CN111643525A - Application of inducing immunological rejection reaction in tumor treatment and method thereof - Google Patents
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Abstract
The invention belongs to the technical field of biomedical engineering, and particularly relates to application of an immunological rejection reaction in tumor treatment and a method thereof. Method for tumor therapy by eliciting an immune rejection response, comprising in particular sorting CD4+T cells and injection of CD4+T cells. The immune rejection therapy skillfully utilizes the immune rejection reaction of an organism to realize the treatment effect on the tumor, and provides a new method for treating the tumor. The cells required by the invention are simple to prepare, do not need to be amplified and cultured in vitro, and the allogeneic transplantation exogenous cells basically cause the generation of a receptorImmunological rejection, high curative effect and universality.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering, and particularly relates to application of an immunological rejection reaction in tumor treatment and a method thereof.
Background
In recent years, the incidence and mortality of cancer has increased rapidly worldwide, becoming one of the leading causes of death in most regions. In 2018, the research data of 185 countries and 36 tumors by the international cancer research institution show that about 1810 thousands of new cancer cases and 960 thousands of cancer death cases exist around the world, which is one of the problems to be overcome by human beings. The traditional method for treating the tumor mainly comprises operations, chemotherapy, radiotherapy and tumor targeted drug treatment. The primary and metastatic focus of some solid tumors can be directly excised by surgery, but the metastasis of tumor cells and the recurrence of tumors cannot be fundamentally controlled. Radiotherapy and chemotherapy respectively utilize radioactive rays and chemical drugs to kill tumor cells, the curative effect of the radiotherapy and the chemotherapy depends on the radiosensitivity and the chemo-therapeutic drug sensitivity of the tumor cells, although the tumor cells sensitive to treatment can be directly killed, the radiotherapy and the chemotherapy often cause the damage to normal tissue cells of an organism which is difficult to control in the treatment process, and also can generate serious toxic and side effects and drug resistance, so that the treatment window is narrow and the effectiveness is low, and a tumor patient suffers great pain in the treatment process.
The tumor immunotherapy is a new tumor treatment method developed in recent years, and has the advantages of good curative effect, high safety, small side effect, low drug resistance and the like. The current tumor immunotherapy mainly comprises immune checkpoint blocker therapy, adoptive cell therapy, tumor vaccine and the like. However, the following problems still exist in this field: 1) due to the characteristics of individual difference, tumor heterogeneity, tumor microenvironment difference and the like of patients, the overall effective rate of tumor immunotherapy is lower, and the population is less; 2) most of PD-1/PD-L1-based drug combination strategies lack clinical evidence and basic theory support; 3) in addition to the PD-1/PD-L1 signal pathway, the research on other immune escape mechanisms is not deep enough, and the development of therapeutic drugs aiming at the targets has a certain distance. 4) The current tumor immunotherapy has complex process and high treatment cost.
Immune rejection refers to the process by which the body destroys the graft (allogeneic cells, tissues or organs) by a specific immune response. Generally after the transplantationThe recipient recognizes the graft antigen and generates a response, and the immune cells in the graft may also recognize the recipient antigen tissue and generate a response. Autologous or allogeneic normal cells, tissues and organs are frequently used clinically to replace the diseased or functionally defective parts so as to maintain and reconstruct the physiological functions of the body, but the concomitant immune rejection seriously hinders the clinical application of cell, tissue and organ transplantation techniques. In organ transplantation, the survival of organ transplantation is promoted by reducing immune rejection reaction usually through reducing the immunogenicity of donor histocompatibility antigen or reducing the immune intensity of recipient cells, and an effective method is provided for treating chronic terminal diseases such as liver, kidney, heart and the like. Therefore, the current research mainly aims at reducing the immune rejection, develops some immunosuppressive agents and the like, and does not find the positive effect of the immune rejection on the body. Moreover, no research related to tumor treatment by using immunological rejection is found at present, and the invention is used for treating the tumor by inducing the immunological rejection in a body through the transplantation of allogeneic immune cells. In addition to exerting an anti-tumor response via its effector cells, immunological rejection may also be induced by inducing tumor cell-specific CD8+The T cells play a specific anti-tumor immune response, improve the recognition and killing effects of autoimmune cells on tumors, effectively reduce the immune escape of the tumors, and better mobilize the immune system of an organism to directionally participate in tumor treatment.
