Method for amplifying CD8+T cells and cell subpopulations thereof in-vitro
11773373 · 2023-10-03
Assignee
Inventors
Cpc classification
C12N2501/999
CHEMISTRY; METALLURGY
C12N5/0638
CHEMISTRY; METALLURGY
C12N2501/056
CHEMISTRY; METALLURGY
C12N2501/51
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed is a method for rapidly amplifying CD8+T cells and functional cell subpopulations thereof in vitro. A TLR1/2 agonist, a TLR2/6 agonist and a TLR5 agonist or a combination of above agonists is added to a conventional culture system for in-vitro amplification of CD8+T cells. Recombinant cytokines IL-2, IL-7 and IL-15 as well as magnetic beads coated with an anti-human CD3 antibody and an anti-human CD28 antibody can be further added to the culture system for continuous co-stimulation.
Claims
1. A method for inducing in-vitro amplification of PD-1+CD8+T cell subpopulations, comprising adding anti-human CD3 antibody, anti-human CD28 antibody, cytokine IL-7, cytokine IL-15, and a Toll-like receptor agonist chosen from one or more in the group of TLR1/2 agonists, TLR2/6 agonists and TLR5 agonists, to a culture system for the PD-1+CD8+T cell subpopulations, thereby causing the in-vitro amplification and functional repair of the PD-1+CD8+T cell subpopulations thereof.
2. The method according to claim 1, wherein the TLR1/2 agonists comprise Pam3CK4; the TLR2/6 agonists comprise FSL-1, Pam2CGDPKHPKSF, or a combination thereof; and the TLR5 agonists comprise Flagellin.
3. The method according to claim 1, wherein the anti-human CD3 antibody or the anti-human CD28 antibody is coated on magnetic beads.
4. The method according to claim 1, wherein the PD-1+CD8+T cell subpopulations comprise sorted and purified PD-1+CD8+T cells.
5. The method according to claim 4, wherein when the PD-1+CD8+T cell subpopulations are amplified in vitro, the anti-human CD3 antibody and the anti-human CD28 antibody are continuously present in the culture system to continuously stimulate the amplification of the PD-1+CD8+T cell subpopulations.
6. The method according to claim 1, wherein the in-vitro amplification of the PD-1+CD8+T cell subpopulations comprise: a. isolation of peripheral blood mononuclear cells (PBMCs) by labeling PBMCs with an anti-human CD3 antibody, an anti-human CD8 antibody and an anti-human PD-1 antibody with different fluorescent labels; staining the PBMCs at room temperature in the dark, resuspending the cells with a staining buffer, washing the cells by centrifugation and then resuspending the cells with the staining buffer; b. sorting the CD3+CD8+PD-1+T cells by a flow cytometer; c. allowing the cells sorted into a 96-well plate to stand for 1 hr, and adding Pam3CK4, FSL-1, Flagellin, IL-7, IL-2, IL-15 as well as magnetic beads coated with an anti-human CD3 antibody and an anti-human CD28 antibody, wherein the ratio of the magnetic beads to the cells is 1:1; and the cells are placed in an incubator at 37° C. for one week; and d. transferring the cells in the 96-well plate to a 24-well plate in the second week, and then sequentially transferring the cells to a T25 cell culture flask, a T75 cell culture flask and a T175 cell culture flask according to the number of growing cells; and each time a culture dish is replaced, corresponding media and stimulants are supplemented until the desired number of cells is achieved.
7. The method according to claim 1, wherein sources of the PD-1+CD8+T cell subpopulations comprise peripheral venous blood from healthy subjects, HIV-1 infected individuals or tumor patients, lymphocytic leukemia, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, uterine cancer, cervical cancer, renal cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, ovarian cancer, thyroid cancer, rhabdomyoma, leiomyoma and cerebroma, HIV infection, HBV infection, HCV infection, or Mycobacterium tuberculosis infection.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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(7) A is a schematic view of PD-1+CD8+ cells sorted from PBMCs; B is a schematic view of stained CD8+ T cells in the amplified product; C is a schematic view showing the detection of the ability of the amplified product to secrete IFN-γ and CD107a; and D is a schematic view showing the ability of the amplified product to secrete IFN-γ and Granzyme B.
