containing pDC2 at a high concentration and a human CD4 ⁺ T cell line recognizing tetanus toxoid (TT) as an antigen. In addition, using the pDC2 fraction and a human autoreactive CD4 ⁺ T cell clone against topoisomerase I (topoI), a scleroderma-specific self-antigen, pDC
Experiments were also conducted on whether dendritic cell subsets such as 2 could induce tolerance to autoreactive T cell responses.

From the above experiments, the following findings that were not known so far were obtained. When the TT-reactive human CD4 ⁺ T cell line was cultured with autologous pDC2 in the presence of TT for 3 days, it became an anergy state that could not be proliferated even if an appropriate antigen stimulation was added thereafter. p
Induction of T cell anergy by DC2 required the presence of an antigen recognized by T cells. A TT-reactive human CD that has fallen into anergy with pDC2
The 4 ⁺ T cell line is a high concentration of interleukin (IL) −
Proliferative ability was restored by antigen stimulation in the presence of 2. TT-reactive human CD made anergy by pDC2
The 4 ⁺ T cell line produced IL-10 upon antigen stimulation. IL-10 is a cytokine that suppresses the function of APC, and it has been shown that T cells in contact with pDC2 not only fall into anergy themselves but also suppress the function of surrounding APCs. pDC2 similarly induced antigen-specific anergy and differentiation into IL-10-producing cells for autoantigen-specific T cells established from patients with scleroderma, an autoimmune disease.

[0010] The above findings, ie, stimulation of dendritic cell subsets such as pDC2 isolated from peripheral blood with a specific antigen (target antigen) allows angiogenesis to T cells that are immunoresponsive to the target antigen. PDC2 is involved in maintaining immune tolerance in vivo by selectively and antigen-specifically suppressing the immune response (immune reaction) of T cells that react with antigens taken up by pDC2 As a result, the present invention has been completed.

That is, the present invention is derived from human peripheral blood,
A dendritic cell subset having a surface marker of CD4 ⁺ HLA-DR ^{+ on} the cell surface and stimulated with a target antigen in vitro, and contacting with a T cell immunoreactive to the target antigen, The peripheral blood dendritic cell subset characterized by being able to induce anergy in the T cell (claim 1), or the dendritic cell subset derived from peripheral blood is subjected to a treatment for inhibiting differentiation into mature dendritic cells. The peripheral blood dendritic cell subset according to claim 1 or (2), wherein the differentiation suppression treatment into mature dendritic cells is irradiation treatment. The peripheral blood dendritic cell subset according to claim 2 or (Third claim), an angiogenic T
The peripheral blood dendritic cell subset according to any one of claims 1 to 3, wherein the cell has IL-10 producing ability (claim 4), or the dendritic cell subset derived from peripheral blood is CD4. The peripheral blood dendritic cell according to any one of claims 1 to 4, which is a plasmacytoid dendritic cell or a type 2 dendritic cell precursor having a surface marker of ⁺ HLA-DR ⁺ CD11c ^- CD123 ⁺ It relates to a subset (claim 5).

The present invention also provides a method for separating a mononuclear cell fraction from human peripheral blood, removing T cells and monocytes from the mononuclear cell fraction,
CD3, CD14, CD19, CD34 and CD5
CD4, characterized by the removal of cells with these surface markers using a monoclonal antibody against 6
^A method for preparing a subset of peripheral blood dendritic cells having a surface marker of ⁺ HLA-DR ⁺ (Claim 6), or a proliferation step of peripheral blood dendritic cells treated with G-CSF. Method for preparing peripheral blood dendritic cell subsets having the described CD4 ⁺ HLA-DR ⁺ surface marker (claim 7)
About.

Furthermore, the present invention provides an anergy T cell characterized in that an anergy is obtained by contact with the peripheral blood dendritic cell subset according to any one of claims 1 to 5 (claim 8), IL-10. The anergy T cell according to claim 7 or (claim 9) or
5. A composition having a selective immune response inhibitory action comprising the peripheral blood dendritic cell subset according to claim 5 and / or the anergy T cell according to claim 9 (claim 1).
0), a method for treating a disease caused by an excessive immune response (Claim 11), which comprises administering the composition having a selective immune response suppressing action according to Claim 10.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The peripheral blood dendritic cell subset of the present invention is derived from human peripheral blood and has CD4 ⁺ on the cell surface.
A dendritic cell subset having a surface marker of HLA-DR ⁺ and stimulated with a target antigen in vitro,
Particularly, if it is a dendritic cell subset that can induce IL-10 production ability in addition to anergy, preferably anergy in addition to the T cell by contacting with a T cell that is immunoresponsive to the target antigen. As a subset of dendritic cells derived from peripheral blood with the CD4 ⁺ HLA-DR ⁺ surface marker, including, but not limited to, pDC
2, and plasmacytoid dendritic cells having a surface marker of CD4 ⁺ HLA-DR ⁺ CD11c ⁻ CD123 ⁺ can be preferably exemplified. In the present invention, these pDC2 and ^{CD4 + HLA-DR + CD11c -} CD123 + , such as plasmacytoid dendritic cells with surface markers CD4 ⁺ HLA-
Dendritic cell subsets derived from peripheral blood with a DR ⁺ surface marker can be used in a mixed state with other cell populations, but dendritic cells derived from peripheral blood with a CD4 ⁺ HLA-DR ⁺ surface marker The cell subset is preferably 40
%, More preferably 50% or more, particularly 60% or more.

