CN108220234B - In-vitro amplification method of non-sentinel lymph node-derived anti-tumor T cells - Google Patents

In-vitro amplification method of non-sentinel lymph node-derived anti-tumor T cells Download PDF

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CN108220234B
CN108220234B CN201611126773.XA CN201611126773A CN108220234B CN 108220234 B CN108220234 B CN 108220234B CN 201611126773 A CN201611126773 A CN 201611126773A CN 108220234 B CN108220234 B CN 108220234B
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杨�远
修瑾
陈阶
唐景玲
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Abstract

The invention discloses an in vitro amplification method of anti-tumor T cells from non-sentinel lymph nodes, which comprises four steps of antigen sensitization and immunosuppression blocking, T cell subset separation and proliferation stimulation, T cell subset re-stimulation and rapid proliferation and harvesting. The method has the characteristics of facilitating the specific activation of the T cells, improving the anti-tumor capacity of the T cells, increasing the number of the T cells with tumor specificity, along with simple and convenient operation and easy popularization.

Description

In-vitro amplification method of non-sentinel lymph node-derived anti-tumor T cells
Technical Field
The invention belongs to the technical field of medical treatment and health, and particularly relates to an in-vitro amplification method of non-sentinel lymph node-derived anti-tumor T cells.
Background
The tumor immunotherapy is to apply immunological principle and method to raise the immunogenicity and/or the sensitivity to killing lymph cell of tumor cell, excite and strengthen the antitumor immune response of body, and to infuse immune cell and/or effector molecule into host body to cooperate with body's immune system to kill tumor and inhibit tumor growth.
Among the numerous tumor immunotherapy protocols, "adoptive T lymphocyte infusion" is an important one, which refers to the process of infusing autologous or allogeneic anti-tumor T lymphocytes activated, expanded in vitro into a patient, killing the tumor cells in the patient.
In adoptive T cell immunotherapy, the source of T cells is mainly two-fold, peripheral blood and tumor tissue. Among these, peripheral blood T cells are the easiest to collect and isolate, but T cells of this origin are not stimulated by tumors since they are far from the tumor region (here, mainly non-hematologic tumors) and thus have a lower tumor-specific T cell content; meanwhile, most peripheral blood T cells belong to effector T cells at the final stage of differentiation, the service life is short, and the amplification efficiency is poor. Therefore, at present, most of the adopted T cells are tumor infiltrating T cells, namely T cells separated from tumor tissues, the T cells are in close contact with tumors and have partial tumor recognition capability, but the T cells are low in content and difficult to separate, and the anti-tumor capability is inhibited because the T cells are in a tumor microenvironment.
Lymph nodes are secondary immune organs unique to mammals, contain a large number of lymphocytes, and are sites where immune recognition and immune effects occur. After the lymphocyte in lymph node is stimulated, it can produce lots of cell factor, immune activation protein and antibody, etc. and can produce killing action for pathogenic substance or tumor cell. Therefore, in theory, the lymph nodes should be an immune barrier that prevents the migration and spread of tumor cells, and tumor-specific T cells are present in the lymph nodes.
However, with the development of tumor immunity research, it was found that immunosuppression in the tumor microenvironment (a localized region composed of tumor cells together with their surrounding stromal cells and lymphocytes) can prevent lymphocytes from functioning normally. In tumor metastasis lymph nodes, the negative immune regulation produced by tumor cells can induce the differentiation of lymphocytes to immunosuppressive cells, inactivate the lymphocytes by secreting immunodetection point proteins, and even reverse the function of the lymphocytes to promote the growth and metastasis of tumors. It is because of this "acclimation" of the tumor cells to the microenvironment that lymphocytes cannot recognize the tumor cells or lose their intrinsic anti-tumor activity, resulting in the lymph nodes of the immune barrier losing barrier function and even becoming a conduit for aiding tumor metastasis.
Therefore, selection of appropriate lymph nodes is critical to obtain specific anti-tumor T cells.
Research shows that the key to obtaining the good effect of the adoptive T cell immunotherapy is to expand the number of a large number of tumor-specific T cells, and the number of the feedback T cells is in positive correlation with the anti-tumor effect. Therefore, it is a technical problem to be solved in the art to obtain a high efficiency T cell in vitro amplification method.
At present, for the existing in vitro amplification method of T cells, besides the insufficient number of tumor specific T cells obtained in the processes of T cell material drawing, in vitro activation and amplification, the following 3 problems exist:
(1) tumor homogenate lysate is used as an antigen to stimulate the proliferation of T cells, and the components of the tumor homogenate are complex and contain a large amount of factors released by dead cells and immunosuppressive factors, and the components are not beneficial to the specific activation of the T cells;
(2) induced immunosuppressive effects (such as programmed death receptor 1 increase and the like) can be caused in the culture process, and the anti-tumor capacity of the T cells is reduced;
(3) the use of a single cytokine to expand T cells results in polarization of a subset of T cells, making the anti-tumor function of the single subset weaker than the mixed subset.
Disclosure of Invention
The invention aims to overcome the difficulties and provide an in vitro amplification method of the non-sentinel lymph node-derived anti-tumor T cells, which is beneficial to the specific activation of the T cells, can improve the anti-tumor capability of the T cells and the number of the tumor-specific T cells, is simple and convenient to operate and is easy to popularize.