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DETAILED DESCRIPTION OF THE INVENTION
(10) The present invention provides a method for amplifying CD8+ T cells and functional cell subpopulations thereof in-vitro, wherein one or more TLR agonists, recombinant human cytokines IL-7, IL-2 and IL-15 as well as magnetic beads coated with an anti-human CD3 antibody and an anti-human CD28 antibody are used in combination, and the continuous stimulation is maintained by the above stimulants, which can significantly enhance the amplification efficiency and activation of the CD8+ T cells, and enable the amplification of the CD8+ T cell subpopulations (e.g. PD-1+CD8+ T cells and effector memory CD8+ T cells (T.sub.EM)) that are difficult to amplify under conventional culture conditions so as to obtain cells that can maintain the function of the cell subpopulations.
(11) In the present invention, the stimulants for stimulating the CD8+ T cells include TLR1/2 agonists, TLR2/6 agonists and TLR5 agonists, recombinant human cytokines IL-7, IL-2 and IL-15 as well as magnetic beads coated with an anti-human CD3 antibody and an anti-human CD28 antibody. Sources of the CD8+ T cells and the PD-1+CD8+ T cells as well as other functional CD8+ T cell subpopulations include, but are not limited to, peripheral blood from healthy subjects, HIV-1 infected individuals or tumor patients. This amplification technique is also applicable to CD8+ T cells and other functional CD8+ T cell subpopulations obtained by other isolation methods, e.g. sorted and purified CD137+CD8+ T cells, sorted and purified CD160+CD8+ T cells, sorted and purified naive CD8+ T cells (T.sub.naive), sorted and purified central memory CD8+ T cells (T.sub.CM), sorted and purified effector memory CD8+ T cells (T.sub.EM), sorted and purified effector CD8+ T cells (T.sub.EMRA), sorted and purified transitional memory CD8+ T cells (TIM), sorted and purified effector stem cell CXCR5+CD8+ T cells, and sorted and purified tissue-resident memory CD8+ T cells (T.sub.RM).
(12) In order that those skilled in the art better understand the technical solution of the present invention, the present invention will be further described below in detail with reference to specific examples.
Example 1: Isolation of Peripheral Blood Mononuclear Cells
(13) 1. 20 ml of human peripheral blood anticoagulated by heparin was placed in a centrifuge tube. The peripheral blood was diluted with normal saline in a ratio of 1:1 and evenly mixed.
(14) 2. 15 ml of a lymphocyte isolation solution (Ficoll) was added to a new 50 mL centrifuge tube, then the evenly mixed blood dilution solution was slowly added to the upper layer of the lymphocyte isolation solution along the tube wall in a volume ratio of the Ficoll to the blood dilution solution of 1:2 to form a clear separation therebetween, and the resulting solution was centrifuged at 3000 rpm for 30 min.
(15) 3. After centrifugation was completed, a mononuclear cell layer was transferred into a new 50 ml centrifuge tube and washed once with 30 ml of an X-VIVO-15 medium, centrifuged at 800 g for 5 min, and the supernatant was discarded.
(16) 4. 20 ml of the X-VIVO-15 medium was added, the resulting mixture was evenly mixed by blowing and suction, centrifuged at 200 g at room temperature for 10 min, and the supernatant was discarded. 10 ml of the X-VIVO-15 medium was added for resuspending and counting.
Example 2: Proliferation of CD8+ T Cells was Effectively Stimulated by TLR Agonists
(17) 1. Peripheral blood mononuclear cells (PBMC) of healthy subjects were isolated according to Example 1.
(18) 2. CD8+ T cells were sorted using an EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (Stemcell: 19053).
(19) 3. The sorted CD8 cells were stained using Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90).
(20) 4. The cells were placed in a 96-well U-shaped bottom plate with 1×10.sup.5 cells per well.
