JP2019158687A - Detection and preparation of effector control t-cell - Google Patents

Abstract

To provide means for finding a marker molecule having high effector control T-cell selectivity to specifically detect an effector control T-cell.SOLUTION: An effector control T-cell is detected from among samples containing T-cells using, as an indicator, expression of (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive by immunohistochemistry while combining expression of a chemokine receptor CXCR and expression of a transcription factor FOXP3 or CD25.SELECTED DRAWING: None

Description

  The present invention relates to the detection of effector regulatory T cells. Specifically, the present invention relates to a method for detecting effector regulatory T cells using a molecular marker and its use.

  Effector regulatory T cells (eTreg) have a strong immunosuppressive activity and play an important role in suppressing excessive immune responses. It is widely involved in the suppression of immune responses in vivo, such as the suppression of immune responses in infections, suppression of autoimmunity, and suppression of tumor immunity, and is deeply involved in the pathology of various diseases. As a method for measuring eTreg, a method using the expression of FOXP3, which is a Treg master gene product, as an index is widely practiced (see, for example, Non-Patent Document 1). It has been reported that CD4 positive T cells having functions are also expressed (see, for example, Non-Patent Documents 2 and 3), and establishment of a FOXP3 positive subset molecular marker reflecting Treg is important.

Clin Cancer Res. 2012 Jun 1; 18 (11): 3022-9. Immunity. 2009 Jun 19; 30 (6): 899-911. Int J Cancer. 2017 Feb 1; 140 (3): 686-695. Proc Natl Acad Sci U S A. 2015 Jun 9; 112 (23): 7225-30.

  Until now, it has been reported that CD25, CTLA4, CCR4 and the like serve as markers for eTreg (see, for example, Non-Patent Document 4), but the selectivity is not necessarily high. Accordingly, an object of the present invention is to find a marker molecule having high eTreg selectivity and to provide means for specifically detecting eTreg.

In order to solve the above problems, the present inventors have advanced research using clinical specimens (derived from head and neck cancer patients). After intensive studies, we found that the chemokine receptor CXCR6 (CXC chemokine receptor type 6) is an excellent FOXP3-positive subset molecular marker that reflects eTreg. In other words, if CXCR6, a marker specific to eTreg, was successfully identified and FOXP3 was used in combination with CXCR6, eTreg could be detected and identified with high selectivity and specificity from a population of CD4-positive T cells. It became clear that it would be possible. Similarly to FOXP3, CD25 also enables selective / specific detection of eTreg when used in combination with CXCR6. Based on the above results and considerations, the following inventions are provided.
[1] Detecting effector-regulated T cells from a sample containing T cells, characterized by combining the expression of the chemokine receptor CXCR6 (CXC chemokine receptor type 6) with the expression of the transcription factor FOXP3 or CD25 Method.
[2] The effector regulatory T cells are detected using the following marker characteristics, that is, (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive, as an index. Detection method.
[3] The detection method according to [2], comprising the following steps (1) to (3):
(1) identifying and fractionating a lymphocyte population from a sample containing T cells,
(2) identifying and fractionating a cell population containing regulatory T cells and not containing cytotoxic T cells from the fractionated lymphocytes;
(3) detecting a cell exhibiting the following marker characteristics in the cell population: (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive as an effector regulatory T cell.
[4] The detection method according to [3], wherein the cell population specified in step (2) is a cell population showing the following marker characteristics: (i) CD4 positive and (ii) CD8 negative. .
[5] The cell population identified in step (2) is a cell population that exhibits the following marker characteristics: (i) CD4 positive, (ii) CD8 negative, and (iii) CD45RA negative. ] The detection method of description.
[6] The detection method according to [3] or [4], wherein in step (3), cells that further exhibit the following marker characteristics, that is, (iii) CD45RA negative are detected as effector regulatory T cells.
[7] The detection method according to [3] or [4], further comprising a step of confirming that the detected cell is CD45RA negative.
[8] The detection method according to any one of [3] to [7], wherein the steps are performed by flow cytometry.
[9] The detection method according to [1] or [2], wherein the effector regulatory T cells are detected by immunohistochemistry.
[10] The detection method according to [9], wherein staining with CXCR6 as a target antigen and staining with FOXP3 as a target antigen are performed, and cells positive in both stainings are detected as effector regulatory T cells.
[11] A method for preparing an effector regulatory T cell, comprising the step of sorting the effector regulatory T cell detected by the method according to any one of [1] to [8].
[12] Effector-regulated T cells obtained by the preparation method of [11].