The most similar realization scheme at present is adoptive cell therapy in the immunotherapy of tumor, which is a biological method for promoting host anti-tumor immunity by inputting immune cells amplified in vitro or processed to exert direct anti-tumor effect so as to treat advanced refractory tumor. There are currently 3 approaches to adoptive treatment of tumors using T cells: obtaining tumor infiltrating lymphocytes from a tumor tissue specimen of a patient; secondly, transforming TCR-T or CAR-T by a genetic engineering method; and thirdly, separating and amplifying self T cells from the peripheral blood of the patient. The methods are characterized in that T cells separated from the same individual are amplified or modified and then are infused back into the individual to play an anti-tumor role, so that immune rejection caused by adoptive cells is avoided.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a method for directly treating tumor using allogeneic T cells, instead of avoiding, by using immune rejection. The tumor treatment effect is skillfully realized by utilizing the immune rejection of the organism, the method is simple and easy to operate, the defects of the existing method can be avoided, and a new thought and method are provided for the tumor treatment.
In order to achieve the purpose, the invention adopts the following technical scheme:
use of an induced immune rejection in the treatment of a tumour, said induced immune rejection being induced by transplantation of allogeneic immune cells.
A method for treating tumor by inducing immune rejection reaction, comprising the following steps:
1. sorting CD4+T cells
1.1 grinding the spleen to prepare a cell suspension for later use.
1.2 and sorting mouse CD4+Magnetic bead binding of T cells
The cell suspension obtained in 1.1 above was mixed with sorted mouse CD4+And mixing the magnetic beads of the T cells uniformly, incubating on ice for 30 minutes, adding PBS, centrifuging, discarding supernatant, and resuspending for later use.
1.3 rinsing MS sorting column
5ml of PBS was added to the column and the liquid was allowed to flow through naturally.
1.4 sample application
And (3) adding the cell suspension incubated in the step (1.2) into the sorting column rinsed in the step (1.3), enabling liquid to flow through naturally, and adding 5ml of PBS to wash the sorting column after all the sample flows through.
1.5 isolation of bound CD4+T cells
After washing, the column was removed and placed in a new 15ml centrifuge tube, 2.5ml PBS was added and the cells were rapidly eluted with a pusher and then eluted once again.
1.6 centrifuging the cell suspension obtained in 1.5 at 1000rpm for 5min, resuspending and counting the cells, adjusting the cell concentrationDegree 4 x 107/ml。
2. Injection of CD4+T cell: intratumoral injection of CD4 sorted in step 1+T cells, by multiple injections. Or tail vein injection into tumor-bearing mice.
The invention has the beneficial effects that:
1) the cells required by the invention are simple to prepare, do not need to be subjected to in vitro amplification culture, and the allogeneic transplantation exogenous cells basically cause the receptor to generate immune rejection, so the treatment efficiency is high and the universality is realized.
2) The immune rejection therapy skillfully utilizes the immune rejection reaction of an organism to realize the treatment effect on the tumor, and provides a new method for treating the tumor.
Drawings
FIG. 1 is a schematic of an experimental technique;
FIG. 2 is a schematic representation of tumor size;
FIG. 3 is a schematic representation of tumor weight;
FIG. 4 shows CD4 in tumor+T cell scale graph;
FIG. 5 shows CD8 in tumor+T cell scale graph;
FIG. 6 is CD8 in peripheral blood+T cell ratio.
Detailed Description
For the convenience of understanding, the technical scheme of the invention is further described in detail by the implementation case in combination with the attached drawings:
as shown in fig. 1 to 6, a method for treating tumor by inducing immune rejection comprises the following steps:
1. recovering mouse colorectal cancer MC38 cell line, carrying out passage and inoculation
Taking out the freezing tube from the liquid nitrogen container, quickly immersing the freezing tube into warm water at 37 ℃, and shaking from time to melt the freezing tube as soon as possible; after melting, quickly transferring to an ultra-clean workbench, disinfecting the surface by using 75% alcohol, opening a cover, sucking out cell suspension, transferring to a sterile centrifuge tube, and adding 10 times of 1640 culture medium containing 10% fetal calf serum; centrifuging at 1000rpm for 5min after mixing; discarding the supernatant, adding 10%Re-suspending the cells in 1640 culture medium of fetal calf serum, uniformly mixing, and transferring to a 10cm culture dish; standing and culturing at 37 ℃ in a 5% CO2 incubator; the culture solution was changed the next day and the culture was continued. Observing the state of the cells, and carrying out passage 2-3 times after the cells are fully plated so as to enable the cells to be in the optimal state. Cells were digested with pancreatin containing 0.25% EDTA, washed twice with PBS, all media removed, resuspended in PBS and cell counted. Adjusting the cell concentration to 2 x 107And/ml, inoculating 100ul of cell suspension to the back of a C57BL/6 mouse, establishing a tumor-bearing mouse model, wherein the number of inoculated cells is 2 × 106A/only.