The invention relates to an in vitro amplification method of non-sentinel lymph node derived anti-tumor T cells, which comprises the following steps:
(2) antigen sensitization and immunosuppression blocking:
a. mixing lymphocyte culture medium and fetal calf serum at a volume ratio of 100:2-5 to obtain lymphocyte culture medium and fetal calf serum mixture; adding interleukin 2 into a lymphocyte culture medium and a fetal calf serum mixture, and adjusting the concentration of the interleukin 2 in the lymphocyte culture medium and the fetal calf serum mixture to be 100-1000 units/ml to obtain a culture solution A for later use;
b. adding non-sentinel lymph node-derived anti-tumor T cells into a part of the culture solution A, and adjusting the volume of the culture solution to make the density of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution A be 1 × 106-2*106Culturing for 12-24 hr per ml;
c. adding an anti-immunosuppressive antibody into another part of the culture solution A to perform immunosuppressive blocking to obtain a culture solution B for later use;
d. sucking all the non-sentinel lymph node-derived anti-tumor T cells obtained after culturing for 12-24 hours in the culture solution A, transferring the cells into a centrifuge tube, and centrifuging for 3-5 minutes at the centrifugal force of 300-350 g; discarding the upper culture solution after the centrifugation is finished;
e. adding a culture solution B into the non-sentinel lymph node-derived anti-tumor T cells from which the upper culture solution is discarded after the centrifugation is completed, and adjusting the concentration of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution B to 0.5 x 106-1*106Culturing for 24-48 h, adding the conventional artificially synthesized tumor polypeptide for cell culture, adjusting the concentration of the conventional artificially synthesized tumor polypeptide for cell culture in the added system to be 0.5-1 microgram/ml, and continuously culturing for 24-48 h to obtain the anti-tumor T cell culture derived from non-sentinel lymph nodes;
(2) t cell subset isolation and stimulation of proliferation:
performing subgroup sorting on the non-sentinel lymph node-derived anti-tumor T cell culture by adopting a conventional sorting method to obtain a CD4+, a CD 25-helper T cell subgroup and a CD8+ killer T cell subgroup, respectively placing the two subgroups into an X-VIVO culture medium for independent culture, and adding an activating reagent, wherein the culture density is 1 × 105-1*107Each group is subjected to half-amount liquid change every 3 days, and cells are counted after 9-12 days of culture;
wherein, the activating reagent of the CD4+, CD 25-helper T cell subgroup is as follows: CD3/CD28 magnetic beads and interleukin 7, wherein the CD3/CD28 magnetic beads are adjusted to be 1-5 per cell in an added system, and the concentration of the interleukin 7 is 5-20 ng/ml;
the activating agents for the CD8+ killer T cell subset were: anti-CD3, interleukin 7 and interleukin 21, adjusting the concentration of the anti-CD3 in the added system to be 5-50 ng/ml, the concentration of the interleukin 7 to be 2-20 ng/ml and the concentration of the interleukin 21 to be 1-5 ng/ml;
(3) restimulation and rapid proliferation of T cell subsets:
after the upper stage culture is finished, adding peripheral blood mononuclear cells irradiated by 15-35 kilogray X rays, wherein the number ratio of the peripheral blood mononuclear cells to cells obtained after culturing for 9-12 days by CD4+, CD 25-helper T cell subset and CD8+ killer T cell subset is 100-500:1, adding conventional artificially synthesized tumor polypeptides for cell culture, adjustingThe concentration of the artificially synthesized tumor polypeptide in the added system for cell culture is 0.5-3 micrograms/ml, the artificial synthesized tumor polypeptide is continuously cultured for 12-15 days, and the culture density is controlled to be 1 x 10 in the culture process6-2*106Per milliliter;
(4) harvesting:
after the culture is finished, respectively sampling and culturing cells from the CD4+, CD 25-helper T cell subset and the CD8+ killer T cell subset, carrying out microorganism, endotoxin, flow cytometry and cytokine secretion analysis, and mixing the two subsets of cells to obtain the compound.
In the method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, the anti-immunosuppressive antibody is anti-programmed death receptor 1, and the concentration of the anti-programmed death receptor 1 in the culture solution a is adjusted to 5 to 30 μ g/ml.
In the method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, the anti-immunosuppressive antibody is anti-cytotoxic T lymphocyte-associated antigen 4, and the concentration of the anti-cytotoxic T lymphocyte-associated antigen 4 in the culture solution a is adjusted to 5-30 μ g/ml.
In the method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, the anti-immunosuppressive antibody is anti-T cell immunoglobulin mucin 3, and the concentration of the anti-T cell immunoglobulin mucin 3 in the culture solution a is adjusted to 5-30 μ g/ml.
In the method for in vitro amplification of the non-sentinel lymph node-derived anti-tumor T cell, the anti-immunosuppressive antibody is a combination of any two of anti-programmed death receptor 1, anti-cytotoxic T lymphocyte-associated antigen 4 and anti-T cell immunoglobulin mucin 3 in a volume ratio of 1:1, and the concentrations of the anti-programmed death receptor 1, the anti-cytotoxic T lymphocyte-associated antigen 4 and the anti-T cell immunoglobulin mucin 3 in the culture solution a are adjusted to be 5-30 micrograms/ml respectively.
In the method for in vitro amplification of the non-sentinel lymph node-derived anti-tumor T cell, the anti-immunosuppressive antibody is a combination of an anti-programmed death receptor 1, an anti-cytotoxic T lymphocyte-associated antigen 4 and an anti-T cell immunoglobulin mucin 3 in a volume ratio of 1:1:1, and the concentrations of the anti-programmed death receptor 1, the anti-cytotoxic T lymphocyte-associated antigen 4 and the anti-T cell immunoglobulin mucin 3 in the culture solution a are adjusted to be 5-30 micrograms/ml respectively.
In the method for in vitro amplification of non-sentinel lymph node-derived anti-tumor T cells, the non-sentinel lymph node-derived anti-tumor T cells are tumor draining lymph nodes located between sentinel lymph nodes and distal lymph nodes without infiltration of tumor cells.