Analysis of effector-regulated T cells (eTreg) in lymph node lymphocytes derived from head and neck cancer 1 Analysis of effector-regulated T cells (eTreg) in head and neck cancer-derived lymph node lymphocytes 2

  The first aspect of the present invention relates to a method for detecting effector regulatory T cells (sometimes abbreviated as “eTreg”) (hereinafter also referred to as “the detection method of the present invention”). An “effector regulatory T cell” is an activated regulatory T cell and exhibits a strong immunosuppressive activity. The detection method of the present invention is characterized in that expression of the chemokine receptor CXCR6 (C-X-C chemokine receptor type 6) and expression of the transcription factor FOXP3 or CD25 are used in combination. In other words, there are roughly two types of marker molecules: CXCR6 (referred to as “first marker molecule” for convenience) and FOXP3 or CD25 (referred to as “second marker molecule” for convenience). One of the greatest features of the present invention is that eTreg is detected by using. As shown in the examples described later, as a result of the study of the present invention, the combined use of CXCR6 and FOXP3 or the combined use of CXCR6 and CD25 becomes a subset molecular marker specific to eTreg, CXCR6 positive and FOXP3 strong positive, It was revealed that eTreg can be detected with high selectivity using CXCR6 positive and CD25 strong positive as indicators. In addition to this finding, based on the fact that the expression of FOXP3 and the expression of CD25 are correlated, the detection method of the present invention is an indicator that (i) CXCR6 positive, and (ii) FOXP3 strong positive or CD25 strong positive. ETreg is detected as

  The expression state of each marker molecule can be detected or confirmed using a marker molecule specific antibody. For example, labeling with a marker molecule-specific labeled antibody and detection of the label, or two-step labeling using a marker molecule-specific unlabeled antibody (primary antibody) and a secondary antibody (labeled antibody) and By detecting the label, the expression of each marker molecule can be detected.

  In the detection method of the present invention, FOXP3 is preferably used as the second marker molecule from the viewpoint of accuracy, specificity, etc. (that is, eTreg is detected using both CXCR6 expression and FOXP3 expression). On the other hand, when the cells (eTreg) are separated after detection (ie, the preparation method described later) and used for other purposes (preparation of transplantation material, research, etc.), CD25 may be used as the second marker molecule. (That is, eTreg is detected using both CXCR6 expression and CD25 expression and fractionated). Unlike FOXP3 detection, CD25 detection does not require cell membrane permeabilization. Therefore, when CD25 is employed as the second marker molecule, it is possible to recover eTreg that is not damaged by membrane permeabilization.

  CD25 is one of the subsets of IL-2 receptors and constitutes interleukin 2 receptor together with CD122 which is an interleukin 2 receptor β chain and CD132 which is an interleukin 2 receptor γ chain. CD25 is expressed in activated T cells, activated B cells, activated macrophages and the like, and is also known as a marker for regulatory T cells (Treg).

  In the detection method of the present invention, a sample containing T cells is used, and eTreg is detected from the sample. For example, a mononuclear cell fraction (PBMC) prepared from a body fluid such as peripheral blood, ascites, pleural effusion, a tissue such as a tumor tissue or a lymph node is used as a sample. In addition, PBMC can be obtained by removing plasma components, red blood cells, platelets and granulocytes from blood collected from a living body, for example, by density gradient centrifugation. PBMC is composed of lymphocytes such as T cells, B cells, NK (natural killer) cells, monocytes, and dendritic cells.

