CN108165529B - Central memory T cell body and its in vitro culture method - Google Patents
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Abstract
The invention discloses an in vitro culture method of central memory T lymphocytes, which comprises the following steps of adding IL-21 into an initial culture medium, wherein the oxygen concentration in a culture environment is 1 +/-0.1%; on day 7 of culture, IL-21 and rapamycin were added to the re-expansion medium. The invention discloses an in vitro culture amplification method which can improve the proportion of central memory T cells, inhibit the differentiation of the central memory T cells to effector cells and increase the killing effect of the central memory T cells to tumor cells by combining rapamycin, interleukin 21 and a low-oxygen environment in a proper culture process for the first time.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a central memory T lymphocyte and an in-vitro culture method thereof.
Background
Adoptive immune cell therapy refers to the infusion of in vitro activated autologous or allogeneic immune effector cells into a patient to kill tumor cells in the patient. Adoptive immune cell therapy is an emerging therapeutic means with significant efficacy in current tumor immunotherapy. Among them, adoptive immunotherapy of T cells is the most widely used immunotherapy means with the most remarkable effect internationally at present, and its action principle is to utilize specific T cells activated by in vitro amplification to implement the killing function to antigen.
Specific T cells are largely divided into two groups, CD4+T cells and CD8+T cells. In adoptive cellular immunotherapy, CD4+The T cells can not only kill the cells themselves, but also assist the immune cells of the body and the co-adoptive immune cells, have strong universality and can be combined with CD8+T cells complement each other. CD4+T lymphocytes can be divided into three subpopulations, naive T lymphocytes, effector T lymphocytes, and memory T lymphocytes. Memory T cells are classified into central memory T Cells (TCM) and effector memory T cells (TEM) according to whether they express the homing receptors CCR7 and CD 62L. TCM cells express CCR7 and CD62L, and are distributed mainly in lymph nodes and tonsils, etc., while TEM is distributed mainly in peripheral tissues. TEM cells have a short life in vivo and cannot achieve long-term therapeutic effects despite a certain clearance effect, while TCM cells maintain self-renewal in vivo to produce a durable killing effect, and have gained more and more attention in immunotherapy.
TCM cells are often used as genetically engineered seed cells in the current international center immune cell therapy technology for modification processing, so that the ability of the TCM cells to kill tumor cells is enhanced. For example, a chimeric antigen receptor technology (CAR-T) or a T cell receptor (TCR-T) technology is used for modifying TCM cells, so that novel immune cells with high killing property and durability can be obtained, and the novel immune cells can be further applied to treatment of malignant tumors.
TCMs are present in smaller proportions and quantities in the body, and as in vitro expansion proceeds TCMs become extremely vulnerable to terminally differentiated effector cells. Therefore, how to obtain TCM cells with high purity, high activity and high amplification capacity in vitro is a hot spot of cellular immunotherapy and a problem to be solved urgently for clinical application of immunotherapy.
Disclosure of Invention
In order to solve the problems of low purity of in-vitro amplification of central memory T cells and easy transformation into effector cells, the invention aims to provide an in-vitro culture method of the central memory T cells, which improves the proportion and functionality of the central memory T cells.
The above purpose of the invention is realized by the following technical scheme:
an in vitro culture method of central memory T lymphocyte comprises the following steps:
in cell culture plates, CD3 antibody was coated;
adding an initial culture medium and the selected T cells for culture, wherein the oxygen concentration in the culture environment is 1 +/-0.1%; IL-21 is added into the initial culture medium;
continuing culturing, and supplementing an initial culture medium every two days;
on day 6, the cells were aspirated into centrifuge tubes, centrifuged, supernatant removed, resuspended in initial medium, and then transferred to cell culture plates for continued culture;
on day 7, the culture flask is taken and coated with the CD3 antibody; transferring the T cells into a centrifuge tube, centrifuging, removing supernatant, resuspending with a re-amplification culture medium, and transferring into a T25 culture flask for continuous culture; IL-21 and rapamycin are added into the re-amplification culture medium;
the culture is continued, and the re-expansion culture medium is supplemented every two days until the 14 th harvest of the central memory T lymphocyte cells.
In one embodiment, the basal medium is AIM V medium.
In one embodiment, the concentration of rapamycin in the re-amplification medium is 5-30 ng/ml.
In one embodiment, the rapamycin is preferably present at a concentration of 10 ng/ml.
In one embodiment, the concentration of interleukin 21 in the initial medium is 25-75 ng/ml.
In one embodiment, the optimal concentration of interleukin 21 is 50 ng/ml.
In one embodiment, the T cells are magnetic bead sorted CD4+ T cells; the T cells are derived from PBMC cells isolated from peripheral blood or cord blood.
