WO2004059319A1 - Procedes individuels de mesure d'une fonction lymphocytaire specifique - Google Patents

Procedes individuels de mesure d'une fonction lymphocytaire specifique Download PDF

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Publication number
WO2004059319A1
WO2004059319A1 PCT/CN2003/001120 CN0301120W WO2004059319A1 WO 2004059319 A1 WO2004059319 A1 WO 2004059319A1 CN 0301120 W CN0301120 W CN 0301120W WO 2004059319 A1 WO2004059319 A1 WO 2004059319A1
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cell
antigen
mhc
hla
tumor
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PCT/CN2003/001120
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English (en)
French (fr)
Inventor
Ge Chen
Xiangjun Liu
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Pel-Freez Biotechnology (Beijing) Ltd.
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Priority to AU2003292883A priority Critical patent/AU2003292883A1/en
Publication of WO2004059319A1 publication Critical patent/WO2004059319A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing

Definitions

  • the invention relates to a method for determining specific cellular immune function. This method is particularly useful for monitoring organ transplant recipients 'specific cellular immune function against transplants and tumor patients' body specific anti-tumor cell immune functions. Background technique
  • the immune system of a normal biological body is composed of humoral immunity and cellular immunity.
  • the precise coordination and mutual regulation between the two play an extremely important role in maintaining the normal life function and anti-disease immunity.
  • the central role of various lymphocytes, especially T lymphocytes, in the immune system has received widespread attention in recent years. T lymphocytes have been widely accepted as the "axis" of the body's anti-disease immunity and rejection immunity.
  • the prerequisite for specific immune response is specific immune recognition, which in turn depends on the integrity of the "dual signaling" mechanism.
  • the material basis for the "dual signal” recognition is:
  • the first signal of specific recognition is composed of the MHC (Most Histocompatibility Complex) gene expression product MHC molecules and specific peptide complexes; and the co-stimulatory factors (B7 family; B7-1 B7-2, etc.), and cell adhesion factors (CD40, etc.) constitute the second signal of immune recognition.
  • the activation, activation, regulation and immune tolerance of the immune response against disease or anti-graft rejection of the organism is closely related to the integrity of the above-mentioned "dual signal" system.
  • the non-existent antigen is first degraded into specific peptides by Antigen Presenting Cells (APC), and then assembled with the corresponding self-MHC molecules to form MHC -Specific peptide complexes, presented on the cell surface, and then co-stimulated by specific T lymphocyte clone specific receptors (T Cell Receptor; TCR) and co-stimulation with co-stimulatory factors and cell adhesion factors on the membrane Recognition and binding of factor receptors (CD28, etc.); thereby specifically activating T lymphocytes and their subpopulations, inducing comprehensive specific immunity in the organism Answer.
  • T lymphocyte clone specific receptors T Cell Receptor; TCR
  • CD28 factor receptors
  • Activated CD8 positive T lymphocytes are induced to form cytotoxic T Lympholytes (CTL) or killer T cells by MHC-I-polypeptide complex, which are used for cells or tumors infected with foreign antigens (such as viruses) Cells or organ transplant donor cells have direct specific killing effect; and MHC-II-peptide complex-induced and activated CD4 positive T lymphocytes or helper T lymphocytes (T Helper; TH) can secrete each This cytokine enhances or regulates the specific killing effect of CTL and other immune cell (such as macrophage activation) functions, and at the same time can stimulate B lymphocyte proliferation, differentiate into plasma cells, and secrete specific antibodies to initiate a humoral immune response.
  • CTL cytotoxic T Lympholytes
  • MHC-I-polypeptide complex-induced and activated CD4 positive T lymphocytes or helper T lymphocytes T Helper; TH
  • This cytokine enhances or regulates the specific killing effect of CTL and other immune
  • T cells especially CD4-positive T lymphocytes, have an "axial" role in the immune system of intact organisms; 2. Specific immune recognition of organisms is specific The basics and prerequisites of the immune response; 3. The completeness of the "dual signal” recognition mechanism determines the sexual shield of the specific immune response (ie, the specific immune "tolerance,” or "attack” effect).
  • transplant rejection When allogeneic tissues and organs are transplanted, the recipient's immune system often produces a transplant rejection (transplant rejection). This is a very complex immunological phenomenon involving multiple immune damage mechanisms mediated by cells and antibodies. Both are responses to the human major histocompatibility complex MHC (ie, HLA, Human Leucocyte Antigen) in the graft. The degree of difference in HLA between the donor and recipient determines the severity of the rejection. With the exception of monozygotic twins, the tissue matching of two individuals with identical HLA systems is almost non-existent, but choosing donors and recipients to match as closely as possible is the key to successful allogeneic organ transplantation.
  • MHC human major histocompatibility complex
  • HLA Human Leucocyte Antigen
  • the HLA system is currently the most complex gene group-HLA complex known to humans, or the coding product of the MHC gene. The presence of multiple alleles at each locus results in a high degree of polymorphism in the HLA system.
  • HLA-I class II genes HLA-I, class II antigens. All nucleated cells can express HLA-I antigens. HLA-II antigens are found in antigen-presenting cells, B cells, and T helper cells.
  • vascular endothelial cells, fibroblasts, and renal tubular epithelial cells can also express class II antigens if they are induced by IFN-Y.
  • T cell-mediated delayed Sensitization and cytotoxicity play an important role in graft rejection.
  • immunological workers from various countries have established a variety of methods and techniques for detecting the immunological functions of biological organisms. It mainly includes various humoral immune function detection methods characterized by measuring the production of specific antibodies, and cellular immune function detection methods that determine the number, activity, and proliferation of various types of immune cells.
  • the function of B lymphocytes can be detected by measuring the level of specific antibodies in body fluids.
  • the function of T lymphocytes against specific antigens is difficult to detect, because T cells have multiple subtypes with different functions, and their functions are also different.
  • Existing methods for detecting cellular immune function include: methods based on immune cell counting; methods based on detecting immune cell proliferation; methods based on detecting cytotoxic activity or secreting cytokines; and in vivo methods such as skin tests and adoptive transfer.
  • U.S. Patent No. 5,773,232 discloses a method for rapidly analyzing the activation of lymphocyte function.
  • the method includes stimulating a lymphocyte-containing sample in vitro with an antigen, and then isolating and lysing a specific subset of lymphocytes. Functional activation of lymphocytes.
  • the document mentions the use of tumor cell proteins or graft-derived proteins as stimulating antigens, but does not disclose the use of tumor cells or lysates of the individual to be tested or cells or lysates or MHC of the donor of the individual to be tested The molecule acts as a stimulating antigen. Therefore, this prior art method is not an individualized method.
  • MLC mixed lymphocyte culture
  • the invention provides a method for monitoring an individual's specific immune cell function to an organ transplant and a tumor in vitro.
  • the method includes 1) combining a test sample of the individual containing immune cells with an antigen from an organ transplant donor or an MHC / HLA molecular library containing the donor's MHC / HLA type, or with the individual's own tumor antigen Or similar tumor antigen libraries together for a period of time sufficient to activate immune cells, 2) isolating and / or classifying the cultured immune cells and determining their activation index; wherein the antigen from the organ transplant donor or the individual
  • the self-tumor antigen is a whole tumor cell or a cell extract containing most cellular material; the method further includes setting a control using the individual's own antigen or a non-tumor antigen as a stimulating antigen.
  • a MHC / HLA molecular library comprising a donor MHC / HLA type is used as a stimulating antigen, and the molecular library is preferably a MHC / HLA class I molecular library, a MHC / HLA class II molecular library, or MHC / HLA A hybrid molecular library of Class I and II molecules.
  • a homogeneous tumor antigen bank is used as the stimulating antigen, and the antigen bank is preferably a whole tumor cell antigen bank, Tumor subcellular antigen library or tumor antigen library composed of single tumor antigen or multiple purified components. More preferably, the antigen library includes common antigens of a certain type of tumor (such as adenocarcinoma, melanoma, etc.).