The enriched fraction of dendritic cell subsets derived from peripheral blood having such a CD4 ⁺ HLA-DR ⁺ surface marker is obtained by separating a mononuclear cell fraction from human peripheral blood, Min T cells and monocytes, and then CD3,
CD4 ⁺ HL of the present invention, characterized in that cells having these surface markers are removed using monoclonal antibodies against CD14, CD19, CD34 and CD56
It can be prepared by a method for preparing a subset of peripheral blood dendritic cells having a surface marker of A-DR ⁺ . Separation of the mononuclear cell fraction from the human peripheral blood can be performed by a known method, for example, specific gravity centrifugation. As a method for removing T cells from the human peripheral blood mononuclear cell (PBMC) fraction,
A known method, for example, a method of removing T cells from the PBMC fraction by rosetting with sheep erythrocytes treated with neuraminidase can be mentioned. Examples of the method for removing monocytes from the human PBMC fraction include a known method, for example, a method for removing monocytes from the PBMC fraction by removing T cells and then attaching them to a Petri dish coated with human Ig. it can. P
After removing T cells and monocytes from the BMC fraction, anti-human C
D3 monoclonal antibody, anti-human CD14 monoclonal antibody, anti-human CD19 monoclonal antibody, anti-human CD
The 34 monoclonal antibody and the anti-human CD56 monoclonal antibody were used respectively for these CD3, CD14, C
As a method of removing cells expressing the CD antigens of D19, CD34 and CD56 on the surface, a known method,
For example, a monoclonal antibody combined with magnetic beads as each of the above human monoclonal antibodies can be used to remove magnetically or to remove each CD antigen by sorting by flow cytometry.

According to the method for preparing a subset of peripheral blood dendritic cells having the CD4 ⁺ HLA-DR ⁺ surface marker of the present invention, pDC present in the peripheral blood in an amount of 0.5% or less
Peripheral blood dendritic cell subsets with surface markers of CD4 ⁺ HLA-DR ⁺ such as 2 can be enriched, eg 4
A concentrated fraction of 2 to 68% can be prepared. A value of 42-68% in this enriched fraction indicates human pD
Report of O'Doherty et al. On the preparation of C (J. Exp. Med.
178, 1067-1078, 1993), which is a little more than 2 to 4 times larger than the value of 9-27%, indicating that the method for preparing a subset of peripheral blood dendritic cells of the present invention is excellent, as well as such enrichment. Most of the fractions are pDC2 and CD4 ⁺ HLA
It is occupied by plasmacytoid dendritic cells having a surface marker of −DR ⁺ CD11c ⁻ CD123 ⁺ , and an anergy can be efficiently induced in T cells by using this concentrated fraction. In addition, as a G-CSF treatment method in the method for preparing a peripheral blood dendritic cell subset having a surface marker of CD4 ⁺ HLA-DR ⁺ of the present invention, for example, human G-
Examples thereof include a method using human peripheral blood pre-administered with CSF, and the yield of the desired peripheral blood dendritic cell subset can be increased. When using recombinant human G-CSF, it is preferable to use G-CSF at a concentration of 5 to 20 μg / kg for 3 to 10 days.

By contacting the peripheral blood dendritic cell subset of the present invention, that is, a T cell that is immunoresponsive to a specific antigen (target antigen) of interest for selectively suppressing an immune response, Peripheral blood dendritic cell subsets capable of inducing IL-10 production ability in addition to anergy, preferably anergy in the T cells are pDC2 and CD4 ⁺ HLA-DR ⁺ CD11c derived from the above human peripheral blood.
^- CD123 ⁺ dendritic cell subsets or enriched fraction thereof with a CD4 ⁺ HLA-DR ⁺ surface markers on the cell surface such as plasmacytoid dendritic cells with surface markers, were stimulated in vitro with the target antigen Preferably, it can be prepared by carrying out a treatment for inhibiting differentiation into mature dendritic cells. Examples of the target antigen include mites, pollen,
Antigens that cause allergic diseases such as food and drugs,
Examples include antigens that cause autoimmune diseases such as platelet membrane proteins and donor antigens that cause rejection of transplanted organs that occur after transplantation of organ transplants. Depending on the type of antigen protein, etc., concentration 1-20
It is preferable to stimulate once with μg / mL of antigen. In addition, when the precursor cells such as pDC2 are activated at an inflammatory site or the like, the above-described treatment for suppressing differentiation into mature dendritic cells differentiates into a mature type that induces an immune response through activation of T cells. Since there is a possibility, it is very preferable. As the treatment for suppressing differentiation into such mature dendritic cells, irradiation treatment,
Examples of the treatment include treatment with an anti-CD40 ligand antibody, treatment with a drug such as mitomycin, etc. Among them, irradiation treatment is preferable, and specifically, a total radiation dose of 40G
A radiation irradiation treatment with y can be suitably exemplified.

The anergy T cell of the present invention is particularly limited as long as it is an anergy T cell that has become anergy by contact with a subset of peripheral blood dendritic cells such as pDC2 stimulated with the target antigen of the present invention. However, Anergy T cells having IL-10 production ability are preferred. IL-10 is a cytokine that suppresses the function of APC, and pDC2
T cells that have come into contact with a subset of peripheral blood dendritic cells such as these have the potential to inhibit not only themselves but also the function of surrounding APCs. Further, a subset of peripheral blood dendritic cells stimulated with the target antigen of the present invention or an enriched fraction thereof,
As an in vitro contact method with T cells having immunoreactivity for a target antigen, R supplemented with 8% autologous plasma was used.
A specific example is a method of co-culturing in PMI1640 medium at 37 ° C. and 5% CO ₂ for ₂ to 3 days.

The composition having a selective immune response suppressing action of the present invention includes the peripheral blood dendritic cell subset of the present invention,
That is, T having immune responsiveness to a specific target antigen (target antigen) for selectively suppressing the immune response
A peripheral blood dendritic cell subset capable of inducing IL-10 production ability in addition to anergy, preferably anergy in addition to an anergy,
The composition is not particularly limited as long as it contains an anergy T cell having 10 production ability and has an action of suppressing an immune response by a T cell selectively and antigen-specifically. Illness caused by the response,
For example, allergic diseases typified by atopic dermatitis and hay fever, autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis, rejection of transplanted organs after transplantation, etc. It can be advantageously used as a therapeutic agent. In addition, the composition having a selective immune response suppressing action of the present invention includes an additive usually used for this kind of therapeutic agent, and anti-CD4 as an inhibitor of differentiation into mature dendritic cells.
A 0 ligand antibody or the like can be blended.