The method for in vitro amplification of the non-sentinel lymph node-derived anti-tumor T cells, wherein the non-sentinel lymph node T cells are isolated by the following method:
(1) selecting lymph nodes from the middle section of the draining lymph node:
taking an isolated tumor tissue, injecting patent Blue V into the isolated tumor tissue at 4 points, namely the upper, the lower, the left and the right of the isolated tumor tissue, wherein each point is injected with 0.2-1 ml, and after injection, the redundant patent Blue at the injection point is sucked by gauze;
searching blue-stained lymph nodes, namely tumor draining lymph nodes TDLNs according to the trend of blue-stained lymph vessels, wherein the first or a plurality of blue-stained lymph nodes are sentinel lymph nodes;
taking a primary tumor as a center, taking 3-4 tumor draining lymph nodes which are positioned between a sentinel lymph node and a distal lymph node, namely 5-7 cm away from the center of the primary tumor, wherein each tumor draining lymph node takes the maximum diameter as an axis;
(2) removing fat tissue attached to lymph nodes by flow cytometry identification and separation method:
cutting the tumor draining lymph node into small pieces of 1-3 mm, adding DNase and collagenase of which the volume is 3-5 times that of the tumor draining lymph node, digesting the small pieces at 36.5-37.5 ℃ for 30-60 minutes, and blowing and sucking the small pieces for 3-4 times by using a syringe needle in the digestion process, wherein the DNase and the collagenase are mixed according to the volume ratio of 1: 1;
after digestion, sieving, taking cell sap, carrying out flow cytometry identification after staining the cell sap by using a tumor cell specific staining antibody, and culturing cells with negative staining results for 8-15 hours;
(3) enzyme-linked immunosorbent assay (ELISA) identification:
and (3) performing enzyme-linked immunosorbent assay spot identification on the negative cells cultured for 8-15 hours by adopting a gamma interferon enzyme-linked immunosorbent assay spot detection kit, and selecting the cells with positive identification results to obtain the non-sentinel lymph node source anti-tumor T cells.
The in vitro amplification method of the anti-tumor T cells derived from the non-sentinel lymph nodes is characterized in that the tumor cell specific staining antibody is composed of an epithelial cell adhesion molecule, cytokeratin 20, a carcinoembryonic antigen and a cell glycoprotein 199 according to a volume ratio of 1:1:1: 1.
In the method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, the syringe needle is a 21-gauge syringe needle.
Compared with the prior art, the invention has obvious beneficial effects, and the technical scheme can show that: the in vitro amplification method of the non-sentinel lymph node source anti-tumor T cells provided by the invention supplements each technical step, and the determined culture reagent combinations and process parameters are necessary conditions for obtaining high-quality and high-quantity tumor specific T cells.
Firstly, the invention adopts non-sentinel lymph nodes to resist tumor-derived T cells as the starting material for in vitro amplification, and has the following advantages: (1) the lymph nodes are rich in T cells, are organs of normal organisms and have no difference among different tumors, so that the lymph nodes are a stable T cell source; (2) lymph nodes in the tumor area (including tumor cell-infiltrated lymph nodes and non-infiltrated lymph nodes) are stimulated by tumor antigen, and tumor-specific T cells exist; (3) the least specific anti-tumor capability of the lymph nodes closest to the primary tumor (especially those infiltrated by tumor cells); lymph nodes too far from the primary tumor cannot receive tumor antigen signals due to the distance, and therefore have no specific anti-tumor capacity; the non-tumor infiltrating lymph nodes (i.e. non-sentinel lymph node-derived anti-tumor T cells) in the middle are not domesticated by tumor cells, so that the immunosuppression is not strong, and the lymph nodes are positioned in the areas capable of receiving tumor antigen signals, so that the lymph nodes with the strongest specific anti-tumor capability are obtained.
In addition, the invention combines an immunodetection point inhibitor in the culture process, breaks through the tumor immunosuppressive effect, and can effectively maintain the antitumor activity of T cells.
Thirdly, the invention adopts the artificially synthesized polypeptide segment to stimulate the proliferation of the T cells, on one hand, the tumor specificity of the T cells is enhanced, and on the other hand, the purified polypeptide does not cause the immunosuppressive effect.
Finally, the invention separates the sub-groups of CD4+, CD 25-and CD8+ T cells after immune recognition, respectively adopts two different culture schemes to carry out targeted amplification, and the two sub-groups of cells are mixed after amplification, so that the cells of the two sub-groups can be respectively proliferated, and the anti-tumor effect of 1+1 & gt 2 is exerted.
The method has the characteristics of facilitating the specific activation of the T cells, improving the anti-tumor capacity of the T cells, increasing the number of the T cells with tumor specificity, along with simple and convenient operation and easy popularization.
Detailed Description
The advantageous effects of the amplification method of the present invention are further illustrated by the following experimental examples.
Examples of the experiments
The comparison of the number, proliferation efficiency, expression of immunosuppressive molecules and immunoactivating molecules and antitumor capability of the non-sentinel lymph node-derived antitumor T cells obtained by amplification in example 3 of the present invention with those obtained by amplification by a conventional method specifically includes:
1. regarding the number of cells:
taking the number of 1.6 x 106The non-sentinel lymph node-derived anti-tumor T cells were amplified in vitro by the method described in example 3 of the present invention, and counted after completion of the culture, so that the total amount of the non-sentinel lymph node-derived anti-tumor T cells was 413 × 106And (4) respectively.
Taking the same number as 1.6 x 106The individual T cells derived from non-sentinel lymph nodes were amplified in vitro by a conventional method, and counted after completion of the culture, and the total amount of the obtained anti-tumor T cells derived from non-sentinel lymph nodes was 45.6 × 106And (4) respectively.
The comparative experiment results show that:
as the invention adopts a specific culture scheme for different subgroups of lymphocytes, a larger number of cells can be obtained compared with the conventional method, and the ratio of the proliferation fold of the cells is 258: 28.
2. Regarding the proliferation efficiency:
the anti-tumor T cells derived from non-sentinel lymph nodes obtained by amplification in example 3 of the present invention and obtained by amplification in a conventional method were subjected to flow cytometry, and the percentage of proliferated activated T lymphocytes obtained by amplification in example 3 of the present invention was 83.2%, and the percentage of proliferated activated T lymphocytes obtained by amplification in a conventional method was 24.4%.