(21) 5. The following stimulants and TLR agonists (invivoGen: tlr-kit1hw, human TLR1-9 Agonist KIT) were added in groups:
(22) (1) blank control;
(23) (2) anti-human CD3 antibody;
(24) (3) anti-human CD3 antibody+anti-human CD28 antibody;
(25) (4) anti-human CD3 antibody+TLR1/2 agonist-Pam3CSK4 (0.1-1 μg/ml);
(26) (5) anti-human CD3 antibody+TLR2 agonist-HKLM (10.sup.8 cells/ml);
(27) (6) anti-human CD3 antibody+TLR3 agonist-Poly(I:C) (10 ng-10 ug/ml);
(28) (7) anti-human CD3 antibody+TLR3 agonist-Poly(I:C) LMW (30 ng-10 ug/ml);
(29) (8) anti-human CD3 antibody+TLR4 agonist-E. coli K12 LPS (10 ng-10 μg/ml);
(30) (9) anti-human CD3 antibody+TLR5 agonist-S. typhimurium Flagellin (10 ng-10 μg/ml);
(31) (10) anti-human CD3 antibody+TLR6/2 agonist-FSL-1 (1 ng-1 μg/ml);
(32) (11) anti-human CD3 antibody+TLR7 agonist-Imiquimod (0.25-10 ug/ml);
(33) (12) anti-human CD3 antibody+TLR8 agonist-ssRNA40 (0.25-10 ug/ml); and
(34) (13) anti-human CD3 antibody+TLR9 agonist-ODN2006 (5 μM).
(35) (The anti-human CD3 antibody and the anti-human CD28 antibody used were magnetic beads coated with the antibodies (CELLection Pan Mouse IgG Kit, Invitrogen: 11531D), wherein the ratio of the cells to the magnetic beads was 1:1)
(36) 6. The cells were cultured in the 96-well U-shaped bottom plate, and cultured in a cell incubator for 7 days.
(37) 7. The proliferation of the CD8+ T cells was detected by a flow cytometer under the stimulation conditions of different TLR agonists.
Example 3: Proliferation of CD8+ T Cell Subpopulations was Effectively Stimulated by TLR Agonists
(38) TLR agonists were capable of effectively stimulating the proliferation of CD8+ T cell subpopulations, i.e. central memory T cells (T.sub.CM) and effector memory T cells (T.sub.EM).
(39) 1. Peripheral blood mononuclear cells (PBMC) of healthy subjects were isolated.
(40) 2. CD8+ T cells were sorted using an EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (stem cell: 19053).
(41) 3. The sorted CD8 cells were stained using Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90).
(42) 4. The cells were placed in a 96-well U-shaped bottom plate with 1×10.sup.5 cells per well.
(43) 5. The following stimulants and TLR agonists (invivoGen: tlr-kit1hw, human TLR1-9 Agonist KIT) were added in groups:
(44) (1) anti-human CD3 antibody;
(45) (2) anti-human CD3 antibody+TLR1/2 agonist-Pam3CSK4 (0.1-1 μg/ml);
(46) (3) anti-human CD3 antibody+TLR6/2 agonist-FSL-1 (1 ng-1 μg/ml); and
(47) (4) anti-human CD3 antibody+TLR5 agonist-S. typhimurium Flagellin (10 ng-10 μg/ml).
(48) (The anti-human CD3 antibody used was magnetic beads coated with the antibody (CELLection Pan Mouse IgG Kit, Invitrogen: 11531D), wherein the ratio of the cells to the magnetic beads was 1:1)
(49) 6. The cells were cultured in the 96-well U-shaped bottom plate, and cultured in a cell incubator for 7 days.
(50) 7. The cells were labeled with antibodies CD8 (BD: 561423), CD45RA (eBioscience: 25-0458-42) and CCR7 (eBioscience: 17-1979-42), and the influence of different TLR agonists on the proliferation of CD8+ T subpopulations (T.sub.Naive: Naive T cells, T.sub.CM: central memory T cells, T.sub.EM: effector memory T cells) was detected by flow cytometry.
(51) 8. The results showed that compared with cell proliferation stimulated by magnetic beads coated with an anti-human CD3 antibody alone, the proliferation of central memory CD8+ T cells (T.sub.CM) and effector memory T cells (T.sub.EM) could be effectively stimulated by a TLR1/2 agonist (Pam3CK4), a TLR2/6 agonist (FSL-1) or a TLR5 agonist (Flagellin) in combination with an anti-human CD3 antibody (as shown in
Example 4: Proliferation of CD8+ T Cell Subpopulations of HIV-1 Infected Individuals was Stimulated by TLR Agonists
(52) TLR agonists were capable of effectively stimulating the proliferation of CD8+ T cell subpopulations, i.e. central memory T cells (T.sub.CM) and effector memory T cells (T.sub.EM) of HIV-1 infected individuals.