As a method for treating a disease caused by an excessive immune response according to the present invention, the above-mentioned composition having a selective immune response suppressing action is used, for example, an allergic disease represented by atopic dermatitis or hay fever, The method is not particularly limited as long as it is administered to a patient in need of treatment of autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis and rejection of organ transplanted at the time of organ transplantation. However, it is preferred to administer the composition prepared from the patient's own peripheral blood. In addition, according to the treatment method of the present invention, an immune response to a specific target antigen can be selectively suppressed, so that the immune response antigen associated with a pathological condition is known with little influence on normal immune function. For example, it can be applied to any disease, and since the cells in the peripheral blood are used as they are, the manipulation is simple, such as the need for genetic manipulation or treatment with drugs, etc. There are many advantages, such as the identification of the required T cell epitopes is unnecessary and all patients can be treated independently of the patient's HLA type.

[0021]

The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples. Example 1 [Materials and Methods] (1-1: Culture Medium and Antigen) RPMI164 supplemented with 8% heat-inactive human AB serum (sigma, St. Louis, MO)
0, 10 mM HEPES, 2 mM L-glutamine,
50 U / ml penicillin and 50 μg / ml streptomycin were used for cell isolation and culture. Tetanus Toxoid (TT) is Massachusetts Public Health Biolog
Purchased from ical Laboratories (Boston, MA). Human topoI polypeptide fragment (amino acid residue)
209-386) was expressed in E. coli as a fusion protein with maltose binding protein (MalBP) (Clin. Immunol. Immunopathol. 76, 266-278, 199).
Five).

(1-2: Human subjects) Five healthy subjects and previous studies (J. Immunol. 158, 485-491, 1997, J. Immun
ol.164, 6138-6146, 2000), peripheral blood samples were obtained multiple times from one scleroderma patient who was also a collaborator. All specimens were collected after the subject signed informed consent in accordance with Institutional Review Board guidelines.

(1-3: Concentration of pDC obtained from peripheral blood)
Some modifications were made to the method described in the literature (J. Exp. Med. 178, 1067-1078, 1993), and pDC was isolated from peripheral blood. The method is shown below. T cells were removed from peripheral blood mononuclear cells (PBMC) by rosetting with sheep erythrocytes (Cosmo Bio Inc., Tokyo, Japan) treated with neuraminidase. Furthermore, human Ig (Sigma, St. Lou
monocytes were removed from the T cell depleted fraction by attaching them to a Petri dish coated with is, MO). Hank's cells containing 1% BSA and 1 mM EDTA and no Ca ²⁺ and Mg ²⁺
Resuspended in balanced salt solution (HBSS), CD3,
CD14, CD19, CD34 (2 × 10 ⁸ beads, Dyn
al, Oslo, Norway) and CD56 (5 × 10 ⁸ , PerSe)
ptive Diagnostics, Cambridge, MA) by mixing magnetic bead-bound monoclonal antibodies against each CD antigen and then removing the bead-bound cells magnetically.
A fraction enriched in DC was obtained.

(1-4: Other professional APCs
Preparation of monocytes was performed by performing a metrizamide concentration gradient method on plastic adherent cells derived from PBMC according to the method described in the literature (J. Immunol. 154, 3821-3835, 1995). After two-stage purification using a nylon wool column, CD3 ⁺ T cells were removed magnetically to obtain a fraction rich in B cells (J. Immunol. 16).
4, 6138-6146, 2000). By culturing under the following series of culture conditions, from plastic-attached PBMC to mature D
C1 was obtained (J. Immunol. Methods, 196, 121-135, 199
6). 1000 U / ml granulocyte-macrophage colony stimulating factor (GM-CSF) and 800 U / ml IL
-4 (Genzyme, Cambridge, MA), the adherent cells were cultured for 7 days, and the cultured cells were transferred to a new culture dish.
The cells were further cultured in a monocyte conditioned medium prepared by culturing adherent cells on a Petri dish coated with 00 μg / ml human Ig (Sigma) for 24 hours in the presence of the antigen. As measured by flow cytometry, the monocyte fraction is greater than 95% CD14 ⁺ cells, the B cell fraction is greater than 90% CD19 ⁺ B cells and 2
Contained less than% CD3 ⁺ T cells, and more than 50% CD83 ⁺ HLA-DR ⁺ cells and less than 2% CD14 ⁺ cells, respectively, in the DC1 cell fraction. 50
pDC cultured for 3 days in the presence of ng / ml IL-3 (Genzyme) is more than 30% CD83 ⁺ HLA-DR ⁺
Contained cells.

(1-5: Flow cytometry analysis) H
LA class I, HLA-DR, CD3, CD4, CD1
1c, CD14, CD19, CD34 (Becton Dickins
on, San Jose, CA), CD40, CD54, CD80,
CD86 (Ancell, Bayport, MN), CD56 (Leinco
Technologies, Ballwin, MO), CD123 (BD PharMi
ngen, San Diego, CA), CD28, CD83 and CD
Cells were stained with fluorescein isothiocyanate (FITC) and / or PE-conjugated mouse monoclonal antibody against 154 (Immunotech, Marseille, France). Using CellQuest software,
FACS Caliber Flow Cytometer (Becton Dicki
nson).

(1-6: Antigen-specific CD4⁺T cell line)
In the method described in the literature (J. Immunol. 158, 485-491, 1997)
Repeat antigen stimulation, limit dilution, and
Tetanus toxoid (TT) specific CD4 from two examinees⁺
A T cell line was obtained. Three that proliferate specifically by TT stimulation
CD4⁺T cell lines (K1, K3 and H1) were established
It was. K1 and K3 are HLA-DR restricted and Th0
Has a cytokine profile, while H1 is HLA
-Restrictive to both DR and HLA-DQ, Th1
The phenotype of was shown. Two types of topoI from scleroderma patients
Specific CD4⁺T cell clones (T2-5 and T2-
6) has already been established and detailed (J. Immunol. 15
8, 485-491, 1997, J. Immunol. 164, 6138-6146, 200
0). These T cell clones are HLA-DR restricted T
Has a profile of h0 cytokines and in vitro
Anti-topoI self from autologous peripheral blood B cells in an antigen-dependent manner
Antibody production was induced. This antigen-specific CD4 ⁺T cell
The strain was divided into antigen, IL-2 (50 U / ml), and radiation irradiation.
Shooting self LBL (Epstein-Barr virus-transformed l
ymphoblastoid B cell line cells) for 7-10 days
Maintained by stimulating at intervals.