The comparative experiment results show that:
the invention uses artificially synthesized polypeptide for stimulation and combines with immunosuppressive blocking antibody, thereby improving the proliferation efficiency of T lymphocytes; the proliferation activated T lymphocyte obtained by the invention is 3.41 times of that obtained by the conventional culture method.
3. Regarding the expression of immunosuppressive and immunoactivating molecules:
the expression of immunosuppressive molecules and immunoactivation molecules of the non-sentinel lymph node-derived anti-tumor T cells obtained by amplification in example 3 of the present invention and by conventional amplification were measured by flow cytometry, and the expression percentage of immunosuppressive molecule programmed death receptor 1, the expression percentage of immunosuppressive molecule cytotoxic T lymphocyte-associated antigen 4, and the expression percentage of immunoactivation molecule CD69 in the non-sentinel lymph node-derived anti-tumor T cells obtained by amplification in example 3 of the present invention were 2.11%, 2.3%, 14.51%, and the expression percentage of programmed death receptor 1, 22.62%, and 5.67% of immunoactivation molecule CD69 in the non-sentinel lymph node-derived anti-tumor T cells obtained by conventional amplification.
The comparative experiment results show that:
the invention adds the antibody of the immune suppression molecule in the process of the expansion of the T lymphocyte, thereby leading the expression of the immune suppression molecule in the product after the expansion to be lower than that of the conventional expansion method; at the same time, the proportion of T lymphocyte activating molecules is increased.
4. With respect to antitumor ability:
the antitumor T cells derived from non-sentinel lymph nodes obtained by amplification in example 3 of the present invention and obtained by amplification in a conventional method were measured for their antitumor ability by ELISPOT, i.e., enzyme-linked immunospot assay, and the number of γ -interferon-secreting cells in the cells obtained by amplification in example 3 of the present invention was 467 and the number of γ -interferon-secreting cells in the cells obtained by amplification in a conventional method was 189.
The comparative experiment results show that:
the specific anti-tumor capability of the expanded non-sentinel lymph node source anti-tumor T cells is 2.46 times that of the traditional method.
Example 1
An in vitro amplification method of anti-tumor T cells derived from non-sentinel lymph nodes, comprising the following steps:
(1) antigen sensitization and immunosuppression blocking:
a. mixing the lymphocyte culture medium and fetal calf serum in a volume ratio of 100:2 to obtain a lymphocyte culture medium and fetal calf serum mixture; adding interleukin 2 into a lymphocyte culture medium and a fetal calf serum mixture, and adjusting the concentration of the interleukin 2 in the lymphocyte culture medium and the fetal calf serum mixture to be 100 units/ml to obtain a culture solution A for later use;
b. adding non-sentinel lymph node-derived anti-tumor T cells into a part of the culture solution A, and adjusting the volume of the culture solution to make the density of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution A be 1 × 106Culturing for 12 hours per milliliter;
c. adding an anti-programmed death receptor 1 into another part of the culture solution A for immunosuppression blocking, and adjusting the concentration of the anti-programmed death receptor 1 in the culture solution A to be 5 micrograms/ml to obtain a culture solution B for later use;
d. sucking all the non-sentinel lymph node-derived anti-tumor T cells obtained after culturing for 12 hours in the culture solution A, transferring the cells into a centrifuge tube, and centrifuging for 3 minutes with the centrifugal force of 300 g; discarding the upper culture solution after the centrifugation is finished;
e. adding a culture solution B into the non-sentinel lymph node-derived anti-tumor T cells from which the upper culture solution is discarded after the centrifugation is completed, and adjusting the concentration of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution B to 0.5 x 106A step of culturing for 24 hours, adding a conventional artificially synthesized tumor polypeptide for cell culture, adjusting the concentration of the conventional artificially synthesized tumor polypeptide for cell culture in an added system to be 0.5 micrograms/ml, and continuously culturing for 48 hours to obtain an anti-tumor T cell culture derived from non-sentinel lymph nodes;
(2) t cell subset isolation and stimulation of proliferation:
performing subgroup sorting on the non-sentinel lymph node-derived anti-tumor T cell culture by adopting a conventional sorting method to obtain a CD4+, a CD 25-helper T cell subgroup and a CD8+ killer T cell subgroup, respectively placing the two subgroups into an X-VIVO culture medium for independent culture, and adding an activating reagent, wherein the culture density is 1 × 105Each group is subjected to half-amount liquid change every 3 days, and cells are counted after 9 days of culture;
wherein, the activating reagent of the CD4+, CD 25-helper T cell subgroup is as follows: CD3/CD28 magnetic beads and interleukin 7, wherein the number of the CD3/CD28 magnetic beads in the added system is adjusted to be 1 per cell, and the concentration of the interleukin 7 in the added system is adjusted to be 5 nanograms per milliliter;
the activating agents for the CD8+ killer T cell subset were: anti-CD3, interleukin 7 and interleukin 21, wherein the concentration of the anti-CD3 in the added system is adjusted to be 5 ng/ml, the concentration of the interleukin 7 in the added system is adjusted to be 2 ng/ml, and the concentration of the interleukin 21 in the added system is adjusted to be 1 ng/ml;
(3) restimulation and rapid proliferation of T cell subsets:
after the upper stage culture is finished, adding peripheral blood mononuclear cells irradiated by 15 kilogray X rays, wherein the number ratio of the peripheral blood mononuclear cells to cells obtained by culturing CD4+, CD 25-helper T cell subset and CD8+ killer T cell subset for 9 days is 100:1, and adding the cellsCulturing with conventional artificially synthesized tumor polypeptide, adjusting the concentration of the artificially synthesized tumor polypeptide in the added system for cell culture to 0.5 microgram/ml, continuously culturing for 12 days, and controlling the culture density to 1 × 106Per milliliter;
(4) harvesting:
after the culture is finished, respectively sampling and culturing cells from the CD4+, CD 25-helper T cell subset and the CD8+ killer T cell subset, carrying out microorganism, endotoxin, flow cytometry and cytokine secretion analysis, and mixing the two subsets of cells to obtain the compound.