In one embodiment, the CD3 antibody is coated at a concentration of 20. + -. 0.2. mu.g/mL in the amplification method described above.
In one embodiment, CD28 antibody is also added to the primary and/or re-amplification media at a concentration of 2. + -. 0.2. mu.g/mL.
In one embodiment, IL-2 is also added to the initial medium and/or the re-amplification medium at a free concentration of 10. + -. 0.5 ng/mL.
Another object of the present invention is to provide central memory T lymphocytes obtained by the above culture method.
The invention has the beneficial effects that:
the invention discloses an in vitro culture amplification method which can improve the proportion of central memory T cells, inhibit the differentiation of the central memory T cells to effector cells and increase the killing effect of the central memory T cells to tumor cells by combining rapamycin, interleukin 21 and a low-oxygen environment in a proper culture process for the first time. The culture method is simple, easy to operate, safe, reliable, low in cost and convenient to popularize.
Drawings
FIG. 1 is a graph showing the proportion of central memory T cells among CD4+ T cells sorted on the first day;
FIG. 2 is a graph showing the ratio of central memory T cells of different treatment groups measured on day 7 of culture under hypoxic conditions;
FIG. 3 is a graph showing the ratio of central memory T cells of different treatment groups measured on day 14 of culture under hypoxic conditions;
FIG. 4 shows the comparative killing of the tumor cells HCC827 by central memory T cells of different treatment groups.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
Example 1: method for expanding central memory T cells
Isolation of PBMC and obtaining of autologous supernatant
Volunteers were recruited and 50ml of fresh blood was drawn into the collection tube by a collection nurse using standard blood drawing procedures. Whole blood was pipetted into 2 50ml centrifuge tubes previously filled with 14ml of lymphocyte separation medium, 25ml of whole blood per tube. Setting the temperature of the centrifuge to 20 ℃, the centrifugal force to 400g, accelerating for 1, decelerating for 5, and centrifuging for 30 min. After the centrifugation is finished, the autologous plasma on the upper layer is sucked and frozen. Sucking the middle tunica albuginea cells into a new centrifuge tube filled with 40ml DPBS, uniformly blowing, setting the temperature of the centrifuge at 20 ℃, 300g, accelerating 5, decelerating 5, and centrifuging for 8 min. After completion of centrifugation, the supernatant was removed, the cell pellet in the centrifuge tube was scraped off, 40ml of DPBS solution was added, and centrifugation was carried out at 300g for 8 min. After centrifugation, the supernatant was removed, resuspended in DPBS solution and counted.
CD4+ T cell sorting
CD4+ T cell sorting was performed using the Dynalbeads CD4Positive Isolation Kit from Thermo fisher, Inc. (cat # 11331D). First, the magnetic beads were vortexed and 100. mu.L (4X 10) of the mixture was added7The amount of PBMC cells) in a tube (2 mLEp tubes), 4mL Buffer1 was added and mixed, the tube was placed on a magnetic stand for 1min, and the supernatant was removed. Add 100. mu.L Buffer1 resuspension magnetic beads for use; PBMC were resuspended with Buffer1 to a density of 1X 107Per mL; adding 25 mu L of washed magnetic beads into 1mL of PBMC, incubating for 30min at 2-8 ℃, and placing on a shaking table for inclined rotation; placing the tube on a magnet for 2min, and gently blotting off the supernatant; removing the test tube, adding 1mL of Buffer1, blowing, uniformly mixing, placing on a magnetic bead for 2min, and removing the supernatant; repeating the above steps; add 100 u L Buffer 2 heavy suspension cells, add 10 u L DETACHABEAD, room temperature incubation for 45min to make cells from the magnetic beads release; placing the test tube on a magnetic frame for 1min, transferring the cell-containing supernatant into a new test tube, adding 500 μ L Buffer 2 to wash the magnetic beads for 2-3 times, and collecting the supernatant; 4mL of Buffer 2 was added, 400g was centrifuged for 6min, the supernatant was removed, and the cells were resuspended in Buffer 2 and counted.
T cell activation and expansion
A24-well cell culture plate was first coated with 300. mu.l of 20. mu.g/mL CD3 antibody at 37 ℃ for 1-2 h. Then preparing an initial culture medium: AIM V medium (Thermo Fisher company, cat # 12055091) + 5% autologous plasma + 2. mu.g/mL CD28 antibody +10ng/mL IL-2+50ng/mL IL-21. After coating was complete, excess CD3 antibody was removed and cultured by adding 500. mu.l of the starting medium and 80 ten-thousandths of the selected T cells in an oxygen concentration of 1%. After two days of culture, the cells appeared to be conglomerated, and the cells were blown off by gentle blow, and then 500 microliters of initial medium was supplemented to continue the culture. On day 4, cells were pipetted into centrifuge tubes, centrifuged at 300g for 5min, the supernatant removed, resuspended in 2ml of medium, and then transferred to 12-well plates for further culture. On day 6, cells were pipetted into centrifuge tubes, centrifuged at 300g for 5min, the supernatant removed, resuspended in 4ml of initial medium, and then transferred to 6-well plates for further culture.