2. Sorting CD4+T cells
2.1 grinding spleen
C57BL/6 and BALB/C mouse spleens were removed from the clean bench, placed in sterile petri dishes, 2ml of pre-cooled PBS was added and triturated. The milled cell suspension was filtered through a 200-mesh filter into a 15ml centrifuge tube, 10ml of PBS was added, and the tube was centrifuged at 1000rpm for 5 min. Discarding supernatant, adding 1ml erythrocyte lysate, mixing uniformly by vortex, lysing for 3-5min, adding 10ml PBS to terminate the lysis reaction, mixing uniformly, centrifuging at 1000rpm for 5min, discarding supernatant, and adding 150ul PBS to resuspend cells for later use.
2.2 and sorting mouse CD4+Magnetic bead binding of T cells
The cell suspension obtained in 2.1 above was mixed with sorted mouse CD4+T cells were mixed with beads (20 ul per mouse) and incubated on ice for 30min with shaking every 10 min. 10ml PBS was added, centrifuged at 1000rpm for 5min, the supernatant was discarded, and 5ml PBS was added for resuspension.
2.3 rinsing MS sorting column
The MS separation column was vertically attached to a MiniMACS separator, and a 15ml centrifuge tube was placed below to collect the waste. 5ml of PBS was added to the column and the liquid was allowed to flow through naturally.
2.4 sample application
And adding the cell suspension incubated in the step 2.2 into the sorting column rinsed in the step 2.3, enabling the liquid to flow through naturally, and adding 5ml of PBS to wash the sorting column after the sample flows through completely.
2.5 isolation of bound CD4+T cells
After washing, the column was removed and placed in a new 15ml centrifuge tube, 2.5ml PBS was added and the cells were rapidly eluted with a pusher and then eluted once again.
2.6 the cell suspension obtained in 2.5 was centrifuged at 1000rpm for 5min, the cells were resuspended and counted, and the cell concentration was adjusted to 4 x 107The/ml was used in the subsequent experiments.
3. Tumor-bearing mice were grouped and injected with CD4+T cells
C57BL/6 tumor-bearing mice were randomly grouped, control group: intratumoral injection of CD4 sorted from spleen of C57BL/6 mice+T cells (IM-WT CD4+T cell), intervention group 1: intratumoral injection of CD4 sorted from spleens of BALB/c mice+T cells (IM-BALB/c CD 4)+T cell), intervention group 2: tail intravenous injection of CD4 sorted from spleen of BALB/c mice+T cells (IV-BALB/c CD 4)+T cell). CD4 injected per mouse+T cell number 2 × 10650ul of liquid, wherein the intratumoral injection group was performed by multipoint injection. The specific procedure is shown in fig. 1, with MC38 cells seeded on day 0 and tumor size measurements started on day 9 and were taken once a day. Injection of CD4 on days 9 and 11+T cell, mice were treated on day 13 and tumor tissue was removed.
4. Flow cytometry detection of tumor tissue CD4+T cell, CD8+T cell proportion and CD8 in peripheral blood+T cell ratio
4.1 detection of CD4 in tumor tissue+T cell, CD8+Digestive juice prepared according to T cell ratio of 4.1.1
Preparing digestive juice containing 0.1% -0.2% collagenase by using 1640 culture medium without serum and double antibody, and then adding nuclease (1:5000 dilution)
4.1.2 digestion of tumor tissue
The soybean particle-sized tumor tissue is taken and placed in a 1.5ml centrifuge tube, 500ul of digestive juice is added, the digestive juice is cut into pieces with scissors (the smaller the tissue piece is better), then the pieces are transferred into a 15ml centrifuge tube, 10ml of digestive juice is added, and the mixture is placed in a constant temperature shaking table at 37 ℃ and digested for 1 hour. After digestion, the mixture is filtered into a new centrifugal tube by a 200-mesh filter screen, centrifuged at 1000rpm for 5min, and the supernatant is discarded. The cells were washed once with PBS containing 5% serum, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Resuspend the cells in PBS containing 5% serum, filter once with 200 mesh filter, and dispense into flow tubes for subsequent flow antibody labeling.