The method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, wherein: the non-sentinel lymph node derived anti-tumor T cells refer to tumor draining lymph nodes which are not infiltrated by tumor cells and are positioned between sentinel lymph nodes and distal lymph nodes; is obtained by adopting the following method for separation:
(1) selecting lymph nodes from the middle section of the draining lymph node:
taking an isolated tumor tissue, injecting patent Blue V into the isolated tumor tissue at 4 points, namely the upper, the lower, the left and the right of the isolated tumor tissue, injecting 0.2 ml of the injection at each point, and sucking redundant patent Blue at the injection point by gauze after the injection;
searching blue-stained lymph nodes, namely tumor draining lymph nodes TDLNs according to the trend of blue-stained lymph vessels, wherein the first or a plurality of blue-stained lymph nodes are sentinel lymph nodes;
taking a primary tumor as a center, taking 3 tumor draining lymph nodes which are positioned between a sentinel lymph node and a distal lymph node, namely, are 5 centimeters away from the center of the primary tumor, wherein each tumor draining lymph node takes the maximum diameter as an axis;
(2) removing fat tissue attached to lymph nodes by flow cytometry identification and separation method:
cutting the tumor draining lymph node into small pieces of 1 mm, adding DNase and collagenase with the volume 3 times that of the tumor draining lymph node, digesting the small pieces of the tumor draining lymph node for 30 minutes at 36.5 ℃, blowing and sucking the small pieces of the tumor draining lymph node for 3 times by using a syringe needle in the digestion process, and mixing the DNase and the collagenase according to the volume ratio of 1: 1;
after digestion, passing through a 40-micron screen, taking cell sap, carrying out flow cytometry identification after staining the cell sap by using a tumor cell specific staining antibody, and culturing the cells with negative staining results for 8 hours;
(3) enzyme-linked immunosorbent assay (ELISA) identification:
and (3) performing enzyme-linked immunosorbent assay spot identification on the negative cells cultured for 8 hours by adopting a gamma interferon enzyme-linked immunosorbent assay kit, and selecting the cells with positive identification results to obtain the non-sentinel lymph node-derived anti-tumor T cells.
Example 2
An in vitro amplification method of anti-tumor T cells derived from non-sentinel lymph nodes, comprising the following steps:
(1) antigen sensitization and immunosuppression blocking:
a. mixing the lymphocyte culture medium and fetal calf serum in a volume ratio of 100:5 to obtain a lymphocyte culture medium and fetal calf serum mixture; adding interleukin 2 into a lymphocyte culture medium and a fetal calf serum mixture, and adjusting the concentration of the interleukin 2 in the lymphocyte culture medium and the fetal calf serum mixture to be 1000 units/ml to obtain a culture solution A for later use;
b. adding non-sentinel lymph node-derived anti-tumor T cells into a part of the culture solution A, and adjusting the volume of the culture solution to make the density of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution A2 x 106Culturing for 24 hours per milliliter;
c. adding an anti-cytotoxic T lymphocyte-associated antigen 4 into another part of the culture solution A to perform immunosuppression blocking, and adjusting the concentration of the anti-cytotoxic T lymphocyte-associated antigen 4 in the culture solution A to be 30 micrograms/ml to obtain a culture solution B for later use;
d. sucking all the non-sentinel lymph node-derived anti-tumor T cells obtained after culturing in the culture solution A for 24 hours, transferring the cells into a centrifuge tube, and centrifuging for 5 minutes at a centrifugal force of 350 g; discarding the upper culture solution after the centrifugation is finished;
e. adding culture solution B into non-sentinel lymph node-derived anti-tumor T cells from which the upper culture solution is discarded after centrifugation is completed, and adjusting the non-sentinel lymph node-derived anti-tumor T cells toThe concentration in culture solution B was 1 x 106Culturing for 48 hours, adding the conventional artificially synthesized tumor polypeptide for cell culture, adjusting the concentration of the conventional artificially synthesized tumor polypeptide for cell culture in an added system to be 1 microgram/ml, and continuously culturing for 24 hours to obtain an anti-tumor T cell culture derived from non-sentinel lymph nodes;
(2) t cell subset isolation and stimulation of proliferation:
performing subgroup sorting on the non-sentinel lymph node-derived anti-tumor T cell culture by adopting a conventional sorting method to obtain a CD4+, a CD 25-helper T cell subgroup and a CD8+ killer T cell subgroup, respectively placing the two subgroups into an X-VIVO culture medium for independent culture, and adding an activating reagent, wherein the culture density is 1 × 107Each group is subjected to half-amount liquid change every 3 days, and cells are counted after 12 days of culture;
wherein, the activating reagent of the CD4+, CD 25-helper T cell subgroup is as follows: CD3/CD28 magnetic beads and interleukin 7, wherein the number of the CD3/CD28 magnetic beads in the added system is adjusted to be 5 per cell, and the concentration of the interleukin 7 in the added system is adjusted to be 20 nanograms per milliliter;
the activating agents for the CD8+ killer T cell subset were: anti-CD3, interleukin 7 and interleukin 21, wherein the concentration of the anti-CD3 in the added system is adjusted to be 50 ng/ml, the concentration of the interleukin 7 in the added system is adjusted to be 20 ng/ml, and the concentration of the interleukin 21 in the added system is adjusted to be 5 ng/ml;
(3) restimulation and rapid proliferation of T cell subsets:
after the culture of the upper stage is finished, adding peripheral blood mononuclear cells irradiated by 35 kilogray X rays, wherein the number ratio of the peripheral blood mononuclear cells to cells obtained after culturing for 12 days by using CD4+, CD 25-helper T cell subset and CD8+ killer T cell subset is 500:1, adding conventional artificially synthesized tumor polypeptide for cell culture, adjusting the concentration of the artificially synthesized tumor polypeptide for cell culture in the added system to be 3 micrograms/ml, continuing to culture for 15 days, and controlling the culture density to be 2X 10 in the culture process6Per milliliter;
(4) harvesting:
after the culture is finished, respectively sampling and culturing cells from the CD4+, CD 25-helper T cell subset and the CD8+ killer T cell subset, carrying out microorganism, endotoxin, flow cytometry and cytokine secretion analysis, and mixing the two subsets of cells to obtain the compound.