  • the antigen from the donor of the graft, the MHC / HLA molecular library, the auto-tumor antigen and the tumor antigen library of the present invention can be prepared at the time of use, or they can be prepared at one time. It is preferably prepared and stored at one time for convenient use at any time.
  • the test sample of the individual is peripheral whole blood, bone marrow, and any biological fluid containing immune cells.
  • the culture time between the test sample and the antigen is preferably 20 minutes to 24 hours, and more preferably 30 minutes to 6 hours.
  • the antigen used for the control is prepared in parallel with the stimulating antigen.
  • Acute, acute and chronic rejection is mainly a humoral immune response caused by antibodies and complements already present in the recipient against the donor HLA molecule.
  • Acute and chronic rejection is mainly mediated by the recipient's cellular immunity against the donor graft.
  • the recipient's T cells specifically and directly recognize the donor's HLA by direct and indirect means, and then activate and proliferate, inducing the recipient's specific rejection response against the graft.
  • the inventors of the present invention have discovered that donor cells for organ transplantation and all information including the donor containing the donor antigen or the graft antigen. Therefore, the antigen as a specific stimulator of an in vitro cellular immunoassay can more accurately reflect the activity of immune cells in an organ transplant recipient. Therefore, in the method for monitoring an individual's specific immune cell function to organ transplants and tumors in vitro of the present invention, donor cells or donor cell extracts containing donor MHC / HLA or donor MHC / HLA type MHC / The HLA molecular antigen library serves as an individualized specific stimulus. The actual results show that: for the recipient's immune cells, including the donor's immunogen stimulation properties, both can specifically activate the recipient's antigen-specific T cells. Cell activation.
  • the activation marker of the immune cells of the recipient is measured after being stimulated by the donor-specific antigen or MHC / HLA molecule, which can reflect the specific rejection status of the recipient's immune cells to the donor graft, thereby achieving rejection.
  • the antigen from the donor of the organ transplant is any tissue fine of the donor.
  • Subcellular components are components containing donor MHC / HLA molecules obtained by processing tissue cells through physical, chemical, biochemical, enzymatic, and other methods.
  • the subcellular component is a crude extract of a donor cell, such as a cell lysate, a cell lysate supernatant, a cell lysate precipitate, and the like.
  • an MHC / HLA molecular library containing a donor MHC / HLA molecule type is used as an antigen to stimulate the immune cells of the recipient.
  • the molecular library includes all and most of the graft's MHC / HLA molecules, which can be MHC / HLA class I molecular libraries, MHC / HLA class II molecular libraries, or molecules that are a mixture of MHC / HLA class I and MHC / HLA class II molecules Library.
  • MHC / HLA class I molecules can be used as stimulators to measure the function of the receptor CD8 + T cells (CTL), and MHC / HLA class II molecules can be used as stimulators to measure the function of the receptor CD4 + T cells (Th).
  • MHC / HLA molecular libraries can be obtained through a variety of methods and technologies from a variety of biological cells and biological materials.
  • phage and bacteria display biotechnology, affinity chromatography technology, DNA recombination technology, and DNA transfection technology.
  • Phage display has proven to be an advantageous technique with which a library of tens or even hundreds of millions of different peptides or proteins can be obtained, and has been applied to affinity screening of recombinant peptide libraries.
  • MHC / HLA proteins in the molecular library similar to the donor's immune phenotype can act directly on the T Cells can also be identified through indirect pathways: APC cells of recipients can process MHC / HLA molecules and present them to corresponding T lymphocytes (CTL, TH1, TH2, etc.) to induce their activation.
  • CTL T lymphocytes
  • the tested immune cells are the first contact with the donor's MHC / HLA molecules, their activation degree, response intensity is relatively low and weak within the set time; if the tested immune cells in the detection system have been used by the donor's MHC / HLA class I or II molecules, then those immune cells will be short-term It activates rapidly within time, and its activation degree and reaction are relatively strong. This is particularly significant in organ transplant rejection.
  • the immune cells of the transplant recipient are sensitized by the donor MHC / HLA molecule and are in a highly sensitive state. In an in vitro reaction system, immune cells again encounter the same type of MHC / HLA molecules, which is quickly activated and measured. Therefore, according to the presence or absence of the recipient's immune cells against the transplant-specific rejection response of the donor immune cells before and after transplantation, the strength and weakness.
  • the culture time of the test sample and the antigen is not particularly limited in the present invention, and may be 20 minutes to 7 days according to the activation index of the immune cells to be measured. In most and preferred applications of the method of the present invention, the culture time is relatively short, preferably 20 minutes to 24 hours, and more preferably 30 minutes to 6 hours. Determine early activation of immune cells. ,,,,,,,,
  • the lymphocytes in his body should never have been in contact with the donor ’s antigen and be in a “resting,” or non-sensitized state; if the donor ’s MHC / HLA Molecules stimulate recipient immune cells in vitro to induce slow but weak (relative to transplantation) activation of immune cells, which is equivalent to the first contact between the immune system and non-antigen.
  • the method described in the present invention can be used as one of the basis for selecting an organ transplant donor.
  • the method of the present invention is used for dynamic tracking and determination of immune cell functions of recipients of organ transplants, especially for dynamic monitoring before and after transplantation and before and after immune administration.
  • the drug selection and dosage for immunosuppressive therapy Adjustment and individualized treatment plan design provide objective reference indicators.
  • the method of the present invention is used to monitor the cellular immune activity of a tumor patient to tumor cells in order to, for example, evaluate the effect of tumor immunotherapy and guide the administration of tumor immunotherapy.
  • the tumor antigen of the individual tumor patient used in the method of the present invention is a whole tumor cell or a cell extract containing most tumor cell material.
  • the cell extract may be a component obtained by treating tumor tissues or cells by physical, chemical, biochemical, enzymatic methods, etc., and is preferably a crude cell extract, such as a cell lysate, a cell lysate supernatant, and a cell lysate. Liquid precipitation and so on.
  • individual tumor cells or cell extracts of a tumor patient are used to prepare individualized specific excitons to determine the specific anti-tumor cell immune activity of the tumor patient.
  • the present invention preferably uses a plurality of homogeneous tumor cells to prepare multivalent tumor antigens (tumor antigen pools) as stimuli for immune cells to measure the anti-tumor cell immune function of the corresponding patient.
  • the homogeneous tumor antigen library used in the method of the present invention is preferably a tumor antigen library composed of a whole cell tumor antigen library, a subcellular tumor antigen library, or a single tumor antigen or a plurality of purified components of the same type as the patient tumor.
  • the tumor antigen library preferably includes common antigens of a certain type of tumor (such as adenocarcinoma, melanoma, etc.).
  • the tumor antigen library can be prepared from homogeneous tumor tissues or tumor cell lines of multiple biological individuals by various methods such as physical, biochemical, conventional chromatography, immunoaffinity chromatography, density gradient centrifugation, and the like.
  • the immune cells detected in the method of the present invention are all cells that directly or indirectly participate in the immune response of the biological body, preferably T, B lymphocytes, NK cells, and various antigen presenting cells (APC).
  • T lymphocytes include cytotoxic T lymphocytes (CTL), helper T lymphocytes (THO; TH1; T 2), memory T lymphocytes, and suppressor T lymphocytes, etc .
  • APC includes dendritic cells, macrophages , B lymphocytes, interstitial cells, fibroblasts, fibroblasts, etc.
  • the immune cells in the present invention also include platelets, red blood cells, various bone marrow cells, various stem cells, and various immune cell precursor cells.
  • the immune cell subgroups to be detected can be isolated first, and then the antigen is used to stimulate the detection; or the mixed immune cells are first stimulated with the antigen, and then the subgroups are detected.
  • the latter is preferred.
  • total T lymphocytes CD3 +
  • CTL CD8 +
  • TH CD4 +
  • NK cells CD16 +, CD56 +
  • 8 lymphocytes 0019+ or .020+
  • APC cells CD80 + / CD83 + / CD86 + / HLA-DR +).