  On the other hand, in the present invention, a tissue piece (eg, tumor tissue, lymph node) collected from a living body, a structure obtained by culturing a tissue piece collected from a living body ex vivo (ie, tissue culture), or collected from a living body. Structure obtained by culturing cultured cells (cultivation under specific conditions after harvesting, processing of genetic manipulation (gene introduction, gene recombination, etc.) etc.) with other cells as necessary A body or the like may be used as a sample. In this case, preferably, eTreg in the sample is detected using immunohistochemistry. Details of detection using immunohistochemistry will be described later.

In the case of a sample containing a suspended or dispersed cell population such as a mononuclear cell fraction, typically, the following steps (1) to (3) are performed to detect eTreg in the sample.
(1) identifying and fractionating a lymphocyte population from a sample containing T cells
(2) identifying and fractionating a cell population that contains regulatory T cells and does not contain cytotoxic T cells from the fractionated lymphocytes
(3) detecting a cell exhibiting the following marker characteristics in the cell population: (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive as an effector regulatory T cell (eTreg)

step 1)
In this step, a lymphocyte population is identified and fractionated from samples prepared in advance containing T cells. Although not limited to this, flow cytometry may be used for each step of the present invention, including this step, because it can be performed with a simple operation and high accuracy can be obtained. Flow cytometry is one of the analytical methods (cytometry) for quantitatively measuring a large number of cells in a short time. The cell suspension / suspension is passed through a capillary tube to detect scattered light and fluorescence. It is a technique for analyzing cells. Flow cytometry operation and features, for example, experimental medicine separate lab must-have flow cytometry Q & A editor Michio Tomura is detailed on November 20, 2017.

  In flow cytometry, a sample is irradiated with a predetermined laser beam, and forward scattered light (FSC) and side scattered light (SSC) are detected. A population of lymphocytes can be identified based on the intensity of forward scattered light and the intensity of side scattered light. For example, when PBMC prepared from human peripheral blood is a sample, when a two-parameter histogram (cytogram) of FSC and SSC is created, it is divided into a lymphocyte population, a monocyte population, and a granulocyte population. Lymphocytes that are relatively small and have a simple internal structure are cell populations in which the intensity of forward scattered light and the intensity of side scattered light are both low. Therefore, in this step, the cell population is usually designated (gating). The lymphocytes thus fractionated are subjected to the next analysis (step (2)).

  Although it is not essential to detect the forward scattered light and the side scattered light at the same time, they are usually detected at the same time from the viewpoint of easy operation and efficient detection. According to the existing flow cytometer, it is possible to simultaneously detect forward scattered light and side scattered light and analyze the intensity of each.

Step (2)
In step (2) following step (1), a cell population containing regulatory T cells and not containing cytotoxic T cells is identified and fractionated from the fractionated lymphocytes. In other words, a cell population that excludes cytotoxic T cells from the lymphocytes fractionated in step (1) and does not contain cytotoxic T cells (usually in addition to regulatory T cells, helper T cells Also). When using flow cytometry, for example, step (2) can be performed using CD4 expression and CD8 expression as indices. Typically, a cytogram is created by developing CD4 and CD8, and a CD4 positive and CD8 negative cell population is designated (gating). The cell population showing the CD4 positive and CD8 negative fractionated in this manner is subjected to the next analysis (step (3)). In one embodiment, in this step (2), CD45RA, which is used as an eTreg marker, is also used to improve the uniformity or purity of the detected eTreg. Specifically, a CD4 positive, CD8 negative, and CD45RA negative cell population is identified, and the cell population is subjected to the next analysis (step (3)). In this case, for example, the target cell population can be specified by the following two-stage fractionation. First, the lymphocytes fractionated in step (1) are expanded with CD45RA and side scattered light, and a cell population with negative CD45RA negative and low side scattered light intensity is designated. Next, the designated cell population is expanded with CD4 and CD8, and a CD4 positive CD8 negative cell population is specified.