The method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, wherein: the non-sentinel lymph node derived anti-tumor T cells refer to tumor draining lymph nodes which are not infiltrated by tumor cells and are positioned between sentinel lymph nodes and distal lymph nodes; is obtained by adopting the following method for separation:
(1) selecting lymph nodes from the middle section of the draining lymph node:
taking an isolated tumor tissue, injecting patent Blue V into the isolated tumor tissue at 4 points, namely the upper, the lower, the left and the right of the isolated tumor tissue, injecting 1 ml of the injection at each point, and sucking redundant patent Blue at the injection point by gauze after the injection;
searching blue-stained lymph nodes, namely tumor draining lymph nodes TDLNs according to the trend of blue-stained lymph vessels, wherein the first or a plurality of blue-stained lymph nodes are sentinel lymph nodes;
taking a primary tumor as a center, taking 4 tumor draining lymph nodes which are positioned between a sentinel lymph node and a distal lymph node, namely, are 7 centimeters away from the center of the primary tumor, wherein each tumor draining lymph node takes the maximum diameter as an axis;
(2) removing fat tissue attached to lymph nodes by flow cytometry identification and separation method:
cutting the tumor draining lymph node into 3 mm small pieces, adding DNase and collagenase with the volume 3-5 times of that of the tumor draining lymph node, digesting for 60 minutes at 37.5 ℃, blowing and sucking for 4 times by using a syringe needle in the digestion process, and mixing the DNase and the collagenase according to the volume ratio of 1: 1;
after digestion, passing through a 70-micron screen, taking cell sap, carrying out flow cytometry identification after staining the cell sap by using a tumor cell specific staining antibody, and culturing cells with negative staining results for 15 hours;
(3) enzyme-linked immunosorbent assay (ELISA) identification:
and (3) performing enzyme-linked immunosorbent assay spot identification on the negative cells cultured for 15 hours by adopting a gamma interferon enzyme-linked immunosorbent assay kit, and selecting the cells with positive identification results to obtain the non-sentinel lymph node-derived anti-tumor T cells.
Example 3
An in vitro amplification method of anti-tumor T cells derived from non-sentinel lymph nodes, comprising the following steps:
(1) antigen sensitization and immunosuppression blocking:
a. mixing the lymphocyte culture medium and fetal calf serum in a volume ratio of 100:3 to obtain a lymphocyte culture medium and fetal calf serum mixture; adding interleukin 2 into a lymphocyte culture medium and a fetal calf serum mixture, and adjusting the concentration of the interleukin 2 in the lymphocyte culture medium and the fetal calf serum mixture to be 500 units/ml to obtain a culture solution A for later use;
b. adding non-sentinel lymph node-derived anti-tumor T cells into a part of the culture solution A, and adjusting the volume of the culture solution to make the density of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution A be 1.5 x 106Culturing for 18 hours per milliliter;
c. adding anti-T cell immunoglobulin mucin 3 into another part of the culture solution A, and adjusting the concentration of the anti-T cell immunoglobulin mucin 3 in the culture solution A to be 15 micrograms/ml to obtain a culture solution B for later use;
d. sucking all the non-sentinel lymph node-derived anti-tumor T cells obtained after culturing for 18 hours in the culture solution A, transferring the cells into a centrifuge tube, and centrifuging for 4 minutes at the centrifugal force of 325 g; discarding the upper culture solution after the centrifugation is finished;
e. adding a culture solution B to the non-sentinel lymph node-derived anti-tumor T cells from which the upper culture solution was discarded after the centrifugation, and adjusting the concentration of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution B to 0.7 x 106Culturing for 36 hours, adding the conventional artificially synthesized tumor polypeptide for cell culture, adjusting the concentration of the conventional artificially synthesized tumor polypeptide for cell culture in the added system to be 0.7 micrograms/ml, and continuously culturing for 36 hours to obtain the non-sentinel lymph node source anti-tumor T cell culture；
(2) T cell subset isolation and stimulation of proliferation:
performing subgroup sorting on the non-sentinel lymph node-derived anti-tumor T cell culture by adopting a conventional sorting method to obtain a CD4+, a CD 25-helper T cell subgroup and a CD8+ killer T cell subgroup, respectively placing the two subgroups into an X-VIVO culture medium for independent culture, and adding an activating reagent, wherein the culture density is 1 × 106Each group is subjected to half-amount liquid change every 3 days, and cells are counted after 10 days of culture;
wherein, the activating reagent of the CD4+, CD 25-helper T cell subgroup is as follows: CD3/CD28 magnetic beads and interleukin 7, wherein the number of the CD3/CD28 magnetic beads in the added system is adjusted to be 3 per cell, and the concentration of the interleukin 7 in the added system is adjusted to be 15 ng/ml;
the activating agents for the CD8+ killer T cell subset were: anti-CD3, interleukin 7 and interleukin 21, wherein the concentration of the anti-CD3 in the added system is adjusted to be 25 ng/ml, the concentration of the interleukin 7 in the added system is adjusted to be 11 ng/ml, and the concentration of the interleukin 21 in the added system is adjusted to be 3 ng/ml;
(3) restimulation and rapid proliferation of T cell subsets:
after the culture of the upper stage is finished, adding 25 kilogray X-ray irradiated peripheral blood mononuclear cells, wherein the number ratio of the peripheral blood mononuclear cells to cells obtained after culturing for 10 days by using CD4+, CD 25-helper T cell subset and CD8+ killer T cell subset is 300:1, adding conventional artificially synthesized tumor polypeptide for cell culture, adjusting the concentration of the artificially synthesized tumor polypeptide for cell culture in the added system to be 1.5 micrograms/ml, continuing to culture for 14 days, and controlling the culture density to be 1.5X 10 in the culture process6Per milliliter;
(4) harvesting:
after the culture is finished, respectively sampling and culturing cells from the CD4+, CD 25-helper T cell subset and the CD8+ killer T cell subset, carrying out microorganism, endotoxin, flow cytometry and cytokine secretion analysis, and mixing the two subsets of cells to obtain the compound.