  • the immune function status of a living body is a comprehensive reflection of various immune cells' communication, contact, interaction, and regulation. Therefore, when testing the immune function of a cell population in vitro, as far as possible, ensure that other cells that directly or indirectly affect a particular immune function of the population coexist in the same measurement system to avoid the one-sidedness and non-objectivity of the test results.
  • the immune function response system should preferably include T lymphocytes. Therefore, the present invention particularly preferably uses an original sample as a measurement target.
  • the original sample is defined as a body fluid sample containing immune cells directly from the organism with minimal or no treatment, mainly including whole peripheral blood, whole bone marrow, a mixed population of nucleated cells from which red blood cells have been removed, peritoneal fluid, pleural fluid, joint fluid Or peritoneal fluid, pleural fluid, cerebrospinal fluid, synovial fluid obtained after centrifugation.
  • the cell separation described in the present invention can use any physical, immune, biological, and other methods capable of separating a specific population of mixed cells, including but not limited to centrifugation, density gradient centrifugation, natural sedimentation, non-specific adsorption, and solid phase.
  • the immune cell activation indicators determined in the method of the present invention may be changes in cell morphology, changes in the number of cells, changes in cell enzymes (such as tyrosine protein kinases; PTKs; Ras-MAP kinases; ZAP-70 kinases, etc.); intracellular ions Concentration change (such as Ca ++ concentration), intracellular and extracellular receptor changes (such as IL-2 receptor, etc.), specific ligand expression (CD40 ligand), activation expression of NF-KB, NFAT, AP-1, and Fas in cells , Cell membrane permeability changes, cell swallowing Phage function changes, cell activation marker protein expression (such as CD molecules: CD69, CD25, CD71, CD95, CD40, etc., MHC-I molecules, MHC-II molecules, etc.); intracellular energy changes (such as changes in ATP, AMP concentration, etc.) Changes in various biological factors secreted by cells (IL-2, IL-12, TNF; INF ⁇ ; IL- 4, etc
  • the activated immune cells can undergo the following major cellular biological changes: enzymatic changes, ion concentration changes, membrane receptor changes, intracellular ATP concentrations increase; the expression of various CD molecules (CD69, CD25, CD71, CD95,), MHC molecule expression (MHC-I molecule, MHC-II molecule), expression of various cytokines (IL-2, IL-4, IL-12, INFr, etc.), increased DNA synthesis (Increased 3H-TdR incorporation), increased cell proliferation and number (CFSE staining flow cytometry), enhanced CTL cytotoxicity and killing function ( 51 Cr-release test, MTT test, etc.).
  • the detection method in the present invention is any detection method capable of correspondingly measuring the above-mentioned activation indicators, including but not limited to: ELISA, Cellular ELISA, ELISPOT, and luminescence detection methods (bioluminescence, chemiluminescence, time-delayed luminescence, etc.) ), Fluorescence detection methods (fluorescence analyzer, fluorescence microscope counting, flow cytometry detection methods, etc.), isotopic detection (such as 51 Cr release, 3 H-TdR incorporation, etc.), cell smear detection, cell immunohistochemistry Detection, cytochrome (MTT) detection, etc.
  • ELISA ELISA
  • Cellular ELISA ELISA
  • ELISPOT and luminescence detection methods
  • luminescence detection methods bioluminescence, chemiluminescence, time-delayed luminescence, etc.
  • Fluorescence detection methods fluorescence analyzer, fluorescence microscope counting, flow cytometry detection methods, etc.
  • the method of the present invention is used for the detection of cellular immune function
  • it is preferable to determine early activation indicators of immune cells such as the expression of various molecular markers, changes in cell function, secretion of various cytokines, intracellular ion concentration or Changes in energy levels, etc.
  • no isotope reagent is used in the method of the present invention.
  • a self-antigen is set as a control for the stimulus.
  • the control antigen is an antigen of the individual to be tested in the case of a graft, and a non-tumor antigen of the individual to be tested in the case of a tumor.
  • a prominent problem is that a variety of factors can directly or indirectly affect the reactivity of immune cells in the test sample, resulting in non-specific activation of immune cells, thus making • Results analysis is difficult to judge.
  • a self-control is set in the detection to eliminate interference of non-specific activation signals, and accurate measurement of specific activation signals is achieved, for example, in the method of the present invention, the ratio of the signal after specific antigen stimulation to the signal after self antigen stimulation is measured.
  • This ratio basically depends on the stimulation of individual immune cells by specific antigens, and is basically not affected by non-specific stimulating factors. Therefore, the setting of an autoantigen control is very important for the dynamic detection of specific cellular immune functions. However, the lack of such controls in the prior art methods cannot accurately determine the specific cellular immune functions.
  • a blank control and an autoantigen control are preferably provided.
  • Blank control No stimulus is added to reflect the functional status of the tested immune cells without the stimulus. It is used to exclude the effects of unknown stimuli other than the above non-specific and specific antigens.
  • Autoantigen control The whole cell or subcellular component of the subject organism is used as the self-control antigen stimulator, preferably peripheral blood nucleated cells. It is preferable that the preparation method and the use amount thereof are exactly the same as those of the specific stimulus. This autoantigen control can better reflect the effect of non-specific antigens on the recipient's immune function.
  • the method of the present invention can more accurately measure the changes in immune cell functions of an individual against a specific antigen, and is suitable for tracking and detecting such changes in immune cell functions of an individual.
  • lyophilized nucleated cell components using repeated freeze-thaw methods freeze cells below 0 ° C, then thaw at room temperature or 37 ° C, repeat 1-5 times; or use ultrasonic disruption or homogenization method to lyse the above cells To prepare subcellular antigens;
  • the subcellular antigens prepared above will be used as self-controls and individualized specific stimuli for the determination of immune cell function;
  • the above-mentioned subcellular antigen components can be stored at low temperature ( ⁇ 20 ° C) for repeated use in dynamic monitoring and analysis.
  • Platelets can be used directly as immunostimulants containing MHC / HLA class I molecules to test immune cell function, or further purified to obtain platelet extracts or MHC / HLA class I molecules.
  • MHC / HLA type in peripheral blood nucleated cells, or platelets, or cell lines expressing MHC molecules, or transgenic cell lines (expressing specific known types) MHC / HLA molecules) as the source of MHC / HLA molecular library preparation to meet the statistical requirements for various types of MHC / HLA types (theoretically covering 100% of the biological population).
  • MHC / HLA Class I and Class II molecules Purification and identification of MHC / HLA Class I and Class II molecules according to the methods provided in Example 3 and Example 4, respectively, MHC / HLA Class I molecular libraries (covering all currently known Class I molecular types) Types), MHC / HLA Class II molecular libraries (covering all currently known MHC / HLA Class II molecular types), full MHC / HLA molecular libraries (including all MHC / HLA Class I molecular types).
  • the above three MHC / HLA molecular libraries can be selected separately as needed.
  • the MHC / HLA class I molecular library is used to detect the CTL cell function of CD8 +; the MHC / HLA class II molecular library is used to detect the CD H + cell function of CDH.
  • MHC / HLA molecular libraries for other MHC types: For example, a small number of MHC (Minor MHC) molecular libraries can also be prepared using the above principles and methods to determine certain other immune functions of the organism.
  • MHC auto-tumor specific antigen
  • Tumor cells in inactivated blood can be used directly as specific antigen stimuli;
  • the above cells can be prepared by the aforementioned freeze-thaw method, detergent lysis method, homogenization, and ultrasonic lysis.
  • Example 7 Preparation of a multivalent tumor antigen library:
  • homogeneous tumors from multiple individuals or multiple homogeneous tumor cell lines can be used as a source for preparing multivalent tumor antigens, such as adenocarcinoma, squamous cell carcinoma, each Leukemia-like cells, melanoma tumor cells, etc .;
  • antigen libraries of different types of tumors can be prepared, and the specific anti-tumor immune function of a specific tumor patient can be determined by using the corresponding types of tumor antigen libraries.
  • the peripheral blood of the organ transplant recipient was extracted and an appropriate amount of heparin was added for anticoagulation.
  • the test blood samples were divided into three equal parts, and each was diluted with RPMI1640 at a ratio of 1: 5.