(1-7: Induction of anergy in antigen-specific T cell lines) The inducing ability of T cell anergy in various types of APCs is described in the literature (J. Exp. Med. 178, 1753-176).
3, 1993, J. Immunol. 159, 4772-4780, 1997). Evaluation was performed after primary culture of T cells with APC pulsed with antigen,
The T cells were cultured in a medium containing IL-2, and then re-challenge in a secondary culture with optimal antigen stimulation. T cells were allowed to stand for 7-10 days and then pulsed with antigen pDC2, monocytes, peripheral blood B cells, DC1, I
Along with L-3 treated pDC2 or LBL, these APs
The cells were cultured in the presence of IL-2 (50 U / ml) for 3 days so that the ratio of C to T cells was 1: 3 unless otherwise specified. Monocytes, B cells, pDC2, IL-3 treated pDC2, and LBL were pulsed with antigen for 2 hours and irradiated before mixing with T cells. In some experiments, pDC2 was used without being irradiated. For DC1, in the final maturation process, the antigen was added to the monocyte conditioned medium, and the cells were irradiated before mixing with T cells. After primary culture, the recovered viable T cells are treated with IL-2.
(50 U / ml) was further re-cultured for 3 days, and antigen-induced T cell proliferation analysis, cytokine production analysis, and / or in vitro self-antigen production induction ability analysis was performed. In some experiments, anti-HLA-DR monoclonal antibodies, anti-HLA-, and so on to a final concentration of 2 μg / ml, unless otherwise stated, during primary culture of antigen-pulsed pDC2.
DQ monoclonal antibody or isotype matched control monoclonal antibody (Leinco Technologies) was added. The fraction enriched in pDC2 was separated from anti-CD4 or anti-CD8 monoclonal antibody-conjugated magnetic beads (Dyna
l) to react with CD4 ⁺ cells or CD8 ⁺
Each cell was removed. Furthermore, CD11c ⁺ cells or CD123 ⁺ cells were also anti-CD11c or anti-CD1 added with goat anti-mouse IgG antibody-conjugated magnetic beads (Dynal).
It was removed by reacting with 23 monoclonal antibodies.
After this treatment, less than 2% of the cells were positive for these markers.

(1-8: T cell proliferation analysis) Literature (J. Imm
unol. 158, 485-491, 1997), T cell proliferation induced by antigen-specific or non-antigen-specific stimulation was measured by ³ H-thymidine incorporation. Varying proportions of T cell lines and irradiated APC were cultured for 48 hours in the presence or absence of antigen and then further ³ H-thymidine (0.5 μCi / well; DuPont NEN,
In the presence of Boston, MA), the cells were cultured for 16 hours. Next, the cultured cells were collected, and the amount of ³ H-thymidine incorporated was measured using a TopCount microplate-scintillation counter (Packard, Meriden, CT). TT is 5 μg /
Used at a concentration of ml, topoI and MalBP (maltose binding protein) were used at a concentration of 10 μg / ml. PMA (25 ng / ml) and ionomycin (1
μg / ml; BD PharMingen) or anti-CD3
A mixture of monoclonal antibody (30 ng / ml) and anti-CD28 monoclonal antibody (1 μg / ml; BD PharMingen) was used to stimulate T cells. All culture experiments were performed 3 or 2 times at the same time, and the results were expressed as mean ± standard deviation (SD) (SD is less than 15% unless otherwise specified).

In addition, TT (5 μg / ml) and irradiated autologous B for 7 days in the presence of pDC2 culture supernatant.
Cells or anti-CD3 monoclonal antibody (30 ng
The proliferation of peripheral blood T cells stimulated with / ml was measured. The pDC2 culture supernatant is TT-specific T cell line K1 and pD
Stimulate C2 in the presence of TT or IL-3 (50
ng / ml) in the presence of 3 days.
After removing cellular components by centrifugation, the undiluted culture supernatant was directly used for T cell culture.

(1-9: Autoantibody production analysis in vitro) Anti-t of a topoI-specific CD4 ⁺ T cell clone
The literature (J. Immunol. 164, 6
138-6146, 2000). All experiments were performed twice at the same time, and the value obtained by subtracting the average value of the reference blank from the average value was used as the measurement result. Unless otherwise noted, SD is less than 15% of the average value, or 0.010 (OD
₄₀₅ ).

(1-10: Cytokine production analysis) CD
To measure the amount of cytokines produced from 4 ⁺ T cells, T cells (2 × 10 ⁵ ) were treated with anti-CD3 monoclonal antibody (30 ng / ml) and PHA (1 μg / ml).
Or PMA (25 ng / ml) and ionomycin (1
(μg / ml) for 48 hours. In some experiments,
T cells were cultured with irradiated autologous LBL (10 ⁴ ) and antigen for 48 hours. Culture supernatant is collected and human IFN-γ, IL-2, IL-4, and IL-1 in the culture supernatant
0 amount of ELISA kit (R & D Systems, Minneapoli
s, MN and BioSource International, Camerillo, CA)
Was measured according to the manufacturer's usage.