Step (3)
In step (3) following step (2), eTreg is roughly detected by two types of markers. Specifically, CXCR6 is used as the first marker molecule, FOXP3 or CD25 is used as the second marker molecule, and CXCR6 positive and FOXP3 strong positive in the cell population fractionated in step (2) (second marker molecule) As ET cells) or CD25 strong positive cells (when CD25 is used as the second marker molecule) is detected as eTreg. CXCR6 is expressed mainly in cells of the immune system such as T cells, NK cells, and B cells, shows chemotaxis to the ligand CXCL16, and is used to migrate and infiltrate these immune system cells into various tissues. Involved. FOXP3 is a Treg master gene and is essential for Treg differentiation and functional expression. CD25 is selectively expressed on Treg, but is also expressed on activated T cells, NK cells, and B cells, and is involved in the differentiation and proliferation of these immune system cells.

  In step (3), CD45RA is also used as a marker, and in addition to (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive, (iii) CD45RA negative cell population is detected as eTreg. Also good. Rather than using CD45RA in step (3), it may be confirmed after step (3) that the detected cells are CD45RA negative. When CD45RA is used in step (3) or after that, normally, CD45RA is not used in step (2).

  When flow cytometry is used, labeling necessary for performing each step is usually performed prior to steps (1) to (3). A specific example of the labeling operation is shown below. First, a fluorescently labeled anti-CD4 antibody, a fluorescently labeled anti-CD8 antibody, and a fluorescently labeled anti-CXCR6 antibody are added to a sample prepared in advance and incubated (labeling by contact with the labeled antibody). When CD45RA is also used as a marker, a fluorescently labeled anti-CD45RA antibody is also added, and labeling is performed targeting CD45RA. Further, when CD25 is employed as the second marker molecule, a fluorescently labeled anti-CD25 antibody is also added, and labeling is performed using CD25 as a target. On the other hand, when FOXP3 is employed as the second marker molecule, after the above-mentioned labeling, fixation with formalin or the like and subsequent membrane permeation treatment are performed, and then a fluorescently labeled anti-FOXP3 antibody is added and incubated.

Examples of fluorescent molecules used for fluorescent labeling of each antibody are APC-Cy ^™ 7, BV511 ^™ , PerCP, BV421 ^™ , PE, Alexa488. Fluorescent molecules are selected so that they can be detected and identified by flow cytometry. Various combinations are possible, but examples of combinations are given below.
Anti-CD4 antibodies of the label BV421 ^TM of labeled BV511 ^TM using anti-CD8 antibody of PerCP to label use anti-CD25 antibody labeled using PE anti CXCR6 antibody labeling using anti-CD45RA antibody APC-Cy ^TM 7 Use Alexa488 to label anti-CD25 or anti-FOXP3 antibodies

The APC-C ^™ y7-labeled anti-CD45RA antibody is, for example, from BD Bioscience, the BV511 ^™ -labeled anti-CD4 antibody is, for example, from BD Bioscience, the PerCP-labeled anti-CD8 antibody is, for example, from BD Bioscience, and the BV421 ^™ -labeled CXCR6 antibody is, for example, from BD Bioscience. PE-labeled CD25 antibody is available from, for example, BD Bioscience, and Alexa488-labeled anti-FOXP3 antibody is available from, for example, BD Bioscience.

Here, a specific example (one example) of the procedure and operation of detection using flow cytometry is shown below.
(1) Separate a mononuclear cell fraction from a biological sample (peripheral blood, body fluid such as ascites, pleural effusion, tissue, etc.). (2) Separate the mononuclear cell fraction with labeled antibodies (BV511 ^™ labeled anti-CD4 antibody, PerCP labeled anti-CD8 antibody, BV421 ^™ labeled anti-CXCR6 antibody and APC-Cy ^™ 7 labeled anti-CD45RA antibody) at 4 ° C for 20 minutes. React.
(3) Wash once with PBS (0.2% BSA).
(4) Incubate for 30 minutes at 4 ° C using a commercially available fixative.
(5) Wash twice with a commercially available cell penetrant.
(6) In a commercially available cell permeation solution, the immobilized mononuclear cells are reacted with Alexa488-labeled anti-FOXP3 antibody at 4 ° C. for 30 minutes.
(7) After washing once with a commercially available cell penetrant, measure using a flow cytometer.