  • One sample was added with the donor subcellular antigen stimulus in Example 2, one sample was added with the recipient autoantigen control stimulus in Example 2, and one sample was not added with any stimulus.
  • At 37 ° C, 5% C0 2 saturated humidity and incubated overnight.
  • Add an appropriate amount of erythrocyte lysate to the above sample to lyse red blood cells, centrifuge to remove erythrocyte lysate fragments; resuspend the pelleted cells with PBS buffer.
  • Each sample was divided into two equal parts.
  • mice anti-human CD3 FITC-labeled monoclonal antibody (2 ⁇ g / ml)
  • mouse anti-human CD4 (or CD8) PE-labeled monoclonal antibody 2 ⁇ g / ml
  • mouse anti-human CD69 PerCP-labeled monoclonal antibody (2 g / ml)
  • another portion was added with an appropriate amount of isotype control antibody, and incubated at 4 ° C in the dark for 30 minutes. Centrifuge at 1,000 g for 5 minutes and wash the cells with a fluorescent washing solution containing 10% FCS and 0.1% BSA. This centrifugation and washing process was repeated three times.
  • Example 9 Measurement of a patient's specific immune function against a tumor
  • Peripheral blood was collected from tumor patients, and appropriate heparin was added to anticoagulate.
  • the test blood samples were divided into three equal portions, and each was diluted with RPMI1640 at a ratio of 1: 5.
  • the subcellular components of the auto-tumor cells prepared in Example 6, the subcellular components of the patient's own peripheral blood nucleated cells, and no stimulants were added separately.
  • 37 ° C, 5% C0 2 saturated humidity and incubated overnight (Note: Add subcellular components corresponding to the number of tumor cells and peripheral blood nucleated cells of the same).
  • mice anti-human CD3 FITC-labeled monoclonal antibody (2 ⁇ g / ml) and mouse anti-human CD4 (or CD8) PE-labeled monoclonal antibody ( 2 ⁇ g / ml) and mouse anti-human CD69 PerCP-labeled monoclonal antibody (2 ⁇ g / ml); another portion was added with an appropriate amount of isotype control antibody, and incubated at 4 ° C in the dark for 30 minutes. Centrifuge at 1000g for 5 minutes and wash the cells with a fluorescent washing solution containing 10% FCS and 0.1% BSA. This centrifugation and washing process was repeated three times.

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Description

一种个体化的测定特异性免疫细胞功能的方法 技术领域
本发明涉及一种测定特异性细胞免疫功能的方法。 该方法尤其可 用于监测器官移植受者针对移植物的特异性细胞免疫功能和肿瘤患 者机体特异性抗肿瘤细胞的免疫功能。 背景技术
正常生物机体免疫系统由体液免疫和细胞免疫所组成， 二者之间 的精确配合和相互调节在维持正常机体生命机能和抗疾病免疫中发 挥着极其重要的作用。 各种淋巴细胞， 尤其是 T淋巴细胞在免疫系统 中的中心作用近年来受到广泛地关注。 T淋巴细胞作为生物机体抗疾 病免疫和排斥免疫的 "轴心" 概念得到了普遍的认同。
随着人类对生物机体免疫机制理解的不断深化， 近年来尤其是在 特异性免疫识别应答机制方 -面的研究， 取得了重要进展。 特异性免疫 应答的前提条件是特异性免疫识别， 而特异性免疫识别又取决于 "双 信号" 机制的完整性。 組成 "双信号" 识别的物质基础是： 由 MHC ( Major Histocompatibility Complex )基因的表达产物 MHC分子和特 异性多肽复合物组成特异性识别的第一信号； 而协同刺激因子 (B7家 族； B7-1 ; B7-2等）、 细胞粘附因子 (CD40等)则组成免疫识别的第二 信号。生物体抗疾病免疫反应或抗移植物排斥免疫反应的激发、活化、 调节以及免疫耐受的产生与上述 "双信号" 系统的完整性有着极其密 切的关联。 外来抗原（非已抗原）或移植物抗原成分进入生物体后， 首 先通过抗原提呈细胞 (Antigen Presenting Cells; APC)将非已抗原降解 为特异性多肽, 然后与相应的自身 MHC分子组装形成 MHC-特异性 多肽复合物， 并提呈于细胞表面， 然后与膜上的协同刺激因子、 细胞 粘附因子等共同被特定 T 淋巴细胞克隆的特异性受体（T Cell Receptor; TCR)以及协同刺激因子受体 (CD28等）所识别、 结合； 从而 特异性地激活 T淋巴细胞及其亚群，诱发生物机体全面的特异性免疫 应答。 通过 MHC-I -多肽复合物诱导激活的 CD8阳性 T淋巴细胞形 成细^毒性 T细包 (Cytotexic T Lympholytes; CTL)或称杀伤性 T细 胞 , 对感染外来抗原（如病毒等)的细胞或肿瘤细胞或器官移植供者细 胞等产生直接的特异性杀伤作用； 而 MHC-II -多肽复合物诱导、 活 化的 CD4阳性 Τ淋巴细胞或称辅助性 Τ淋巴细胞 (T Helper; TH)可通 过分泌各种细胞因子强化或调节 CTL 的特异性杀伤作用和其他免疫 细胞 (如巨噬细胞活化)功能， 同时可刺激 B淋巴细胞增殖、 分化成浆 细胞， 分泌特异性抗体启动体液免疫应答。 综上所述， 可获得以下重 要结论： 1、 T细胞、 尤其是 CD4阳性的 T淋巴细胞在完整生物机体 免疫系统中具有 "轴心" 作用； 2、 生物机体的特异性免疫识别是特 异性免疫应答的基础和前提条件； 3、 "双信号" 识别机制的完备与 否决定着特异性免疫应答的性盾（即特异性免疫 "耐受，， 或 "攻击" 效应）。