(53) 1. Peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals were isolated.
(54) 2. CD8+ T cells were sorted using an EasySep™ Negative Selection Human CD8+ T cell Enrichment kit (Stemcell: 19053).
(55) 3. The sorted CD8 cells were stained using Cell Proliferation Dye eFluor 670 (ebioscience: 65-0840-90).
(56) 4. The cells were placed in a 96-well U-shaped bottom plate with 1×10.sup.5 cells per well.
(57) 5. The following stimulants and TLR agonists (invivoGen: tlr-kit1hw, human TLR1-9 Agonist KIT) were added in groups:
(58) (1) anti-human CD3 antibody;
(59) (2) anti-human CD3 antibody+TLR1/2 agonist-Pam3CSK4 (0.1-1 μg/ml);
(60) (3) anti-human CD3 antibody+TLR6/2 agonist-FSL-1 (1 ng-1 μg/ml);
(61) (4) anti-human CD3 antibody+TLR5 agonist-S. typhimurium Flagellin (10 ng-10 μg/ml);
(62) (5) anti-human CD3/28 antibodies;
(63) (6) anti-human CD3/28 antibodies+TLR1/2 agonist-Pam3CSK4 (0.1-1 μg/ml);
(64) (7) anti-human CD3/28 antibodies+TLR6/2 agonist-FSL-1 (1 ng-1 μg/ml); and
(65) (8) anti-human CD3/28 antibodies+TLR5 agonist-S. typhimurium Flagellin (10 ng-10 μg/ml).
(66) (The anti-human CD3 antibody and the anti-human CD28 antibody in the form of coated magnetic beads (CELLection Pan Mouse IgG Kit, Invitrogen: 11531D) were used to stimulate the cells, wherein the ratio of the cells to the magnetic beads was 1:1)
(67) 6. The cells were cultured in the 96-well U-shaped bottom plate, and cultured in a cell incubator for 7 days.
(68) 7. The cells were labeled with antibodies CD8 (BD: 561423), CD45RA (eBioscience: 25-0458-42) and CCR7 (eBioscience: 17-1979-42), and the influence of different TLR agonists on the proliferation of CD8+ T subpopulations (T.sub.Naive: Naive T cells, T.sub.CM: central memory T cells, T.sub.EM: effector memory T cells) was detected by a flow cytometer.
(69) 8. The results showed that compared with cell proliferation stimulated by magnetic beads coated with an anti-human CD3 antibody alone, the proliferation of central memory CD8+ T cells (T.sub.CM) and effector memory T cells (T.sub.EM) could be more effectively stimulated by adding a TLR1/2 agonist (Pam3CK4), a TLR2/6 agonist (FSL-1) or a TLR5 agonist (Flagellin) (as shown in
Example 5: Sorting of CD8+ T Cells and PD-1+CD8+ T Cells
(70) 1. Peripheral blood mononuclear cells (PBMC) of healthy subjects were isolated according to Example 1.
(71) 2. 1×10.sup.7 isolated PBMCs were taken and labeled with an anti-human CD3 antibody, an anti-human CD8 antibody and an anti-human PD-1 antibody with different fluorescent labels.
(72) 3. The PBMCs were stained at room temperature in the dark for 20 min, and the cells were resuspended with a staining buffer (1×PBS+2% FBS), centrifuged at 500 G for 5 min, washed twice and then resuspended with the staining buffer to a concentration of 1×10.sup.7 cells/ml.
(73) 4. CD3+CD8+ T cells and CD3+CD8+PD-1+ T cells were sorted by a flow cytometer into a 96-well plate, and 100 μl of an X-VIVO15 medium was preliminarily placed in the 96-well plate. 1-3×10.sup.4 cells were sorted per well.
Example 6: TLR Agonists were Capable of Effectively Enhancing the Proliferation of PD-1+CD8+ T Cells Stimulated by Anti-Human CD3/CD28 Antibodies
(74) 1. Peripheral blood mononuclear cells (PBMC) of healthy subjects were isolated.