Example 2 [Results] (2-1: Concentration of pDC and its characteristics) To enrich human pDC in the PBMC fraction, known T cells, monocytes, B cells, NK cells and CD34 ⁺ hematopoietic stem cells are known. Cells with the following phenotype were removed by a series of procedures. As a result, CD3, CD14, CD19, CD34 or CD5
Cells positive for 6 consistently fell below 2% of the collected fraction. As shown in FIG. 1a, the main cells in the pDC enriched fraction were CD4 ⁺ HLA-DR ⁺ cells, consistent with the phenotype of pDC freshly isolated from peripheral blood (J. E
xp. Med. 178,1067-1078, 1993). The proportion of CD4 ⁺ HLA-DR ⁺ cells in 9 blood samples is 42%
It was -68% (average 55%). Almost all cells in the fraction enriched in pDC were spherical cells lacking processes, but most cells formed aggregates after culturing for 3 days in the presence of IL-3, and cytoplasmic processes extending from the surface of the aggregates. Some cells formed.

Furthermore, according to FIG. 1b, which shows the results of phenotypic analysis, most of pDCs expressed CD123 strongly although they lacked CD11c expression. The proportion of CD123 ⁺ cells in the CD4 ⁺ cells always exceeded 90%, while C
D11c ⁺ cells were less than 7% and pDC2 was preferentially enriched. The exact reason for selective enrichment of pDC2 is unknown, but pDC1 expresses CD2 (Immuno
logy, 82, 487-493, 1994, J. Exp. Med. 178, 1067-10
78, 1993) removed by E-rosetting or because pDC1 has Ig adhesion (Immunology, 8
2, 487-493, 1994) The possibility of removal by Fc receptor panning is considered. As shown in FIG.
DC2 is HLA class I, HLA-DR, and CD54
Expressed. In addition, about 50% of pDC2 expressed CD40, but pDC2 did not express CD80 and CD86 at all or hardly expressed.

In order to measure the ability of pDC2 to stimulate T cells,
Monocytes, peripheral blood B cells, pDC2, DC1 and LBL (Epste
in-Barr virs-transformed lymphoblastoid B cell lin
Various self-APCs including e cells) were used as stimulating cells, and TT-induced proliferative responses in TT-specific CD4 ⁺ T cell lines K1 or H1 were examined. PDC as shown in FIG.
2 did not induce TT-specific T cell proliferation in various proportions of APC and T cells. This result shows that pDC2 isolated from peripheral blood is allotrophic
ory capacity) previous report (J. Immuno
l.165, 566-572, 2000, Blood, 95, 2484-2490, 200
0, J. Immunol. 163, 3250-3259, 1999).
Peripheral blood B cells, DC1 and LBL promoted the growth of TT-specific T cell lines, whereas monocytes immediately after isolation were poorly stimulating in the culture system described above.

(2-2: TT-specific CD by pDC2
Anergy induction of 4 ⁺ T cell lines) To examine whether antigen presentation by pDC2 induces T cell anergy,
T-specific CD4 ⁺ T cell line K1 was cultured with autologous pDC, monocytes, peripheral blood B cells, DC1 or LBL pulsed and irradiated with TT and re-challenge with autologous LBL in the presence of TT (FIG. 3). ). TT-specific T cells were primary cultured with antigen-pulsed pDC and A
Increase the number of PCs or increase the TT concentration, LBL and TT
No re-challenge was seen at all
(Figure 3a). Monocytes pulsed with TT, peripheral blood B cells, DC1
Alternatively, TT-specific T cells cultured with LBL showed a proliferative response in an antigen concentration-dependent manner (FIG. 3b). TT
Similar results were obtained from the specific CD4 ⁺ T cell lines K3 and H1. In FIG. 3c, non-irradiated pDC2 induced T cell unresponsiveness, indicating that pDC2 irradiation treatment was not necessarily required. By culturing pDC2 in the presence of IL-3, CD40, CD8
0 and CD86 expression is upregulated, IL
-3 treated pDC2 lacked the ability to induce T cell anergy.

(2-3: Intercellular mechanism of T cell anergy induced by pDC2) In order to examine the characteristics of the interaction between T cells and pDC2 generated during primary culture inducing T cell anergy, TT-specific CD4 ⁺ T cell line K
1 and H1 were cultured with pDC2 under various conditions and TT-induced T cell proliferation was assessed by re-challenge with LBL and TT. First, T
T-specific T cells can be transformed into autologous or allogeneic pDC2 (both HLA-D, under various ratios of APC and T cells).
Cultured with RB1 allele incompatibility (FIG. 4a). T
Cell unresponsiveness is indicated when the ratio of APC to T cells is 1: 300.
Was induced by self-pDC2 over a wide range up to. Since allogeneic pDC2 did not induce T cell anergy, TCR-MHC was used to induce T cell anergy.
It was found that contact between cells was necessary.

Next, TT-specific T cells were cultured with autologous pDC2 in the presence or absence of various antigens (FIG. 4).
b). TT-specific T cells cultured with pDC2 pulsed with TT did not proliferate in secondary culture, but TT-specific T cells pretreated with pDC2 without antigen or with pDC2 supplemented with an irrelevant antigen were Remarkable growth was observed in the secondary culture. This indicated that the T cell anergy induced by pDC2 is antigen dependent and antigen specific. Furthermore, the blocking effect of the interaction between TCR and MHC class II molecules generated during T cell anergy induction was examined using a monoclonal antibody specific for HLA-DR or DQ (FIG. 4c). Anti-HLA-DR monoclonal antibody is a TLA in HLA-DR restricted K1 strain.
Although the cellular anergy induction was completely blocked, both HLA-DR and HLA-DQ monoclonal antibodies
Anergy induction in H1 strains that are DR and HLA-DQ restricted was partially blocked, respectively. The inhibitory effect was dependent on the dose as determined using various concentrations (0.1-5 μg / ml) of anti-HLA-DR monoclonal antibodies.

Finally, to examine the possibility of T cell anergy induction by a small number of HLA-DR ⁺ CD4 ⁻ cells or pDC1 in the pDC2 enriched fraction, using magnetic beads conjugated with monoclonal antibodies, CD4 ⁺ cells, CD
8 ⁺ cells, CD11c ⁺ cells or CD123 ⁺ cells
DC2 enriched fraction is removed and the removed fraction is TT-specific T
Involvement of pDC1 or pDC2 in T cell anergy induction was examined by primary culture with cells (FIG. 4).
d). From the pDC2 enriched fraction, CD4 ⁺ cells or CD1
When 23 ⁺ cells were removed, the induction of T cell anergy was completely suppressed, but when CD8 ⁺ cells or CD11c ⁺ cells were removed, the induction was not suppressed. The above experimental results show that CD4 ⁺ HLA-DR ⁺ CD11c ⁻ CD12
It shows that T cell anergy induction is mediated by T cell recognition of antigenic peptides presented by MHC class II molecules on 3 ⁺ pDC2.