在同种异体组织、 器官移植时， 受者的免疫系统常对移植物产生 排异反应 （transplant rejection ) , 这是一个十分复杂的免疫学现象， 涉及细胞和抗体介导的多种免疫损伤机制， 都是针对移植物中的人类 主要组织相容性复合物 MHC (即 HLA， Human Leucocyte Antigen人 白细胞抗原） 的反应， 供者与受者 HLA的差异程度决定了排异反应 的轻或重。 除单卵双生外， 两个个体具有完全相同的 HLA 系统的组 织配型几乎是不存在的， 但选择供者与受者配型尽可能地接近， 是异 体组织器官移植成功的关键。
HLA系统是目前人类已知的最复杂的基因群一 HLA复合体， 或 称 MHC基因的编码产物。每个基因位点存在多种等位基因，导致 HLA 系统的高度多态性。 与移植排斥有关的主要是 HLA- I类、 II类基因 编码的 HLA- I类、 II类抗原。 所有人有核细胞均可以表达 HLA- I类 抗原， HLA- II类抗原见于抗原递呈细胞、 B 细胞及 T辅助细胞。 此 外， 血管内皮细胞、 纤维母细胞及腎小管上皮细胞等如果受到 IFN - Y的诱导， 亦可表达 II类抗原。
在人体和实验性组织、 器官移植中证实， T细胞介导的迟发性超 敏反应与细胞毒作用对移植物的排斥起着重要的作用。
人类对生物免疫系统理解、 认识的深化， 推动了实验免疫学分析 技术的发展； 而新的多样化免疫学技术的出现， 又给免疫学基础理论 研究带来了多角度分析的观测工具， 从而进一步促进了免疫学理论的 完善和科学性， 并成为临床免疫学的坚实基础。 近年来， 各国免疫学 工作者建立了多种检测生物机体免疫学功能的方法和技术。 其主要包 括各种以测定特异性抗体产生为特征的体液免疫功能检测方法和测 定各类免疫细胞数目、 活性、 增殖等指标的细胞免疫功能检测方法。
B淋巴细胞的功能可以通过测量体液中特异性抗体的水平来检测。 而 T淋巴细胞针对特异性抗原的功能较难检测， 因为 T细胞有多种具有 不同功能的亚型， 其发挥功能的方式也不同。
现有的检测细胞免疫功能的方法包括： 基于免疫细胞计数的方 法； 基于检测免疫细胞增殖的方法； 基于检测细胞毒活性或分泌细胞 因子的方法； 以及在体的方法如皮肤试验和过继转移。
上述方法在实际应用中具有各自的优缺点， 但存在的最大不足是 在绝大多数情况下， 以上方法只是对机体非特异性和特异性免疫的混 合测量， 不能有效的反映生物机体的特异性细胞免疫功能， 特别是个 体化特异性免疫活性。
美国专利 5773232号公开了一种快速分析淋巴细胞功能活化的方 法， 该方法包括用抗原体外刺激含淋巴细胞的样品， 然后分离并裂解 特定的淋巴细胞亚群， 通过裂解物中的 ATP 水平来测定淋巴细胞的 功能活化。 该文献中提到了使用肿瘤细胞蛋白或来自移植物的蛋白作 为刺激抗原，但没有公开使用待测个体自身的肿瘤细胞或其裂解物或 待测个体移植物供者的细胞或其裂解物或 MHC分子作为刺激抗原。 因此， 该现有技术的方法并不是一种个体化的方法。
另一种测定细胞免疫活性的现有技术方法是称为 "混合淋巴细胞 培养（MLC ) " 的方法。 该方法是将器官移植供者的淋巴细胞灭活使 其丧失繁殖能力后作为刺激物与器官移植受者的淋巴细胞共培养 3 - 7天，利用 3H-TdR掺入技术测定受检样品中淋巴细胞的 DNA合成情 况， 从而评价受者免疫细胞对供者淋巴细胞的反应性 （ 见
REINSMOEN NL.5 WARD FE. HISTROY OF HLA AND TRANSPLANTATION IMMUNOLOGY, LONDON. UK: IMPERIAL COLLEGE OF PRESS 2001 ) 。 该方法没有提到连续测定以检测器官 移植受者的细胞免疫活性， 而且该方法需要长时间的共培养， 也不适 于细胞免疫活性的监测。 另外， 该方法没有提到设置以器官移植受者 的细胞或细胞提取物作为刺激物的对照。
在器官移植免疫和抗肿瘤免疫中， 受者对相应供者器官的特异性 排斥免疫功能监测以及肿瘤患者对其肿瘤细胞的特异性抗肿瘤免疫 功能监测具有十分重要的临床应用价值。 因此， 需要一种能准确监测 个体对移植物或肿瘤细胞的特异性免疫活性的方法。 发明概述
本发明提供了一种用于体外监测个体对器官移植物、 肿瘤的特异 性免疫细胞功能的方法。 该方法包括 1) 将含有免疫细胞的该个体的 受检样品与来自器官移植物供者的抗原或包含该供者' MHC /HLA类 型的 MHC/HLA分子库、或与该个体自身的肿瘤抗原或同类肿瘤抗原 库一起培养一段足以活化免疫细胞的时间， 2)将培养后的免疫细胞分 离和 /或分类并测定其活化指标；其中所述来自器官移植物供者的抗原 或所述该个体自身的肿瘤抗原是全肿瘤细胞或含有大部分细胞物质 的细胞提取物； 该方法还包括设置以所述个体自身的抗原或非肿瘤抗 原作为刺激抗原的对照。
在本发明的一个实施方案中， 使用包含供者 MHC/HLA 类型的 MHC/HLA分子库作为刺激抗原， 所述分子库优选为 MHC/HLA I类 分子库、 MHC/HLA II类分子库、 或 MHC/HLA I类和 II类分子的混 合分子库。
在本发明的一个实施方案中， 使用同类肿瘤抗原库作为刺激抗 原， 所述抗原库优选为与患者肿瘤相同种类的全肿瘤细胞抗原库、 肿 瘤亚细胞抗原库或单一肿瘤抗原或多种纯化组分所组成的肿瘤抗原 库。 更优选该抗原库包括某一类肿瘤 （如腺癌、 黑色素细胞瘤等） 的 共同抗原。
本发明来自移植物供者的抗原、 MHC/HLA分子库、 自身肿瘤抗 原和肿瘤抗原库可以在使用时制备， 也可以一次性制备。 优选一次性 制备并储存， 方便随时使用。
在本发明的一个优选实施方案中， 所述个体的受检样品为外周全 血、 骨髓和含免疫细胞的任何生物体液。
在本发明的一个优选实施方案中， 受检样品与抗原的培养时间优 选为 20分钟到 24小时， 更优选为 30分钟到 6小时。
优选对照所用的抗原与刺激抗原平行制备。 发明详述 急性、 急性和慢性排斥反应。 超急性排斥反应主要由受者体内已经存 在的抗供者 HLA分子的抗体和补体引起的体液免疫反应。 而急性、 慢性排斥反应主要由受者针对供者移植物的细胞免疫所介导。 受者的 T细胞通过直接和间接的方式特异性识别供者 HLA, 继而活化增殖， 诱发受者抗移植物的特异性排斥反应。