(75) 2. 1×10.sup.7 PBMCs were taken, and an anti-human CD3 antibody (BD: 558117), a CD8 antibody (BD: 561423) and a PD-1 antibody (BD: 565024) were added. The cells were incubated at room temperature in the dark for 20 min, then washed once with 500 g of PBS for 5 min and then resuspended with 1 ml of PBS.
(76) 3. CD3+CD8+PD-1+ T cells were sorted into a 96-well U-shaped bottom plate using a flow cell sorter (BD FACSAria II), with 6,000 cells sorted per well. The cells sorted into the 96-well plate were allowed to stand for 1 hr, and fresh X-VIVO15 medium was added.
(77) 4. The following TLR agonists (invivoGen: tlr-kit1hw, human TLR1-9 Agonist KIT) were added in groups: a TLR1/2 agonist-Pam3CSK4 (0.1-1 μg/ml), a TLR5 agonist-S. typhimurium Flagellin (10 ng-10 μg/ml) and a TLR6/2 agonist-FSL-1 (1 ng-1 μg/ml) in combination with cytokines IL-7 (PeproTech: 200-07-1000, 20 ng/ml-200 ng/ml) and IL-15 (PeproTech:200-15-1000, 10 ng/ml-100 ng/ml) as well as magnetic beads (CELLection™ Dynabeads, Invitrogen: 11531D) co-coated with an anti-human CD3 antibody (BD: 555336) and an anti-human CD28 antibody (BD: 555725).
(78) The following stimulants were added in groups to 5 wells:
(79) (1) CD3/28+IL-7+IL-15+TLR1/2 agonist+TLR2/6 agonist+TLR5 agonist;
(80) (2) CD3/28+IL-7+IL-15+TLR1/2 agonist+TLR2/6 agonist;
(81) (3) CD3/28+IL-7+IL-15+TLR1/2 agonist+TLR5 agonist;
(82) (4) CD3/28+IL-7+IL-15++TLR2/6 agonist+TLR5 agonist; and
(83) (5) CD3/28+IL-7+IL-15.
(84) 5. The cells were cultured in an incubator at 37° C., transferred to a 24-well culture plate on the 8.sup.th day and transferred to a T25 cell culture flask on the 15.sup.th day. Each time a culture flask was replaced, magnetic beads coated with CD3/28 were supplemented to the cells, wherein the ratio of the magnetic beads to the cells was 1:1. The cells were counted each time the medium was replaced.
(85) 6. After culture for 19 days, it was observed that PD-1+CD8+ T cells proliferated at the fastest rate under the culture conditions of CD3/28+IL-7+IL-15+TLR1/2 agonist+TLR2/6 agonist+TLR5 agonist. The experimental results are shown in
Example 7: Amplification of CD8+ T Cells and PD-1+CD8+ T Cells
(86) 1. The cells sorted into the 96-well plate were allowed to stand for 1 hr, and fresh X-VIVO 15 medium was added. A TLR1/2 agonist (Pam3CK4, invivoGen: tlrl-pms, 1-300 ng/ml), a TLR2/6 agonist (FSL-1, invivoGen: tlrl-fsl, 1-100 ng/ml), a TLR5 agonist (Flagellin, invivoGen: tlrl-epstfla-5, 10-100 ng/ml), IL-7 (PeproTech: 200-07-1000, 20 ng/ml-200 ng/ml), IL-2 (PeproTech: 200-02-1000, 10 U/ml-100 U/ml), IL-15 (PeproTech: 200-15-1000, 10 ng/ml-100 ng/ml) as well as magnetic beads (CELLection™ Dynabeads, Invitrogen: 11531D) coated with an anti-human CD3 antibody (BD: 555336) and an anti-human CD28 antibody (BD: 555725) were added at the same time, wherein the ratio of the magnetic beads to the cells was 1:1. The cells were placed in an incubator at 37° C. for one week.
(87) 2. The cells in the 96-well plate were transferred to a 24-well plate in the second week, and then the cells were sequentially transferred to a T25 cell culture flask, a T75 cell culture flask and a T175 cell culture flask according to the number of growing cells.