TT-specific T cell lines K1 and TT or IL
P prepared by stimulating non-irradiated pDC2 with -3
DC2 culture supernatant was used to investigate the role of soluble factors in pDC2-induced T cell anergy. These p
The DC2 culture supernatant did not suppress stimulation by autologous B cells in the presence of TT or the proliferative response of peripheral blood T cells induced by anti-CD3 monoclonal antibodies. Furthermore, peripheral blood T cells primary cultured in pDC2 culture supernatant with autologous B cells and TT proliferated upon re-challenge with B cells pulsed with TT. This means that pDC2
It is shown that the soluble factor produced from is not sufficient to induce T cell anergy.

The TT-specific T cell line K1 was cultured with TT-pulsed autologous pDC for 3 days, and the expression of CD154 (CD40 ligand) in the K1 line was examined before and after the culture. As shown in FIG. 5, pDC2 pulsed with TT
And T cell cultures did not induce up-regulation of CD154, while CD154 expression was significantly upregulated after incubation with TT-pulsed LBL. Furthermore, the expression of CD80 and CD86 in non-irradiated pDC2 under co-culture with TT-specific T cell line K1
Measured in the presence or absence of T. After 3 days of culture, few CD3 ^- live cells were recovered, and no enhanced CD80 and CD86 expression could be detected in the CD3 ^- cell fraction, regardless of the presence or absence of antigen in the primary culture.

(2-4: Recovery of pDC2-induced T cell anergy by IL-2) It has been reported that antigen stimulation in the presence of exogenous IL-2 partially or completely restores the proliferation ability of anergy T cells. (J. Exp. Med. 184, 1-8,
1996, Immunology, 64, 413-417, 1988). To examine whether IL-2 can restore the angiogenic state of T cells induced by antigen-pulsed pDC2 TT
Specific CD4 ⁺ T cell lines K1 and H1 were cultured with autologous pDC2 or LBL pulsed with TT and various concentrations of I
Re-challenge was performed using LBL and TT in the presence of L-2. As shown in FIG. 6a, 400 U / ml
Or higher concentrations of IL-2 induced complete recovery of the anergy state in the K1 strain, but the presence of 50 U / ml IL-2 during the induction of anergy did not affect. This indicates that high concentrations of IL-2 are required to recover pDC2-induced T cell anergy. In contrast, even in the presence of high concentrations of IL-2,
The recovery of the growth disorder of the H1 strain was partial. Anergy T
To examine the effects of various stimuli on cell recovery, TT-specific CD4 ⁺ T cell line K1 cultured with autologous pDC2 pulsed with TT in primary culture was re-challenged in various culture conditions (FIG. 6b). PMA
And ionomycin coexist with the anti-CD3 monoclonal antibody and IL-2 (400 U / ml),
Induced complete recovery of T cell anergy. In contrast, anti-CD28 monoclonal antibodies combined with antigen stimulation or anti-CD3 monoclonal antibodies showed no effect. IL-2 in the absence of stimulation via TCR
Alone did not induce the recovery of Anergy T cells. This result indicates that the recovery of pDC2-induced T cell anergy is TC
It indicates that both an R-mediated activation signal and a high concentration of IL-2 are required. TT-specific CD4
⁺ Similar results were obtained with the T cell line K3.

(2-5: Cytokine profile in anergy T cells) T cell anergy induction is induced by IL-2.
Related to blocking the production of certain cytokines such as J. Exp. Med. 184, 1-8, 1996, I
mmunology 64, 413-417, 1988), p pulsed with antigen
T that became anergy by culturing with DC2
Cytokine production by cells was examined. TT-specific CD
4 ⁺ T cell lines K1 (Th0) and H1 (Th1) were cultured with LBL or pDC2 in the presence of TT and stimulated with anti-CD3 monoclonal antibody and PHA. As shown in FIG. 7, after incubation with pDC2 pulsed with TT,
Production of γ, IL-2 and IL-4 was suppressed. IL-
2 synthesis was not detected by TCR-mediated activation with PMA and ionomycin, and T cells that became anergy when cultured with pDC2 pulsed with antigen lacked IL-2 production ability . In contrast, IL-10 production is maintained, and IL-10 production is Th0.
In the stock K1, it was rather increased.

(2-6: Anergy induction of topoI-specific CD4 ⁺ T cell clones by pDC2) Next, pDC2 induces an anergy state in activated autoreactive CD4 ⁺ T cell clones derived from scleroderma patients. I checked what could be done. TopoI-specific CD4 ⁺ T cell clones are cultured with autologous pDC2, monocytes or LBL pulsed with topoI and then subcultured with LBL and topo.
Re-challenge was performed using I (FIG. 8). TopoI-specific T cell clones T2-5 and T2-6 cultured with pDC2 pulsed with topoI did not proliferate against topoI in secondary cultures, but were pretreated with LBL or monocytes pulsed with topoI T2-5 and T
2-6 grew on rechallenge. T cell anergy induction is induced by CD1 from fractions rich in pDC2.
Instead of removing 1c ⁺ cells, it was inhibited by removing CD123 ⁺ cells. PDC without antigen
PDC2 pulsed with two or irrelevant antigens did not induce T cell anergy. The combination of antigen stimulation and high concentrations of exogenous IL-2 (800 U / ml) in secondary culture restored the anodynic state of topoI specific T cells. During primary culture with pDC2 pulsed with topoI, addition of anti-HLA-DR monoclonal antibody blocked the induction of T cell anergy.