本发明的发明人发现器官移植的供者细胞及其包括供者 含了供者抗原或移植物抗原的全部信息。 因此， 所述抗原作为体外细 胞免疫测定的特异性刺激原可以更准确地反映器官移植受者体内免 疫细胞的活性。 因此， 本发明的体外监测个体对器官移植物、 肿瘤的 特异性免疫细胞功能的方法中，使用供者细胞或含供者 MHC/HLA的 细胞提取物或含供者 MHC/HLA类型的 MHC/HLA分子抗原库作为个 体化特异性刺激原。 实猃表明： 对于受者的免疫细胞， 含供者 免疫原刺激特性，二者均可特异性地激活受者的供者抗原特异性 T细 胞活化。 在本发明的方法中， 经供者特异性抗原或 MHC/HLA分子刺 激后测定受者免疫细胞的活化标志， 可反映受者免疫细胞对供者移植 物的特异性排斥状态， 从而达到对排斥反应的预测和动态监测， 并为 调整临床免疫治疗用药提供参考指标。
本发明的方法中， 来自器官移植物供者的抗原是供者任何组织细 子。 亚细胞成分是通过物理、 化学、 生物化学、 酶学等方法对组织细 胞处理后获得的包含供者 MHC/HLA分子的成分。 优选， 所述亚细胞 成分是供者细胞的粗提取物， 例如细胞裂解液、 细胞裂解液上清、 细 胞裂解液沉淀等。
在本发明的一个优选实施方案中， 使用含有供者 MHC/HLA分子 类型的 MHC/HLA分子库作为抗原刺激受者的免疫细胞。该分子库包 括全部和大部分移植物的 MHC/HLA分子， 可以是 MHC/HLA I类分 子库、 MHC/HLA II类分子库、或 MHC/HLA I类和 MHC/HLA II类分 子混合的分子库。 MHC/HLA I类分子作为刺激原可以测定受体 CD8+ 的 T细胞 （ CTL ) 的功能， MHC/HLA II类分子作为刺激原可以测定 受体 CD4+的 T细胞（Th ) 的功能。 MHC/HLA分子库可以通过多种 方法技术， 以多种生物细胞、 生物材料为来源获得。 例如噬菌体和细 菌展示生物技术、 亲和层析技术、 DNA重组技术、 DNA转染技术等。 噬菌体展示已证明是一种有利的技术， 利用它可以获得容纳几万甚至 几亿的不同肽或蛋白质的库， 并已应用于重组肽库的亲和筛选。
上述三种 MHC分子库在与某一特定生物个体免疫细胞群体进行 温育或共培养时，分子库中与供者免疫表型类似的 MHC/HLA蛋白可 以通过直接识别途经作用于受者的 T 细胞， 也可以通过间接识别途 经： 受者的 APC细胞可将 MHC/HLA分子加工后提呈给相应的 T淋 巴细胞 （CTL, TH1 , TH2等）， 诱导其活化。 如果受检免疫细胞是第 一次接触供者的 MHC/HLA分子， 在设定时间内它们的活化程度、 反 应强度相对低而且弱； 若检测系统中的受检免疫细胞曾经被供者的 MHC/HLA I类或 II类分子所致敏， 则那些被致敏的免疫细胞会在短 时间内迅速活化， 其活化的程度和反应较为强烈。 这在器官移植排斥 反应中尤其显著。 在临床器官移植实践中， 移植手术完成后， 移植受 者的免疫细胞被供者 MHC/HLA分子致敏而处在高敏感状态。在体外 反应系统中， 免疫细胞再次遭遇相同类型的 MHC/HLA分子， 从而迅 速活化并被测量。 因此， 根据移植前后受者免疫细胞对含有与供者相 免疫细胞抗移植物特异性排斥反应的有无和强弱。
本发明中对于受检样品与抗原的培养时间没有特别限制， 根据待 测量的免疫细胞的活化指标不同， 例如可以是 20分钟到 7天。 在本 发明方法的大部分和优选的应用中， 所述培养时间较短， 优选为 20 分钟到 24小时， 更优选为 30分钟到 6小时。 测定免疫细胞早期活化 察。 、 、 、 、 、 、
器官移植受者在接受移植前， 其体内的淋巴细胞应该从未与供者 的抗原接触过， 处在 "静息，， 状态或称非致敏状态； 此时若以供者的 MHC/HLA 分子在体外刺激受者免疫细胞可诱发緩慢而强度较弱（相 对移植后）的免疫细胞活化， 相当于免疫系统与非已抗原的第一次接 触。 根据供者与受者的 MHC/HLA符合程度， 受者细胞免疫反应的强 度也存在不同差异， 即供者与受者 MHC/HLA的符合程度越高则受者 免疫细胞的活化程度越低， 移植后发生排斥反应的发生几率也越低； 反之， 则排斥反应的发生几率将增加。 因此在器官移植前， 本发明所 述及的方法可以作为选择器官移植供者的依据之一。
器官移植后受者需要接受免疫抑制剂的治疗。 如果治疗剂量不 足， 不能防止免疫排斥反应的发生； 如果治疗剂量过大， 会全面抑制 受者的免疫系统， 合并感染等并发症。 免疫功能的测定结果受到个体 差异、 供者与受者的 MHC/HLA符合程度、 免疫抑制药物剂量等多种 因素的影响。 因此， 本发明方法用于对器官移植受者的免疫细胞功能 进行动态跟踪测定， 特别是移植前后和免疫用药前后的动态监测。 从 而做出具有科学依据的正确判断， 为免疫抑制治疗的用药选择、 剂量 调整和个体化治疗方案设计提供客观的参考指标。
在本发明的另一实施方案中， 本发明的方法用于监测肿瘤患者个 体对肿瘤细胞的细胞免疫活性， 以便例如评价肿瘤免疫治疗的效果、 指导肿瘤免疫治疗的用药。 本发明方法中使用的肿瘤患者个体自身的 肿瘤抗原是全肿瘤细胞或含有大部分肿瘤细胞物质的细胞提取物。 所 述细胞提取物可以是经物理、 化学、 生物化学、 酶学等方法对肿瘤组 织或细胞处理后获得的成分，优选为细胞粗提取物，例如细胞裂解液、 细胞裂解液上清、 细胞裂解液沉淀等。 在本发明方法的优选实施方案 中， 利用肿瘤患者自身的肿瘤细胞或细胞提取物制备个体化特异性刺 激原， 来测定肿瘤患者的特异性抗肿瘤细胞免疫活性。
但在临床实践中， 由于种种原因对于许多肿瘤患者不能获得其自 身的肿瘤细胞而不能进行个体化特异性肿瘤抗原制备， 给测定带来困 难。 因此， 针对这种情况， 本发明优选使用多种同类肿瘤细胞制备多 价肿瘤抗原（肿瘤抗原库)作为免疫细胞的刺激物对相应患者的抗肿瘤 细胞免疫功能进行测定。
本发明方法中使用的同类肿瘤抗原库优选为与患者肿瘤相同种 类的全细胞肿瘤抗原库、 亚细胞肿瘤抗原库或单一肿瘤抗原或多种纯 化组分所组成的肿瘤抗原库。 该肿瘤抗原库优选包括某一类肿瘤（如 腺癌、黑色素细胞瘤等）的共同抗原。所述肿瘤抗原库可以通过物理、 生物化学、 常规层析法、 免疫亲和层析法、 密度梯度离心等多种方法 由多个生物个体的同类肿瘤组织或肿瘤细胞系制备。
本发明方法中所检测的免疫细胞为所有直接或间接参与生物机 体免疫反应的任何细胞， 优选 T、 Β淋巴细胞、 ΝΚ细胞和各种抗原 提呈细胞（ APC )。其中 Τ淋巴细胞包括细胞毒性 Τ淋巴细胞（ CTL )、 辅助性 Τ淋巴细胞 (THO; TH1 ; ΤΗ2)、 记忆性 Τ淋巴细胞、 抑制性 Τ 淋巴细胞等； APC包括树突状细胞、 巨噬细胞、 Β淋巴细胞、 间质细 胞、 纤维母细胞、 纤维细胞等。 此外本发明所述的免疫细胞还包括血 小板、 红细胞、 各种骨髓细胞、 各种干细胞、 各种免疫细胞前体细胞 根据实际需要， 可以先分离需要检测的免疫细胞亚群， 然后用抗 原刺激检测； 或先用抗原刺激混合的免疫细胞， 然后分离亚群检测。 后者为优选。例如测定总 T淋巴细胞 (CD3+)、 CTL(CD8+)> TH(CD4+), NK细胞 (CD16+， CD56+)、 ；8淋巴细胞(0019+或。020+)、 APC细胞 (CD80+/CD83+/CD86+/HLA-DR+)等的反应性。
生物机体的免疫功能状态是各种免疫细胞相互联系 (Communication)、接触 (Contaction)、作用 (Interaction)、调节 (Regulation) 的综合体现。 因此， 在体外检测某细胞群体免疫功能时， 应尽可能保 证其它直接或间接影响该群体某种特定免疫功能的细胞共存于同一 测定体系中， 以避免检测结果的片面性和不客观性。 例如， 在检测某 生物体 T淋巴细胞功能时，优选保留相应的抗原提呈细胞存在于同一 检测系统之中， 从而保证 T淋巴特异性识别系统的完整性； 另外， 在 测定 NK细胞或 B细胞免疫功能的反应体系中应优选包含 T淋巴细 胞。 因此， 本发明特别优选使用原始样品作为测定对象。 原始样品定 义为直接来自生物体经最少处理或不经任何处理的含免疫细胞的体 液样品， 主要包括全外周血、 全骨髓、 去除红细胞的有核细胞混合群 体、 腹腔液、 胸腔液、 关节液或经离心后获取的腹腔液、 胸腔液、 脑 脊液、 关节液。