(88) 3. Each time a culture dish was replaced, corresponding media and stimulants were supplemented until the desired number of cells was achieved. Cell proliferation is shown in
Example 8: Detection of Cytokine Secretion Ability of Amplified CD8+ T Cells and PD-1+CD8+ T Cells
(89) The cells amplified in Example 7 were taken and placed in a 96-well plate at 1×106 cells/well. PMA (phorbol myristoyl acetate) (50 ng/ml)/ionomycin (5 μM) and an anti-CD107a antibody (Biolegend: 328616) were added. After 1 hr, GolgiPlug (BD: 555029) was added, and cultured for 5 hrs, and then the cells were stained with IFN-γ (BD: 557763) and Granzyme B (abbreviated as GrB in
Example 9: Culture Supernatants of Amplified CD8+ T Cells and PD-1+CD8+ T Cells were Analyzed for Mycoplasma and Endotoxin
(90) Culture supernatants of the amplified CD8+ T cells and PD-1+CD8+ T cells were analyzed for mycoplasma using a LONZA kit (MycoAlert Mycoplasma DetectionKIT) according to the manufacturer's instructions. The ratio of the detected value B to the detected value A of a microplate reader was calculated. If the ratio was less than 0.9, the result was qualified as valid and there was no mycoplasma contamination. If the ratio was greater than or equal to 0.9, the detection result was disqualified. The ratio in this experiment was less than 0.9, i.e. the amplified CD8+ T cells and PD-1+CD8+ T cells had no mycoplasma contamination.
(91) Culture supernatants of the amplified CD8+ T cells and PD-1+CD8+ T cells were analyzed for endotoxin using a LONZA kit (Limulus Amebocyte Lysate QCL-1000 kit) according to the manufacturer's instructions. The endotoxin content of the sample was calculated according to a standard curve using endotoxin in E. coli O111:B4 as a standard. The detection result was qualified if the endotoxin content was not more than 0.3 EU/ml, but disqualified if the endotoxin content was greater than 0.3 EU/ml. The endotoxin content detected in this experiment was not more than 0.3 EU/ml, i.e. the amplified CD8+ T cells and PD-1+CD8+ T cells were qualified.
Example 10: Comparison of Proliferation of PD-1+CD8+ T Cells Under Intermittent Stimulation and Continuous Stimulation
(92) PD-1+CD8+ T cells exhibited different proliferation ability and different number of functional cells under intermittent stimulation and continuous stimulation.
(93) 1. Peripheral blood mononuclear cells (PBMC) of healthy subjects were isolated.
(94) 2. 1×10.sup.7 PBMCs were taken, and an anti-human CD3 antibody (BD: 558117), a CD8 antibody (BD: 561423) and a PD-1 antibody (BD: 565024) were added. The cells were incubated at room temperature in the dark for 20 min, then washed once with 500 g of PBS for 5 min and then resuspended with 1 ml of PBS.
(95) 3. CD3+CD8+PD-1+ T cells were sorted into a 96-well U-shaped bottom plate by a flow cell sorter (BD FACSAria II), with 10,000 cells per well. The cells sorted into the 96-well plate were allowed to stand for 1 hr, and fresh X-VIVO 15 medium was added.
(96) 4. The following stimulants were used: a TLR1/2 agonist (Pam3CK4, invivoGen: tlrl-pms, 1-300 ng/ml), a TLR2/6 agonist (FSL-1, invivoGen: tlrl-fsl, 1-100 ng/ml), a TLR5 agonist (Flagellin, invivoGen: tlrl-epstfla-5, 10-100 ng/ml), IL-7 (PeproTech: 200-07-1000, 20 ng/ml-200 ng/ml), IL-2 (PeproTech: 200-02-1000, 10 U/ml-100 U/ml), IL-15 (PeproTech: 200-15-1000, 10 ng/ml-100 ng/ml) as well as magnetic beads (CELLection™ Dynabeads, Invitrogen: 11531 D) coated with an anti-human CD3 antibody (BD: 555336) and an anti-human CD28 antibody (BD: 555725). The cells were divided into 5 groups, as shown in Table 1. Taking group 7-2-2 as an example: all the stimulants were added for 7 days of continuous stimulation in the first round, the cells were transferred to a 24-well plate, all the stimulants were added for 2 days of stimulation, then the magnetic beads coated with CD3/28 were removed, the cells were cultured for 5 days and then transferred to a T25 culture flask, all the stimulants and the magnetic beads coated with anti-human CD3/28 were added for 2 days of stimulation, then the magnetic beads coated with CD3/28 were removed, and the cells continued to be cultured for 5 days. The days of stimulation and intermittent days are shown in Table 1.