4.1.3 labeling of flow antibodies
The flow antibodies CD45, CD3, CD4 and CD8 were added to the 4.1.2 cells, incubated at 4 ℃ for 30min, washed once with PBS, centrifuged at 1000rpm for 5min, and the supernatant was discarded. PBS was added to resuspend the cells, which were then filtered into a fresh flow tube and tested on the machine.
4.2 detection of CD8 in peripheral blood+T cell ratio
The peripheral blood of the mouse is taken, red blood cells are cracked by red blood cell cracking liquid for 5min each time and 2-3 times, the red blood cells are centrifuged for 5min at 1000rpm each time, and the supernatant is discarded. The resulting cells were washed once with PBS, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in PBS. CD3, CD4 and CD8 flow antibodies are added, the mixture is incubated at 4 ℃ for 30min, then PBS is added for washing cells once, the mixture is centrifuged at 1000rpm for 5min, and the supernatant is discarded. PBS was added to resuspend the cells, which were then filtered into a fresh flow tube and tested on the machine.
Claims (7)
1. The application of the immune rejection reaction in tumor treatment is characterized in that: the induced immune rejection is induced by transplanting allogeneic immune cells.
2. Use of the method of claim 1 for eliciting an immune rejection response in the treatment of tumors, wherein the method comprises the steps of: the allogeneic immune cells are CD4 from allogeneic sources+T cells.
3. Use of the method of claim 2 for eliciting an immune rejection response in the treatment of tumors, wherein the method comprises the steps of: the immune cells of the allogeneic source are artificially extracted and separated CD4 of the allogeneic source of the same species+T cells.
4. For tumour therapy by eliciting an immune rejection responseThe method is characterized in that: the method specifically comprises the following steps: 1) sorting CD4+T cells, 2) injection of CD4+T cells.
5. The method for the treatment of tumors by eliciting an immune rejection response according to claim 4, wherein: the step 1) specifically comprises the following steps:
1.1 grinding spleen to prepare cell suspension for later use;
1.2 and sorting mouse CD4+Magnetic bead binding of T cells
The cell suspension obtained in 1.1 above was mixed with sorted mouse CD4+Mixing the magnetic beads of the T cells uniformly, incubating on ice for 30 minutes, adding PBS, centrifuging, discarding the supernatant, and resuspending for later use;
1.3 rinsing MS sorting column
Adding PBS into the separation column to allow the liquid to flow through naturally;
1.4 sample application
Adding the cell suspension incubated in the step 1.2 into the sorting column rinsed in the step 1.3, enabling liquid to flow through naturally, and adding PBS to wash the sorting column after all the samples flow through;
1.5 isolation of bound CD4+T cells
After washing is finished, taking down the separation column, putting the separation column into a new centrifugal tube, adding PBS, rapidly eluting cells by using a pusher, and then repeatedly eluting once;
1.6 the cell suspension obtained in step 1.5 is centrifuged, the cells are resuspended and counted, and the cell concentration is adjusted.
6. The method for the treatment of tumors by eliciting an immune rejection response according to claim 4, wherein: the injection method in the step 2) is to directly inject the separated immune cells at the position of the tumor focus or inject the immune cells into the body through vein injection at intervals for a plurality of times.
7. The method for the treatment of tumors by eliciting an immune rejection according to any one of claims 4-6, wherein: and 2) adopting multi-point injection in the injection method in the step 2).
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| CN1981031A (en) * | 2004-03-05 | 2007-06-13 | 宾久法尼亚大学理事会 | Regulatory T cells and their use in immunotherapy and suppression of autoimmune responses |
| CN104394877A (en) * | 2012-05-08 | 2015-03-04 | 约翰·霍普金斯大学 | Methods and compositions for infusion of transiently engrafting, selected populations of allogeneic lymphocytes to treat cancer |
| US20200163997A1 (en) * | 2012-05-08 | 2020-05-28 | The Johns Hopkins University | Cancer immunotherapy using transfusions of allogeneic, tumor-specific cd4+ t cells |
| CN108348552A (en) * | 2015-09-10 | 2018-07-31 | 纪念斯隆-凯特林癌症中心 | The method for treating Huppert's disease and plasma cell leukemia by T cell therapy |
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| HEATHER J. SYMONS ET AL.: "The Allogeneic Effect Revisited: Exogenous Help for Endogenous, Tumor-Specific T Cells", 《BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION》 * |
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Inventor after: Yan Fenglian Inventor after: Zhu Yuzhen Inventor after: Zhang Hui Inventor before: Xiong Huabao Inventor before: Yan Fenglian Inventor before: Zhu Yuzhen Inventor before: Zhang Hui |