A T cell helper function that induces the production of anti-topoI antibodies by autologous B cells was pre-cultured with pDC2, monocytes or LBL pulsed with topoI.
It was examined by culturing oI-specific CD4 ⁺ T cell clones (FIG. 9). TopoI-specific T cell clones lacked their helper function after treatment with antigen-pulsed pDC2, but their helper function was not affected when cultured with antigen-pulsed monocytes or LBL. T cell helper function was restored by removal of CD123 ⁺ cells from the pDC2 fraction, but not by removal of CD11c ⁺ cells. The lack of anti-topoI antibody production in media containing Anergy T cells is due to exogenous IL in secondary cultures.
-2 (800 U / ml) was fully recovered.

The cytokine profile of topoI-specific CD4 ⁺ T cells that became anergy by culturing with pDC2 was examined. TopoI specific CD4
⁺ Th0 clones T2-5 and T2-6
Cultured with LBL or pDC2 in the presence or absence of poI and re-challenge with LBL and topoI.
The results are shown in Table 1. In Table 1, ND indicates that measurement was not performed. After incubation with pDC2 in the presence of topoI, IL-2 production was completely blocked, but I
FN-γ production was not significantly affected. Interestingly, IL-10 production was promoted in Anergy T cells, showing a cytokine profile indicating expression of IFN-γ and IL-10. In addition, activation by PMA and ionomycin without TCR causes IFN-
Expression of γ and IL-10 was induced, but IL-2 synthesis was not induced.

[0046]

[Table 1]

[0047]

According to the present invention, since the immune response to a specific target antigen can be selectively suppressed, it has little influence on normal immune function, and the antigen of the immune reaction associated with the pathological condition is known. For example, it can be applied to any disease, and since the cells in the peripheral blood are used as they are, the manipulation is simple, such as the need for genetic manipulation or treatment with drugs, etc. It is possible to provide a selective immune response suppressing therapeutic that does not require identification of the required T cell epitope and can be treated for all patients regardless of the patient's HLA type.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of analysis by flow cytometry of a fraction enriched in pDC. (A) Cells stained with anti-HLA-DR labeled with FITC and anti-CD4 monoclonal antibody labeled with PE (right) and cells stained with an isotype-matched control monoclonal antibody labeled with FITC and PE (left) are shown. (B, c) shows molecules expressed on the surface of pDC immediately after isolation. Cells in the pDC fraction were stained with anti-CD4 monoclonal antibody and several monoclonal antibodies. The CD4 ⁺ cells were gated and measured by flow cytometry. The expression of surface molecules is shown in the shaded area of the histogram. Uncolored histograms show controls stained with an isotype matched control monoclonal antibody. The results shown here are those obtained from at least two experiments.

FIG. 2 shows the antigen-specific proliferative response of TT-specific CD4 ⁺ T cell lines induced by various autologous APCs. Monocytes, peripheral blood B cells, pDC2, D as stimulating cells for TT-specific T cells K1 and H1 in the presence of TT
C1 and LBL ratios are increased and cultured.
T cell proliferation induced by T was measured by ³ H-thymidine incorporation. One of three experiments showing the same results is shown.

FIG. 3: Various types of irradiated self-AP pulsed with TT
FIG. 2 is a graph showing an antigen-induced proliferation response of a TT-specific CD4 ⁺ T cell line pre-cultured with C AP pulsed with TT
After primary culture with C, the ratio of APC to T cells was 1: 3, and TT and autologous LBL were used to re-challenge the TT-specific T cell line, K1, to increase T cell proliferation by ³ H-thymidine. It was measured by the amount of uptake. (A) T cells with various types of irradiated APC, including monocytes, peripheral blood B cells, pDC2, DC1 and LBL,
The cells were cultured in the presence of TT (5 μg / ml) at various ratios of PC and T cells. (B) T cells are transformed into monocytes, peripheral blood B cells, pDC2 and LB
Along with L, the ratio of APC to T cells was 1: 3 and the cells were cultured in the presence of various concentrations of TT. (C) LBL, monocytes, pDC2, and IL-3 treated pD
T cells with various types of irradiated APC, including C2, were cultured at a ratio of 1: 3 in the presence of TT (5 μg / ml). Non-irradiated pDC2 was also used as APC for primary culture. Furthermore, the ratio of APC to T cells was 1: 3, and T cells were re-challenged in the presence (black box) and absence (white box) of TT.

FIG. 4: TT cultured with pDC2 under various culture conditions
FIG. 2 shows the antigen-induced proliferative response of a specific CD4 ⁺ T cell line. After primary culture, the ratio of APC to T cells was 1:
3 and using TT and autologous LBL, TT-specific T cell lines K1 and H1 are re-challenged and T cell proliferation is increased by ³ H-
It was measured by the amount of thymidine incorporated. The results shown here are those obtained from at least two experiments. (A) T cells are expressed in the ratio of APC and T cells shown in the figure,
Self-LBL pulsed with T, self-pDC2, or similar pD
Incubated with C2. (B) T cells are treated with antigen or TT (1 and 5 μg / m
l), MalBP (10 μg / ml), or topo
Cultured with autologous pDC2 without addition of autologous pDC2 pulsed with I (10 μg / ml) or antigen. (C) T cells were cultured with autologous pDC2 pulsed with TT in the presence or absence of anti-HLA-DR, anti-HLA-DQ, or isotype matched control monoclonal antibody. (D) TT-pulsed self p excluding CD4 ⁺ cells, CD8 ⁺ cells, CD11c ⁺ cells, or CD123 ⁺ cells
T cells were cultured with the DC2 fraction.

FIG. 5: CD154 in TT-specific T cells after culture with autologous LBL and pDC2 pulsed with TT.
FIG. 3 shows the expression of CD28. TT-specific T cell line K1 was cultured for 3 days with autologous LBL or pDC2 pulsed with TT. CD3 ⁺ cells were then collected and subjected to flow cytometric analysis. Molecular surface expression is indicated by the shaded portion of the histogram, and the non-shaded histogram indicates the control stained with the isotype matched control monoclonal antibody. The figure shows a representative of the results of three experiments.