The method for in vitro amplification of anti-tumor T cells derived from non-sentinel lymph nodes, wherein: the non-sentinel lymph node derived anti-tumor T cells refer to tumor draining lymph nodes which are not infiltrated by tumor cells and are positioned between sentinel lymph nodes and distal lymph nodes; is obtained by adopting the following method for separation:
(1) selecting lymph nodes from the middle section of the draining lymph node:
taking an isolated tumor tissue, injecting patent Blue V into the isolated tumor tissue at 4 points, namely the upper, the lower, the left and the right of the isolated tumor tissue, injecting 0.6 ml of the injection at each point, and sucking redundant patent Blue at the injection point by gauze after the injection;
searching blue-stained lymph nodes, namely tumor draining lymph nodes TDLNs according to the trend of blue-stained lymph vessels, wherein the first or a plurality of blue-stained lymph nodes are sentinel lymph nodes;
taking a primary tumor as a center, taking 3 tumor draining lymph nodes which are positioned between a sentinel lymph node and a distal lymph node and are 6 centimeters away from the center of the primary tumor, wherein each tumor draining lymph node takes the maximum diameter as an axis;
(2) removing fat tissue attached to lymph nodes by flow cytometry identification and separation method:
cutting the tumor draining lymph node into small pieces of 2 mm, adding DNase and collagenase with 4 times volume of the tumor draining lymph node, digesting for 45 minutes at 37 ℃, blowing and sucking for 3 times by a No. 21 syringe needle in the digestion process, and mixing the DNase and the collagenase according to the volume ratio of 1: 1;
after digestion, passing through a 70-micron screen, taking cell sap, dyeing the cell sap by using a tumor specific dyeing antibody consisting of epithelial cell adhesion molecules, cytokeratin 20, cancer blank antigen and cell glycoprotein 199 according to the volume ratio of 1:1:1:1, then carrying out flow cytometry identification, and culturing cells with negative dyeing results for 12 hours;
(3) enzyme-linked immunosorbent assay (ELISA) identification:
and (3) performing enzyme-linked immunosorbent assay spot identification on the negative cells cultured for 12 hours by adopting a gamma interferon enzyme-linked immunosorbent assay kit, and selecting the cells with positive identification results to obtain the non-sentinel lymph node-derived anti-tumor T cells.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.

Claims (10)

1. An in vitro amplification method of anti-tumor T cells derived from non-sentinel lymph nodes, comprising the following steps:
(1) antigen sensitization and immunosuppression blocking:
a. mixing lymphocyte culture medium and fetal calf serum at a volume ratio of 100:2-5 to obtain lymphocyte culture medium and fetal calf serum mixture; adding interleukin 2 into a lymphocyte culture medium and a fetal calf serum mixture, and adjusting the concentration of the interleukin 2 in the lymphocyte culture medium and the fetal calf serum mixture to be 100-1000 units/ml to obtain a culture solution A for later use;
b. adding non-sentinel lymph node-derived anti-tumor T cells into a part of the culture solution A, and adjusting the volume of the culture solution to make the density of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution A be 1 × 106-2*106Culturing for 12-24 hr per ml;
c. adding an anti-immunosuppressive antibody into another part of the culture solution A to perform immunosuppressive blocking to obtain a culture solution B for later use;
d. sucking all the non-sentinel lymph node-derived anti-tumor T cells obtained after culturing for 12-24 hours in the culture solution A, transferring the cells into a centrifuge tube, and centrifuging for 3-5 minutes at the centrifugal force of 300-350 g; discarding the upper culture solution after the centrifugation is finished;
e. adding a culture solution B into the non-sentinel lymph node-derived anti-tumor T cells from which the upper culture solution is discarded after the centrifugation is completed, and adjusting the concentration of the non-sentinel lymph node-derived anti-tumor T cells in the culture solution B to 0.5 x 106-1*106Culturing for 24-48 hr, adding the artificially synthesized tumor polypeptide for cell culture, and regulating the content of the artificially synthesized tumor polypeptide for cell culture in the added systemThe concentration of the compound is 0.5-1 microgram/ml, and the culture is continued for 24-48 hours to obtain the non-sentinel lymph node source anti-tumor T cell culture;
(2) t cell subset isolation and stimulation of proliferation:
performing subgroup sorting on the non-sentinel lymph node-derived anti-tumor T cell culture by adopting a conventional sorting method to obtain a CD4+, a CD 25-helper T cell subgroup and a CD8+ killer T cell subgroup, respectively placing the two subgroups into an X-VIVO culture medium for independent culture, and adding an activating reagent, wherein the culture density is 1 × 105-1*107Each group is subjected to half-amount liquid change every 3 days, and cells are counted after 9-12 days of culture;
wherein, the activating reagent of the CD4+, CD 25-helper T cell subgroup is as follows: CD3/CD28 magnetic beads and interleukin 7, wherein the CD3/CD28 magnetic beads are adjusted to be 1-5 per cell in an added system, and the concentration of the interleukin 7 is 5-20 ng/ml;
the activating agents for the CD8+ killer T cell subset were: anti-CD3, interleukin 7 and interleukin 21, adjusting the concentration of the anti-CD3 in the added system to be 5-50 ng/ml, the concentration of the interleukin 7 to be 2-20 ng/ml and the concentration of the interleukin 21 to be 1-5 ng/ml;
(3) restimulation and rapid proliferation of T cell subsets:
after the culture of the upper stage is finished, adding peripheral blood mononuclear cells irradiated by 15-35 kilogray X rays, wherein the number ratio of the peripheral blood mononuclear cells to cells obtained after culturing for 9-12 days by CD4+, CD 25-helper T cell subgroup and CD8+ killer T cell subgroup is 100-1: 500, adding conventional artificially synthesized tumor polypeptides for cell culture, adjusting the concentration of the artificially synthesized tumor polypeptides for cell culture in the added system to be 0.5-3 micrograms/milliliter, continuing to culture for 12-15 days, and controlling the culture density to be 1X 10 in the culture process6-2*106Per milliliter;
(4) harvesting:
after the culture is finished, respectively sampling and culturing cells from the CD4+, CD 25-helper T cell subset and the CD8+ killer T cell subset, carrying out microorganism, endotoxin, flow cytometry and cytokine secretion analysis, and mixing the two subsets of cells to obtain the compound.