本发明中所述的细胞分离可以使用任何能将混合细胞中的某特 定群体分离的物理、 免疫、 生物学等方法， 包括但不限于离心、 密度 梯度离心、 自然沉降、 非特异性吸附、 固相吸附、 免疫磁珠吸附、 聚 苯乙烯微珠免疫吸附、 聚苯乙烯微孔板吸附， 以及使用单或多标记技 术 (如单或多色荧光标记)在相应仪器 (如流式细胞仪、 荧光显微镜等） 上对各种免疫细胞进行分类鉴别的任何方法。
本发明方法中所测定的免疫细胞活化指标可以是细胞形态变化、 细胞数目变化、 细胞酶学变化 (如酪氨酸蛋白激酶； PTKs; Ras-MAP 激酶； ZAP-70激酶等）、 细胞内离子浓度变化 (如 Ca++浓度）、 细胞内 外受体变化（如 IL-2 受体等）、 特异性配体表达 (CD40 配体）、 细胞 NF-KB, NFAT, AP-1 , Fas激活表达、 细胞膜通透性变化、 细胞吞 噬功能变化、细胞活化标志蛋白表达 (如 CD分子： CD69, CD25 , CD71， CD95 , CD40等， MHC-I分子， MHC-II分子等）； 细胞内能量变化 (如 ATP、 AMP浓度变化等）、细胞分泌各种生物因子的变化 (IL-2, IL-12, TNF; INF γ； IL-4等）、 细胞核变化（细胞核数目、 DNA含量变化等）； 细胞功能变化 (杀伤功能、 吞噬功能、 粘附功能等)、 细胞蛋白质浓度 变化等。
免疫细胞受到上述特异性抗原刺激后， 活化的免疫细胞可发生下 列主要细胞生物学变化： 酶学改变， 离子浓度改变、 膜受体改变、 细 胞内 ATP浓度增加；各种 CD分子表达 (CD69, CD25 , CD71 , CD95 , ), MHC分子表达 (MHC-I类分子， MHC-II类分子）， 各种细胞因子表达 (IL-2, IL-4, IL-12, INFr等）， DNA合成增加 (3H-TdR掺入增加）、 细胞增殖及数目增加 （ CFSE染色流式测定） ， CTL细胞毒性杀伤功 能增强（51Cr-释放试验， MTT试验等）。 通过对上述任何一种细胞生物 学变化的测量， 可建立多种抗原特异性细胞免疫功能的测定方法。
本发明中的检测方法为能够对上述活化指标进行相应测量的任 何检测方法， 包括但不限于： ELISA, 细胞 ELISA(Cellular ELISA), ELISPOT, 发光检测方法（生物发光、 化学发光、 时间延迟发光等）， 荧光检测方法（荧光测定仪、 荧光显微镜计数、 流式细胞仪检测方法 等）， 同位素检测（如 51Cr释放、 3H-TdR掺入等）， 细胞涂片染色检测， 细胞免疫组织化学检测， 细胞色素 (MTT)检测等。 考虑到本发明的方 法用于细胞免疫功能检测， 本发明中优选测定免疫细胞的早期活化指 标， 如各种分子标志的表达、细胞功能的变化、各种细胞因子的分泌、 细胞内离子浓度或能量水平的变化等。 考虑到操作的筒便性， 优选本 发明的方法中不使用同位素试剂。
本发明方法的另一个特征是，设置一个自身抗原作为刺激物的对 照。 所述对照抗原在移植物的情况下为待测个体自身的抗原， 在肿瘤 的情况下为待测个体自身的非肿瘤抗原。在特异性细胞免疫功能的检 测方法学中， 一个突出问题是由于多种因素可以直接或间接影响受检 样品中免疫细胞的反应活性， 导致免疫细胞的非特异性激活， 从而使 •结果分析判定困难。 本发明在检测中设置自身对照以排除非特异性活 化信号的干扰， 实现特异性活化信号的准确测定， 例如本发明方法中 测定特异性抗原刺激后的信号与自身抗原刺激后信号的比值。 该比值 基本取决于特异性抗原对个体免疫细胞的刺激，基本不受非特异性刺 激因素的影响。 因此， 设置自身抗原对照对于特异性细胞免疫功能的 动态检测非常重要， 而现有技术方法中由于缺少这种对照而无法准确 测定特异性细胞免疫功能。
本发明的方法中优选设置空白对照和自身抗原对照。
空白对照： 不加入任何刺激原， 反映受检免疫细胞在没有刺激原 时的功能状况， 用以排除上述非特异性和特异性抗原以外的未知刺激 因素的影响。
自身抗原对照： 使用受检生物个体的全细胞或亚细胞成分作为自 身对照抗原刺激物， 优选为外周血有核细胞。 优选其制备方法和使用 量与特异性刺激原完全相同。 这种自身抗原对照可以较好的反映非特 异性的抗原对受者免疫功能的影响。
特异性抗原： 以器官移植供者的全细胞或亚细胞成分、 或包括供 者 MHC分子的 MHC分子抗原库作为刺激原； 或个体自身的肿瘤抗 原、 同类肿瘤抗原库作为刺激原。
综合分析上述三个检测结果， 比较其相互间差异， 从而对受检个 体的特异性细胞免疫功能做出客观合理的评估。
纵上所述， 与现有技术方法相比， 本发明方法能够更准确地测定 个体针对特定抗原的免疫细胞功能变化， 适于跟踪检测个体的这种免 疫细胞功能变化。 本发明的具体实施方案
实施例 1 器官移植免疫功能检测中全细胞抗原刺激物和对照刺 激物的制备
1.抽取器官移植供者和受者外周静脉血各 5-10ml, 分别置于两支 抗凝管中； 2.在上述管中加入适量红细胞裂解液、 裂解红细胞， 离心去除红 细胞裂解碎片；
3.收集有核细胞成分并进行细胞计数， 使上述供者和受者的细胞 具有相同的细胞浓度；
4.使用放射性照射或裂霉素处理有核细胞， 使其丧失分裂活性。 由器官移植受者制备的细胞抗原将作为检测方法中的自身抗原对照 特异性刺激物。
实施例 2 器官移植免疫功能检测中亚细胞抗原刺激物和对照刺 激物的制备
1.同实施例 1中的步骤 1-3;
2.使用反复冻融法裂解有核细胞成分： 即在 0°C以下冻结细胞， 然后在常温或 37°C融解，反复 1-5次；或使用超声破碎法或匀浆方法， 裂解上述细胞， 制备亚细胞抗原；
3.上述制备的亚细胞抗原将分别作为自身对照和个体化特异性刺 激物用于免疫细胞功能的测定；
4.上述亚细胞抗原成分可置低温(<20 °C )保存， 供动态监测分析中 多次使用。
实施例 3 MHC/HLA分子的純化制备：
1.同实施例 1中的步骤 1-3;
2.用含有去垢剂的适当緩冲液溶解有核细胞， 经离心等方法去除 细胞核成分， 保留含有 MHC/HLA 分子的细胞膜和细胞浆可溶性组 份；
3.使用抗 MHC/HLA I类或 /和 II类分子抗体的亲和层析柱， 分离 纯化相应的 MHC/HLA I类或 /和 II类分子组分；
4.利用 SDS-PAGE 技术鉴定上述纯化成分的分子量； 利用抗 MHC/HLA分子抗体 Western Blot技术 (免疫蛋白印迹技术)对純化物 进行特异性纯度鉴定；
5.测定上述纯化物的蛋白浓度，置低温保存备用或直接用于检测。 实施例 4 血小板 MHC/HLA I类分子的制备：
1.同实施例 1中的步骤 1 ;
2.常规离心分离获得相应的血浆组分；
3.进一步分离血小板。血小板可以作为含 MHC/HLA I类分子的抗 原刺激物直接用于检测免疫细胞功能， 或进一步纯化获得血小板提取 物或 MHC/HLA I类分子。
4.利用勾浆、 超声破碎技术或血小板溶解液裂解血小板， 去除不 含 MHC/HLA分子的有形成分。 保留含 MHC/HLA I类分子的组分直 接用于检测。
5.利用 MHC/HLA I类分子抗体的亲和层析柱，对步骤 4获得的血 小板溶解物进行 MHC/HLA I类分子純化；
6.同实施例 3中的步骤 4-5。
实施例 5 MHC分子库的制备:
1.利用多个（100士个体）已知 MHC/HLA型别的生物体外周血有核 细胞、 或 /和血小板， 或表达 MHC分子的细胞系、 或转基因细胞系（表 达特定已知型别的 MHC/HLA分子)作为 MHC/HLA分子库制备来源， 以满足统计学上对各类 MHC/HLA型别的要求 (理论上覆盖 100%的生 物群体）。
2.根据实施例 3、 实施例 4所提供的方法对 MHC/HLA I类、 II类 分子进行纯化、 鉴定， 可分别获得 MHC/HLA I类分子库 (覆盖目前已 知的全部 I类分子型别）、 MHC/HLA II类分子库 (覆盖目前已知的全部 MHC/HLA II类分子型别）、全 MHC/HLA分子库（包括全部 MHC/HLA I类 II类分子型别）。
3.上述三种 MHC/HLA 分子库可 ^据需要分别选用。 例如利用 MHC/HLA I类分子库检测 CD8+的 CTL细胞功能；利用 MHC/HLA II 类分子库检测 CD4+的 TH的细胞功能等。
4.除上述 MHC/HLA 分子库外， 对于其它 MHC 型别： 如少数 MHC(Minor MHC)分子库也可利用上述原理、 方法制备， 用于测定生 物体的某些其它免疫功能。 实施例 6 自身肿瘤特异性抗原的制备：
1.将由外科手术中切除的患者自身肿瘤组织剪成碎块， 或活检所 得肿瘤组织， 用胶原酶、 胰蛋白酶等消化， 梯度密度离心等技术制成 单个肿瘤细胞悬液；
2.经灭活处理的血液中的肿瘤细胞可直接用作特异性抗原刺激 物；
3.同时制备肿瘤患者外周血灭活的有核细胞作为自身抗原对照；
4.上述细胞可使用前述的冻融法、 去垢剂裂解法、 匀浆、 超声裂 实施例 7 多价肿瘤抗原库制备：
1.根据现有各种肿瘤组织学来源、 分类方法， 可利用来自多个个 体的同类肿瘤或多个同类肿瘤细胞系作为制备多价肿瘤抗原的来源， 如腺癌类、 鱗癌类、 各类白血病细胞、 黑色素细胞瘤细胞等；
2.用上述实施例 1 中所提供的方法， 可制备成不同种类肿瘤的抗 原库， 利用相应各类肿瘤抗原库可对某一特定肿瘤患者的特异性抗肿 瘤免疫功能进行测定。
' 实施例 8 测量受者的 CTL对移植物供者抗原的反应性
抽取器官移植受者的外周血， 加入适量肝素抗凝。 测试血样被分 成三等份， 分别用 RPMI1640以 1 : 5的比例稀幹。 一份样本中加入 实施例 2中的供者亚细胞抗原刺激物， 一份样本中加入实施例 2中的 受者自身抗原对照刺激物, 一份样本中不加任何刺激物。 在 37°C、 5 % C02、 饱和湿度条件下温育过夜。 在上述样本中加入适量红细胞裂 解液裂解红细胞， 离心去除红细胞裂解碎片； 用 PBS緩冲液重悬沉淀 的细胞。 每个样本分为相等的两份， 一份加入适量的小鼠抗人 CD3 FITC标记的单克隆抗体（ 2 μ g/ml ) 、 小鼠抗人 CD4 (或 CD8) PE标 记的单克隆抗体（ 2 μ g/ml )和小鼠抗人 CD69 PerCP标记的单克隆抗 体 （2 g/ml ) ； 另一份加入适量同型对照抗体， 在 4°C下避光孵育 30分钟。 lOOOg离心 5分钟， 用含 10 % FCS和 0.1 % BSA的荧光洗液 洗涤细胞。 该离心、 洗涤过程重复三次。 加入 100 μ ΐ固定液（含 1 % 甲醛、 2 %葡萄糖、 0.1 %叠氮钠的 PBS緩冲液）在 4°C下避光固定 15 分钟。 用流式细胞仪检测激活的淋巴细胞及其亚群的活化标志 CD69 的分子表达。 比较供者抗原刺激組、 自身抗原对照组和空白对照組的 结果， 判断受者对移植物的个体化特异性免疫功能状况。
实施例 9 测量患者对肿瘤的特异性免疫功能
抽取肿瘤患者的外周血， 加入适量肝素抗凝。 测试血样被分成三 等份， 分别用 RPMI1640以 1 : 5的比例稀释。 分别加入实施例 6制 备的自身肿瘤细胞的亚细胞成分、 患者自身外周血有核细胞的亚细胞 成分、 以及不加任何刺激物。 在 37°C、 5 % C02、 饱和湿度条件下温 育过夜（注： 加入的亚细胞成分所对应的肿瘤细胞数和外周血有核细 胞数相同） 。 在上述样本中加入适量红细胞裂解液裂解红细胞， 离心 去除红细胞裂解碎片； 用 PBS缓冲液重悬沉淀的细胞。每个样本分为 相等的两份， 一份加入适量的小鼠抗人 CD3 FITC标记的单克隆抗体 ( 2 μ g/ml )、小鼠抗人 CD4 (或 CD8) PE标记的单克隆抗体（ 2 μ g/ml ) 和小鼠抗人 CD69 PerCP标记的单克隆抗体（ 2 μ g/ml ) ； 另一份加入 适量同型对照抗体， 在 4°C下避光孵育 30分钟。 1000g离心 5分钟， 用含 10 % FCS和 0.1 % BSA的荧光洗液洗涤细胞。 该离心、 洗涤过程 重复三次。 加入 ΙΟΟ μ Ι固定液（含 1 %甲醛、 2 %葡萄糖、 0.1 %叠氮 钠的 PBS缓冲液）在 4°C下避光固定 15分钟。 用流式细胞仪检测激 活的淋巴细胞及其亚群的活化标志 CD69的分子表达。 比较肿瘤抗原 刺激组、 自身抗原刺激对照组和空白对照的结果。 判断患者针对肿瘤 的特异性免疫功能状态。

Claims

权 利 要 求
1. 一种用于体外监测个体对器官移植物、 肿瘤的特异性免疫细 胞功能的方法， 其包括 1) 将含有免疫细胞的该个体的受检样品与来 自器官移植物供者的抗原或包含该供者 MHC /HLA类型的 MHC/HLA 分子库、或与该个体自身的肿瘤抗原或同类肿瘤抗原库一起培养一段 足以活化免疫细胞的时间， 2)将培养后的免疫细胞分离和 /或分类并测 定其活化指标； 其中所述来自器官移植物供者的抗原为全细胞或含有 MHC/HLA的亚细胞成分或 MHC/HLA分子， 或所述该个体自身的肿 瘤抗原是全肿瘤细胞或含有大部分细胞物质的细胞提取物； 该方法还 包括设置以所述个体自身的抗原或非肿瘤抗原作为刺激抗原的对照。
2. 权利要求 1的方法，其中所述对照中的抗原与权利要求 1 - 1 ) 中的抗原平行制备。
3. 权利要求 1的方法，其中所述 MHC/HLA分子库为 MHC/HLA I类分子库、 MHC/HLA II类分子库、 或 MHC/HLA I类和 II类分子的 混合分子库。
4. 权利要求 1 的方法， 其中所述肿瘤抗原库为全细胞肿瘤抗原 库、 亚细胞肿瘤抗原库或单一肿瘤抗原或多种纯化组分所組成的肿瘤 抗原库。
5. 权利要求 1的方法， 其中所述培养时间为 20分钟到 24小时。
6. 权利要求 5的方法， 其中所述培养时间为 30分钟到 6小时。
7. 权利要求 1 的方法， 其中所述受检样品为外周全血、 骨髓、 任何生物体液。
8. 权利要求 7 的方法， 其中所述生物体液为关节液、 胸腔液、 腹腔液或髓脊液。
9. 权利要求 1 的方法， 其中所述免疫细胞为一种淋巴细胞或其 亚群、 NK细胞、 抗原提呈细胞、 血小板、 红细胞、 骨髓细胞、 干细 胞； 或免疫细胞前体细胞。
10. 权利要求 1的方法， 其中所述细胞分离 /分类方法为离心、 密 度梯度离心、 自然沉降、 非特异性吸附、 固相吸附、 免疫磁珠吸附、 聚苯乙烯微珠免疫吸附、 聚苯乙烯微孔板吸附， 或任何使用单或多标 记技术对各种免疫细胞进行分类鉴别的任何方法。
11. 权利要求 1的方法， 其中所述活化指标为细胞形态变化、 细 胞数目变化、 细胞酶学变化、 细胞内离子浓度变化、 细胞内外受体变 化、 细胞膜通透性变化、 细胞吞噬功能变化、 细胞活化标志蛋白表达、 细胞内能量变化、 细胞分泌各种生物因子变化、 细胞核变化、 细胞功 能变化、 或细胞蛋白质浓度变化。
12. 权利要求 Γ的方法， 其中所述抗原或肿瘤抗原制备成可以多 次使用的储备物的形式。
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