FIG. 6: TT in anergy using exogenous IL-2
It is a figure which shows the recovery | restoration of a specific T cell line. (A) In order to induce the angiogenic state of TT-specific T cell lines K1 and H1, the ratio of APC to T cells was 1:10 (black squares, black circles) and cultured with autologous pDC2 pulsed with TT. On the other hand, K1 and H1 were cultured with autologous LBL pulsed with TT at a ratio of APC to T cells of 1: 3 (white squares, white circles). Next, under various concentrations of exogenous IL-2, the ratio of APC to T cells was 1: 3, T cells were re-challenged with autologous LBL and TT, and T cell proliferation was increased by ³ H-thymidine. It was measured by the amount taken up. (B) The ratio of APC to T cells is 1:10 and cultured with autologous pDC2 pulsed with TT,
The anergy state of the T-specific T cell line K1 was induced. Next, exogenous IL-2 (400 U / ml) or anti-CD28
AP in the presence or absence of monoclonal antibodies
T cells were re-challenged with autologous LBL and TT so that the ratio of C to T cells was 1: 3. PMA and ionomycin, anti-CD3 monoclonal antibody and anti-CD
28 monoclonal antibody, anti-CD3 monoclonal antibody and IL-2 (400 U / ml), or IL-2 (400
T cells were stimulated with U / ml only. A representative of the results of three experiments is shown in the figure.

FIG. 7 shows the cytokine profile of a TT-specific CD4 ⁺ T cell line that has become anergy by culturing with autologous pDC2 pulsed with TT. TT-specific C
D4 ⁺ T cell lines K1 (Th0) and H1 (Th1)
Is cultured with LBL or pDC2 in the presence of TT,
Next, it stimulated with the anti-CD3 monoclonal antibody and PHA. IFN-γ, IL-2, IL- in the culture supernatant
4, and the amount of IL-10 was measured by ELISA.
One of three experiments showing the same result is shown in the figure.

FIG. 8 topoI-specific CD4 ⁺ cultured with autologous APC pulsed with topoI under various culture conditions.
FIG. 4 shows an antigen-induced proliferative response of a T cell clone. The ratio of APC to T cells is 1: 3, or 1: with the LDC pulsed with topoI, monocytes, pDC2, pDC2 fraction excluding CD123 ⁺ cells, or pDC2 fraction excluding CD11c ⁺ cells. 30
The cells were cultured in the presence or absence of anti-HLA-DR or anti-HLA-DQ monoclonal antibody (primary culture). Next, A
The ratio of PC to T cells was 1: 3 and IL-2 (800 U
/ Ml) in the presence or absence of self LBL and topo
Using I, the T cells were re-challenged and T cell proliferation was measured by the amount of ³ H-thymidine incorporation. One of three experiments showing the same results is shown.

FIG. 9: TopoI-specific CD4 ⁺ T pre-cultured with autologous APC plus topoI under various culture conditions.
It is a figure which shows the inducibility which produces the anti- topoI antibody of a cell clone. APC to T cell ratio of 1: 3 or 1: 3
0, LBL pulsed with topoI, monocyte, pDC
2, pDC2 fraction excluding CD123 ⁺ cells, or CD
T cells were cultured with the pDC2 fraction excluding 11c ⁺ cells (primary culture). Next, T cells were re-challenge using autologous peripheral blood B cells and topoI in the presence or absence of IL-2, and the amount of anti-topoI antibody in the culture supernatant was measured by ELISA. The average amount of anti-topoI antibody in the culture supernatant of only peripheral blood B cells is 0.017
(OD ₄₀₅ ). 1 of 3 experiments showing the same results
Indicates one.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B065 AA94X AB10 AC20 BA21                       BD50 CA44                 4C087 AA01 AA02 BB34 CA04 NA05                       NA14 ZB07 ZB13

Claims (11)

[Claims] 1. Derived from human peripheral blood, CD4 on the cell surface
^A subset of dendritic cells having a surface marker of ⁺ HLA-DR ⁺ and stimulated with a target antigen in vitro,
A peripheral blood dendritic cell subset, wherein an anergy can be induced in the T cell by contacting with the T cell having immunoreactivity to the target antigen. 2. The peripheral blood dendritic cell according to claim 1, wherein the dendritic cell subset derived from peripheral blood is a dendritic cell subset that has been subjected to a treatment for inhibiting differentiation into mature dendritic cells. Subset. 3. The peripheral blood dendritic cell subset according to claim 2, wherein the treatment for inhibiting differentiation into mature dendritic cells is irradiation treatment. 4. Angiogenic T cells are IL-
The peripheral blood dendritic cell subset according to any one of claims 1 to 3, which has 10 production ability. 5. The dendritic cell subset derived from peripheral blood is a plasmacytoid dendritic cell or a type 2 dendritic cell precursor having a surface marker of CD4 ⁺ HLA-DR ⁺ CD11c ⁻ CD123 ⁺ The peripheral blood dendritic cell subset according to any one of claims 1 to 4. 6. Separating a mononuclear cell fraction from human peripheral blood, removing T cells and monocytes from the mononuclear cell fraction,
3, CD4 ⁺ HLA− characterized by removing cells with these surface markers using monoclonal antibodies against CD14, CD19, CD34 and CD56
Preparation of peripheral blood dendritic cell subsets with DR ⁺ surface markers. 7. The CD4 according to claim 6, which comprises a step of proliferating peripheral blood dendritic cells treated with G-CSF.
⁺ Preparation of peripheral blood dendritic cell subsets with HLA-DR ⁺ surface markers. 8. An anergy T cell characterized by an anergy upon contact with the peripheral blood dendritic cell subset according to any one of claims 1 to 5. 9. The anergy T cell according to claim 7, which has IL-10 production ability. 10. A composition having a selective immune response suppressing action, comprising the peripheral blood dendritic cell subset according to any one of claims 1 to 5 and / or the anergy T cell according to claim 9. 11. A method for treating a disease caused by an excessive immune response, comprising administering the composition having a selective immune response suppressing action according to claim 10.