4. Rapamycin addition and central memory T cell re-expansion
On day 7, a T25 flask was removed and 2mL of 20. mu.g/mL CD3 antibody was added to the flask and coated at 37 ℃ for 1-2 h. Preparing a re-amplification culture medium: AIM V medium (Thermo Fisher company, cat # 12055091) + 5% autologous plasma + 2. mu.g/mL CD28 antibody +10ng/mL IL-2+50ng/mL IL-21+10ng/mL Rapamycin (Rapamycin). Excess CD3 antibody was removed, T cells cultured in 6-well plates were transferred to a centrifuge tube, centrifuged at 300g for 5min, the supernatant removed, resuspended in 12ml of re-expansion medium, and then transferred to a T25 flask for continued culture. On day 9, the T25 flask was supplemented with 10ml of re-expansion medium and the cell pellet was pipetted down to homogeneity. On day 11, cells from the T25 flask were transferred to a T75 flask, supplemented with 10ml of re-expansion medium, and then pipetted to homogenize the cell pellet. And on the 13 th day, supplementing 10ml of re-amplification culture medium into a T75 culture bottle, blowing off cell masses evenly by using a pipette, continuously culturing the central memory T cells, and continuously culturing for 14 days to obtain the culture medium.
Example 2: flow assay detection of central memory T cells
The CD4+ T cells sorted in example 1, TCM cells cultured at day 7, and TCM cells cultured at day 14 were subjected to flow detection using CD4 antibody, CD95 antibody, CD45RO antibody, and CD62L antibody. All antibodies were purchased from BD, 30-50 ten thousand cells per tube for the required cell amount, 5 μ l of each antibody was added, stained in the dark at room temperature for 15min, washed and centrifuged 2 times with DPBS solution, and finally resuspended in a machine for detection with 400 μ l DPBS. The central memory T cell is CD45RO+CD62L+CD95+A cell. The detection results are shown in fig. 1, fig. 2 and fig. 3, and it can be seen that the proportion of cultured TCM cells can be increased by adding IL-21 in the early stage of culture, and the proportion of TCM can be maintained by adding rapamycin in the late stage of culture to inhibit the transformation of central memory T cells into effector cells.
FIG. 1A shows a sorted CD4+The proportion of T cells, 98.8%, indicated that substantially all of the cells sorted out were CD4+T cells. FIG. 1B shows a sorted CD4+CD45RO in T cells+CD62L+Cell ratio, FIG. 1C is CD4+CD45RO in T cells+CD95+Cell ratio. Because of CD95 in FIG. 1C+The cell proportion was 99.17%, almost 100%, so CD4+CD45RO in T cells+CD62L+CD95+The cell ratio was 14.9% × 99.17% ═ 14.8%, i.e. initial sortingCD4 (1)+The proportion of central memory T cells in T cells was 14.8%.
FIG. 2 shows the ratio of central memory T cells to which IL-21 was added on day 7 of culture under hypoxic conditions. The proportion of CD45RO + CD62L + cells in the cells of fig. 2A was 75.4%, and the proportion of CD45RO + CD95+ cells in the cells of fig. 2B was 97.7%. Since the proportion of CD95+ cells in fig. 2B was 99.97%, almost 100%, the proportion of CD45RO + CD62L + CD95+ cells in the cells was 75.4% × 99.97% ═ 75.4%, i.e., the proportion of central memory T cells was 75.4%; FIGS. 2C and 2D show the proportion of central memory T cells without IL-21 addition at day 7 under hypoxic conditions. The proportion of CD45RO + CD62L + cells in the cells of fig. 2C was 60.8%, and the proportion of CD45RO + CD95+ cells in the cells of fig. 2D was 95%. Since the proportion of CD95+ cells in fig. 2D was 99.96%, almost 100%, the proportion of CD45RO + CD62L + CD95+ cells in the cells was 60.8% × 99.96% ═ 60.8%, i.e., the proportion of central memory T cells was 60.8%.
Fig. 3A and 3B are graphs of the proportion of central memory T cells on day 14 obtained using re-expansion medium on day 7 under hypoxic conditions, 64.9% x 99.96% to 64.9%; fig. 3C and 3D are graphs of the proportion of central memory T cells obtained on day 14 under hypoxic conditions, with initial medium (i.e. without rapamycin) continued on day 7, at 32.5% x 99.98% to 32.5%.