  By the way, according to the detection method of the present invention, eTreg in a sample can be detected with high selectivity. Therefore, eTreg with high purity can be obtained by separating the detected eTreg. Thus, eTreg with high purity can be prepared by using the detection method of the present invention. Thus, as another aspect, the present invention provides a method for preparing eTreg, which comprises the step of fractionating eTreg detected by the detection method of the present invention. The eTreg obtained by the preparation method of the present invention can be used for its own research (for example, analysis of the characteristics / characteristics of eTreg, immunopathological analysis in various diseases such as cancer). It can also be used as cells used in various assays (for example, assays for evaluating the action / effect of drugs on eTreg) or as transplant materials (for example, application to treatment of autoimmune diseases, post-transplantation GVHD, etc.). .

  As described above, when a tissue piece or the like collected from a living body is a sample, preferably, eTreg in the sample is detected using immunohistochemistry. Even when immunohistochemistry is used, in the present invention, CXCR6 expression and transcription factor FOXP3 or CD25 expression are used in combination (in other words, (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive. ETreg is detected using the indication as an index). Specifically, staining using anti-CXCR6 antibody and staining using anti-FOXP3 antibody or anti-CD25 antibody are used in combination, and double-stained cells are detected as eTreg.

  In immunohistochemistry, either an enzyme antibody method or a fluorescent antibody method may be used. Further, a method (cocktail method) in which primary antibody reactions against a plurality of antigens simultaneously proceed by mixing primary antibodies derived from different animals may be used. The order of staining with CXCR6 as a target antigen and staining with FOXP3 or CD25 as an antigen is not particularly limited.

  Detection by immunohistochemistry can be performed by the following procedure. For convenience of explanation, staining using CXCR6 as an antigen is used as the first stage staining, and staining using FOXP3 or CD25 as an antigen is used as the second stage staining, but, of course, the order may be reversed.

(1) Pretreatment
(1-1) Fixing and embedding in paraffin Fix the sample with formalin, paraformaldehyde, anhydrous ethyl alcohol, etc. Then embedded in paraffin. Generally dehydrated with alcohol, treated with xylene, and finally embedded in paraffin. A specimen embedded in paraffin is sliced into a desired thickness (for example, 3 to 5 μm thick) and spread on a glass slide. In place of the paraffin-embedded specimen, an alcohol-fixed specimen, a dry-sealed specimen, a frozen specimen, or the like may be used.
(1-2) Deparaffinization Generally, it is treated sequentially with xylene, alcohol (for example, ethanol), and water (deionized water or purified water).
(1-3) Pretreatment (antigen activation)
If necessary, enzyme treatment, heat treatment and / or pressure treatment are performed for antigen activation. For example, a specimen (slide glass) is immersed in 10 mM Tris-HCl pH 9.0 and is subjected to autoclaving (105 ° C., 10 minutes). After autoclaving, cool naturally and wash with PBS.
(1-4) Other treatments When peroxidase is used as a labeling substance for staining, endogenous peroxidase activity may be removed by treatment with hydrogen peroxide. On the other hand, non-specific reaction may be inhibited by treatment with bovine serum albumin solution (for example, 1% solution) for several minutes to several tens of minutes. The following primary antibody reaction can be performed using an antibody solution containing bovine serum albumin or the like, and the treatment can be omitted.