2. The method for the in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to claim 1, wherein: the anti-immunosuppressive antibody is anti-programmed death receptor 1, and the concentration of the anti-programmed death receptor 1 in the culture solution A is adjusted to be 5-30 micrograms/ml.
3. The method for the in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to claim 1, wherein: the anti-immunosuppressive antibody is anti-cytotoxic T lymphocyte-associated antigen 4, and the concentration of the anti-cytotoxic T lymphocyte-associated antigen 4 in the culture solution A is adjusted to be 5-30 micrograms/ml.
4. The method for the in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to claim 1, wherein: the anti-immunosuppressive antibody is anti-T cell immunoglobulin mucin 3, and the concentration of the anti-T cell immunoglobulin mucin 3 in the culture solution A is adjusted to be 5-30 micrograms/ml.
5. The method for the in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to claim 1, wherein: the anti-immunosuppressive antibody is formed by combining any two of an anti-programmed death receptor 1, an anti-cytotoxic T lymphocyte-associated antigen 4 and an anti-T cell immunoglobulin mucin 3 according to a volume ratio of 1:1, and the concentrations of the anti-programmed death receptor 1, the anti-cytotoxic T lymphocyte-associated antigen 4 or the anti-T cell immunoglobulin mucin 3 in the culture solution A are respectively adjusted to be 5-30 micrograms/ml.
6. The method for the in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to claim 1, wherein: the anti-immunosuppressive antibody is formed by combining an anti-programmed death receptor 1, an anti-cytotoxic T lymphocyte-associated antigen 4 and an anti-T cell immunoglobulin mucin 3 according to a volume ratio of 1:1:1, and the concentrations of the anti-programmed death receptor 1, the anti-cytotoxic T lymphocyte-associated antigen 4 or the anti-T cell immunoglobulin mucin 3 in a culture solution A are respectively adjusted to be 5-30 micrograms/ml.
7. The method for the in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to claim 1, wherein: the non-sentinel lymph node-derived anti-tumor T cell refers to a tumor draining lymph node which is not infiltrated by tumor cells and is positioned between the sentinel lymph node and a distal lymph node.
8. A method of in vitro expansion of non-sentinel lymph node derived anti-tumor T cells according to any one of claims 1, 2, 3, 4, 5, 6 and 7 wherein: the non-sentinel lymph node source anti-tumor T cell is obtained by adopting the following method for separation:
(1) selecting lymph nodes from the middle section of the draining lymph node:
taking an isolated tumor tissue, injecting patent blue into the isolated tumor tissue at 4 points of the upper, lower, left and right parts of the isolated tumor tissue, injecting 0.2-1 ml of the patent blue into each point, and sucking redundant patent blue at the injection point by gauze after injection;
searching blue-stained lymph nodes, namely tumor draining lymph nodes according to the trend of the blue-stained lymph vessels, wherein the first or a plurality of blue-stained lymph nodes are sentinel lymph nodes;
taking a primary tumor as a center, taking 3-4 tumor draining lymph nodes which are positioned between a sentinel lymph node and a distal lymph node, namely 5-7 cm away from the center of the primary tumor, wherein each tumor draining lymph node takes the maximum diameter as an axis;
(2) removing fat tissue attached to lymph nodes by flow cytometry identification and separation method:
cutting the tumor draining lymph node into small pieces of 1-3 mm, adding DNase and collagenase of which the volume is 3-5 times that of the tumor draining lymph node, digesting the small pieces at 36.5-37.5 ℃ for 30-60 minutes, and blowing and sucking the small pieces for 3-4 times by using a syringe needle in the digestion process, wherein the DNase and the collagenase are mixed according to the volume ratio of 1: 1;
after digestion, sieving, taking cell sap, carrying out flow cytometry identification after staining the cell sap by using a tumor cell specific staining antibody, and culturing cells with negative staining results for 8-15 hours;
(3) enzyme-linked immunosorbent assay (ELISA) identification:
and (3) performing enzyme-linked immunosorbent assay spot identification on the negative cells cultured for 8-15 hours by adopting a gamma interferon enzyme-linked immunosorbent assay spot detection kit, and selecting the cells with positive identification results to obtain the non-sentinel lymph node source anti-tumor T cells.
9. The method of claim 8, wherein said method comprises the steps of: the tumor cell specific staining antibody is composed of epithelial cell adhesion molecules, cytokeratin 20, cancer blank antigen and cell glycoprotein 199 according to the volume ratio of 1:1:1: 1.
10. The method of claim 9, wherein said method comprises the steps of: the syringe needle is a 21 gauge syringe needle.
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