(97) TABLE-US-00001 TABLE 1 cell culture First round of Second round of Third round of culture culture culture Days of Inter- Days of Inter- Days of Inter- stimu- mittent stimu- mittent stimu- mittent Group lation days lation days lation days 7-2-2 7 0 2 5 2 5 7-5-5 7 0 5 2 5 2 7-5-7 7 0 5 2 7 0 7-7-5 7 0 7 0 5 2 7-7-7 7 0 7 0 7 0
(98) 5. After the three rounds of culture and stimulation, the 5 groups of cells were respectively taken and placed in a 96-well plate at 1×10.sup.6 cells/well. 1×10.sup.6 magnetic beads coated with an anti-human CD3 antibody and an anti-human CD28 antibody, and 5 μl of an anti-CD107a antibody (Biolegend: 328616) were added to 100 ul of the system. After 1 hr, GolgiPlug (BD: 555029) was added, and cultured for 5 hrs, then the cells were stained with CD8 (BD: 563677), IFN-γ (Biolegend: 502506), IL-2 (Biolegend: 500307) and Granzyme B (abbreviated as GrB in
(99) 6. The cells of each group were counted, and the proportion and count of different functional cells in each group of cells were calculated. The results are shown in
Example 11: Proliferation of PD-1+CD8+ T Cells of Lung Cancer Patients was Stimulated by TLR Agonists
(100) 1. Peripheral blood mononuclear cells (PBMC) of lung cancer patients were isolated.
(101) 2. 1×10.sup.7 PBMCs were taken, and an anti-human CD3 antibody (BD: 558117), a CD8 antibody (BD: 561423) and a PD-1 antibody (BD: 565024) were added. The cells were incubated at room temperature in the dark for 20 min, then washed once with 500 g of PBS for 5 min and then resuspended with 1 ml of PBS.
(102) 3. CD3+CD8+PD-1+ T cells were sorted into a 96-well U-shaped bottom plate by a flow cell sorter (BD FACSAria II), and 12,000 cells were sorted. The cells sorted into the 96-well plate were allowed to stand for 1 hr, and fresh X-VIVO 15 medium was added.
(103) 4. The following TLR agonists were added to the X-VIVO medium: a TLR1/2 agonist (Pam3CK4, invivoGen: tlrl-pms, 1-300 ng/ml), a TLR2/6 agonist (FSL-1, invivoGen: tlrl-fsl, 1-100 ng/ml), a TLR5 agonist (Flagellin, invivoGen: tlrl-epstfla-5, 10-100 ng/ml), IL-7 (PeproTech: 200-07-1000, 20 ng/ml-200 ng/ml), IL-2 (PeproTech: 200-02-1000, 10 U/ml-100 U/ml), IL-15 (PeproTech: 200-15-1000, 10 ng/ml-100 ng/ml) as well as magnetic beads (CELLection™ Dynabeads, Invitrogen: 11531 D) coated with an anti-human CD3 antibody (BD: 555336) and an anti-human CD28 antibody (BD: 555725), wherein the ratio of the magnetic beads to the cells was 1:1.
(104) 5. The cells in the 96-well plate were transferred to a 24-well plate in the second week according to cell growth, and then the cells were sequentially transferred to a T25 cell culture flask, a T75 cell culture flask and a T175 cell culture flask according to the number of growing cells.
(105) 6. The cells were counted during cell culture. The amplification curve is shown in
(106) The basic principles, main features and advantages of the present invention have been illustrated and described above. Those skilled in the art should understand that the present invention is not limited to the above examples, merely the principles of the present invention are described in the above examples and the description, various changes and improvements can also be made to the present invention without departing from the spirit and scope of the present invention, and these changes and improvements shall fall within the claimed scope of the present invention. The claimed scope of the present invention is defined by the appended claims and equivalents thereof.