(2) First stage staining
(2-1) Primary antibody reaction Anti-CXCR6 antibody diluted to an appropriate concentration is dropped onto a specimen on a slide glass, and then reacted for several tens of minutes to several hours (for example, at room temperature for 1 hour). After completion of the reaction, wash with an appropriate buffer such as phosphate buffer. The washing operation may be performed a plurality of times (for example, three times).
(2-2) Addition of labeling reagent Peroxidase (for example, HRP) and alkaline phosphatase (AP) are frequently used as labeling substances. A secondary antibody to which a labeling substance is bound is dropped onto a specimen on a slide glass, and then reacted for several tens of minutes to several hours (for example, one hour at room temperature). After completion of the reaction, wash with an appropriate buffer such as phosphate buffer. The washing operation may be performed a plurality of times (for example, three times).
(2-3) Coloring reaction Add color reaction and react. DAB (3,3′-diaminobenzidine), Betazoid DAB, Cardassian DAB, Vina Green ^™ , Romulin AEC, Bajoran Purple, Ferangi Blue ^™ , Vulcan Fast Red, Warp Red ^™, etc. can be used as the coloring reagent. For example, when DAB is used, it is colored brown when reacted at room temperature for 5 to 10 minutes. After color development, the specimen is thoroughly washed with tap water to remove the coloring reagent.
(2-4) Removal of antibody Heat treatment (for example, immersing specimen (slide glass) in 10 mM Tris-HCl, pH 9.0 boiled in a microwave oven) The antibody bound on the specimen is removed according to 2-2).

(3) Second stage staining
(3-1) Primary antibody reaction Anti-FOXP3 antibody (when FOXP3 is used as the second marker molecule) or anti-CD25 antibody (when CD25 is used as the second marker molecule) diluted to an appropriate concentration on the slide glass It is dropped on the specimen and then allowed to react for several tens of minutes to several hours (eg, 1 hour at room temperature). After completion of the reaction, wash with an appropriate buffer such as phosphate buffer. The washing operation may be performed a plurality of times (for example, three times).
(3-2) Addition of labeling reagent The secondary antibody is bound in the same manner as in (2-2) above.
(3-3) Coloring reaction Color is developed in the same manner as in (2-3) above. However, the coloring reagent is selected so as to obtain a color that can be distinguished from the color developed by the first-stage staining. An example of a combination of two color developing reagents that enable identification is the combination of DAB and Vina green ^™ . For example, when Vina green ^TM is used, it is colored green when reacted at room temperature for 5 to 10 minutes. After color development, the specimen is thoroughly washed with tap water to remove the color reagent.

(4) Nuclear staining, dehydration / transmission / encapsulation If necessary, nuclear staining is also performed. For example, nuclear staining is performed by reacting Meyer's hematoxylin for several seconds to several tens of seconds. Wash with running water and color (usually for a few minutes). In addition, a dehydration / clearance / encapsulation process is performed as necessary. For example, after dehydrating with alcohol, it is treated with xylene and finally sealed with synthetic resin, glycerin, rubber syrup or the like.

(5) Observation, recording and image analysis
Cells stained with 2 colors (1st stage staining and 2nd stage staining) are detected as eTreg. Used for recording (photographing, etc.) and image analysis as required.

1. Detection of eTreg in clinical specimens (from head and neck cancer patients) (1) Analysis of effector-regulated T cells (eTreg) in lymph node lymphocytes from head and neck cancer 1 (Fig. 1)
Lymphocytes isolated from lymph nodes removed from head and neck cancer patients, 6-fold staining (CD45RA / CD4 / CD8 / CD25 / CXCR6 / FOXP3) using monoclonal antibodies, and eTreg analysis by flow cytometry Went. Gate the lymphocyte fraction (A), expand the cells in the gate two-dimensionally with CD4 / CD8, further gate the CD4-positive cells (B), two-dimensionally the cells in the gate with CD45RA / FOXP3 Expanded. CD45RA ^- FOXP3 ⁺⁺ cell population becomes eTreg fraction (C bold line). Next, the same CD4 positive cell population was developed two-dimensionally with CXCR6 / CD25 and gated at five locations (D, a-e). When two-dimensional expansion was performed with CD45RA / FOXP3 in the gate a (CD25 ⁺⁺ CXCR6 ⁺ ), the cell population was concentrated in the eTreg fraction indicated by C (E). From this, it became clear that eTreg is selectively contained in the lymphocyte subset of CD4 ⁺ CD25 ⁺⁺ CXCR6 ⁺ .