Example 3: comparison of killing effect of TCM cells on tumor cells under different culture conditions
After 14 days of culture of central memory T cells, the HCC827 cell line in culture was taken, digested with 0.05% trypsin at 37 ℃ for 5min, and after termination of digestion with serum, centrifuged at 500g for 5 min. Supernatant removed, DPBS resuspended and counted, 5 x 10 removed6Cells were centrifuged at 500g for 5 min. Supernatant was removed and 500. mu.l of DPBS solution was resuspended. 1.5ml of the other sample was added with 500. mu.l of DPBS solution and 1. mu.l of 5mM CFSE (fluorescent dye) solution, mixed well, and then the 500. mu.l of CFSE solution was gently added to another tube of 500. mu.l of cell suspension, and incubated at 37 ℃ for 10-20min in the dark. Then 2ml FBS serum was added to stop staining and 500g was centrifuged for 5 min. Remove supernatant, add 10ml DPBS solution to wash twice, finally use AIMV + 5% AB serum medium heavy suspension to 1 x 106Cell concentration per mlTaking a 48-hole plate, and adding 5 x 10 of the solution into each hole5Tumor cells, and cells of different treatment groups with effective target ratios of 1:1,10:1 and 20:1, respectively. Wherein each processing group is respectively as follows: control TCM cells at low oxygen concentrations (without IL-21 and rapamycin); TCM cells at low oxygen concentration (1%) (with IL-21 and rapamycin added); control TCM cells at normal atmospheric oxygen concentration (without IL-21 and rapamycin); TCM cells (with IL-21 and rapamycin added) at normal oxygen concentration, the other steps were the same as in example 1. After the cells are mixed uniformly, the cells are placed in an incubator at 37 ℃ for culturing for 16h, then after supernatant is removed, DPBS is washed for 2 times, after 0.05 percent of pancreatin is used for digestion, 5000 microspheres are added into each tube for counting and 5 microliters of 7-AAD fuel is added for dyeing, and finally, 400 microliters of DPBS solution is used for resuspending the cells for flow cytometry.
The results are shown in FIG. 4, Hypoxia Ctrl cells refer to central memory T cells cultured under hypoxic conditions without IL-21 and rapamycin; hypoxia TCM cell refers to central memory T cells added with IL-21 and rapamycin under hypoxic condition; the Normal Ctrl cell refers to the central memory T cell without IL-21 and rapamycin under the condition of Normal oxygen concentration; normal TCM cell refers to central memory T cells supplemented with IL-21 and rapamycin in culture under Normal oxygen concentration conditions. The result shows that the killing effect of the TCM cells cultured in the hypoxic environment on specific tumor cells is greatly enhanced compared with the TCM cells cultured in the normal oxygen concentration.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. An in vitro culture method of central memory T lymphocytes is characterized by comprising the following steps:
in cell culture plates, CD3 antibody was coated;
adding the primary culture medium and the sorted CD4+Culturing the T cells, wherein the concentration of oxygen in the culture environment is 1 +/-0.1%; the initial culture medium is a basic culture medium added with IL-21; the concentration of the interleukin 21 in the initial culture medium is 50 ng/ml;
continuing culturing, and supplementing an initial culture medium every two days;
on day 6, the cells were aspirated into centrifuge tubes, centrifuged, supernatant removed, resuspended in initial medium, and then transferred to cell culture plates for continued culture;
on day 7, the culture flask is taken and coated with the CD3 antibody; transferring the T cells into a centrifuge tube, centrifuging, removing supernatant, resuspending with a re-amplification culture medium, and transferring into a T25 culture flask for continuous culture; the re-amplification culture medium is a basic culture medium added with 50ng/ml of IL-21 and rapamycin; the concentration of rapamycin in the reamplification medium is 10 ng/ml;
the culture was continued, and the re-expansion medium was supplemented every two days until day 14, and central memory T lymphocytes were harvested.
2. The in vitro culture method according to claim 1, wherein the basal medium is AIM V medium.
3. The in vitro culture method according to claim 1, wherein said CD4 is+The T cells are derived from PBMC cells isolated from peripheral blood or cord blood.
4. The in vitro culture method according to claim 1, wherein in the initial medium and/or the re-amplification medium, an antibody CD28 is further added at a concentration of 2 ± 0.2 μ g/mL.
5. The in vitro culture method according to claim 1, wherein IL-2 is further added to the initial medium and/or the re-amplification medium at a free concentration of 10 ± 0.5 ng/mL.
6. The in vitro culture method of claim 1, wherein the CD3 antibody is coated at a concentration of 20 ± 0.2 μ g/mL.
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