(2) Analysis of effector-regulated T cells (eTreg) in head and neck cancer-derived lymph node lymphocytes 2 (Fig. 2)
Lymphocytes were isolated from lymph nodes removed from patients with head and neck cancer, and 6-fold staining (CD45RA / CD4 / CD8 / CD25 / CXCR6 / FOXP3) using monoclonal antibodies was performed, and eTreg was analyzed by flow cytometry. Went. Gate the lymphocyte fraction (A), expand the cells in the gate two-dimensionally with CD4 / CD8, further gate the CD4-positive cells (B), two-dimensionally the cells in the gate with CD45RA / FOXP3 Expanded. CD45RA ^- FOXP3 ⁺⁺ cell population becomes eTreg fraction (C bold line). Next, the CD4 positive cell population was developed two-dimensionally with CXCR6 / FOXP3 and gated at five locations (D, a-e). When two-dimensional expansion was performed with CD45RA / FOXP3 in the gate a (FOXP3 ⁺⁺ CXCR6 ⁺ ), the cell population was concentrated in the eTreg fraction indicated by C (E). From this, it became clear that eTreg is selectively contained in the lymphocyte subset of CD4 ⁺ FOXP3 ⁺⁺ CXCR6 ⁺ .

  Effector-regulated T cells (eTreg) are widely involved in the suppression of immune responses in vivo, and play an important role in the pathogenesis and progression of various diseases or in healing. The present invention that enables specific detection of such important cells can be expected to make a great contribution to the development or establishment of treatment / diagnosis techniques for immune system diseases.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (12)

  A method for detecting effector-regulated T cells from a sample containing T cells, which comprises using expression of a chemokine receptor CXCR6 (C-X-C chemokine receptor type 6) and expression of a transcription factor FOXP3 or CD25.   The detection method according to claim 1, wherein the effector-regulatory T cell is detected using as an index the following marker characteristics: (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive. . The detection method according to claim 2, comprising the following steps (1) to (3):
(1) identifying and fractionating a lymphocyte population from a sample containing T cells,
(2) identifying and fractionating a cell population containing regulatory T cells and not containing cytotoxic T cells from the fractionated lymphocytes;
(3) detecting a cell exhibiting the following marker characteristics in the cell population: (i) CXCR6 positive and (ii) FOXP3 strong positive or CD25 strong positive as an effector regulatory T cell.   The detection method according to claim 3, wherein the cell population identified in step (2) is a cell population exhibiting the following marker characteristics: (i) CD4 positive and (ii) CD8 negative.   4. The cell population identified in step (2) is a cell population exhibiting the following marker characteristics: (i) CD4 positive, (ii) CD8 negative, and (iii) CD45RA negative. Detection method.   The detection method according to claim 3 or 4, wherein in step (3), cells exhibiting the following marker characteristics, namely (iii) CD45RA negative, are detected as effector regulatory T cells.   The detection method according to claim 3 or 4, further comprising a step of confirming that the detected cell is CD45RA negative.   The detection method according to claim 3, wherein each step is performed by flow cytometry.   The detection method according to claim 1 or 2, wherein effector regulatory T cells are detected by immunohistochemistry.   The detection method according to claim 9, wherein staining with CXCR6 as a target antigen and staining with FOXP3 as a target antigen are performed, and cells that are positive in both stainings are detected as effector regulatory T cells.   A method for preparing an effector regulatory T cell, comprising the step of sorting the effector regulatory T cells detected by the method according to any one of claims 1 to 8.   The effector regulatory T cell obtained by the preparation method of Claim 11.

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