CN113150981A - Method for simulating influence of cytokine release on indirect target cell phenotype in vitro - Google Patents

Method for simulating influence of cytokine release on indirect target cell phenotype in vitro Download PDF

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CN113150981A
CN113150981A CN202010075799.6A CN202010075799A CN113150981A CN 113150981 A CN113150981 A CN 113150981A CN 202010075799 A CN202010075799 A CN 202010075799A CN 113150981 A CN113150981 A CN 113150981A
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张斌
孙耀
赵龙
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Fifth Medical Center of PLA General Hospital
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Abstract

The invention discloses a method for simulating influence of cytokine release on indirect target cell phenotype in vitro, relates to the field of immune cell research, and particularly relates to in vitro research on influence of cytokine on indirect target cell phenotype when CAR-T cells and corresponding target cells are incubated together. CRS in vitro model CART cells (A cells) and corresponding target cells (B cells) were added to the upper compartment and indirect target cells (C cells, including HUVEC, PBMCs, CD34+ magnetic bead sorted G-PBMCs) were added to the lower 12-well plates in a Corining Transwell 12-well plate (0.4 μm pore size, 12mm bottom area). Substances with a pore size of less than 0.4 μm can freely pass through (e.g., cytokines), while cells cannot freely pass through, thereby mimicking the effect of cytokine release on the phenotype of indirect target cells such as HUVECs when CAR-T cells are contacted with tumor cells.

Description

Method for simulating influence of cytokine release on indirect target cell phenotype in vitro
Technical Field
The invention relates to the field of cytokine release research in immunotherapy, in particular to in-vitro research on indirect target cells through cytokine release when CAR-T cells and corresponding target cells are incubated together.
Background
Since 2010, Chimeric antigen receptor (CAR-T) therapy has been breaking through, and has shown good therapeutic effects on various hematological tumors such as acute lymphocytic leukemia, B cell lymphoma, multiple myeloma, and the like, and partial solid tumors. In 2017, the U.S. FDA approved two CAR-T drugs to be marketed. The widespread application of CAR-T also brings about a plurality of adverse reactions, wherein the most common and serious phenomenon is Cytokine Release Syndrome (CRS). CRS was first defined in the 90 s treatment with T cell mab OKT 3. The CRS of most of the monoclonal antibodies currently used in the clinic is low in incidence, but is rather high in incidence in T cell-based immunotherapy, which is often in the early stages of clinical development, including bispecific-antibody (DART), T-cell receptor modified-T cells (TCR-T) or CAR-T, where the highest incidence of CRS occurs after CAR-T treatment. CRS after CAR-T treatment mainly shows fever, hypertension, blood coagulation dysfunction, capillary vessel leakage and the like. CRS occurs in about 54-91% of patients receiving treatment, with a severe CRS incidence of 8.3-43%. The primary mechanism of CRS generation is not completely understood at present, mainly because CAR-T recognizes antigen-positive target cells (tumor cells or normal cells) upon reinfusion back into the body or other T cell tumor immunotherapy results in a massive activation, proliferation, killing of target cells and release of cytokines in large amounts. The released cytokines further activate bystander immune cells (monocytes, macrophages, T cells, B cells, etc.) or non-immune cells (e.g., endothelial cells), causing these cells to release a large amount of cytokines, creating a cascade effect that leads to the development of CRS and the corresponding clinical symptoms. [1,2].
Referring to figure 1, after CAR-T cell transfusion, tumor cells are identified, activated, expanded and released cytokines such as IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha, etc., immune cells such as monocytes/macrophages, T cells and DC cells in vivo are activated, and non-immune cells such as endothelial cells, the number and kinds of cytokines released are expanded. In addition, IL-6 and sIL-6R α can act on most cells through gp 130. Adrenaline passes through monocytes/macrophages and T cells, and upon stimulation induction, alpha 1-and alpha 2-adrenergic receptor levels are elevated and cytokine and catecholamines hormones (adrenaline, noradrenaline and dopamine) release is increased, further exacerbating inflammation
In CRS, activated T cells release cytokines and chemokines such as IL-2, sIL-2R alpha, IL-6, sIL-6R, GM-CSF, IFN-gamma, other bystander immune cells such as monocytes/macrophages (releasing IL-1Ra, IL-10, IL-6, IL-8, CXCL10(IP-10), CXCL9(MIG), IFN-alpha, CCL3(MIP-1 alpha), CCL4(MIP-1 beta), and sIL-6R, Dendritic Cells (DC) [3] in recent two years of research shows that activation of endothelial cells and monocytes/macrophages plays an increasingly important role in the development of CRS [1,4-7] further studies changes in the state of CRS in normal cells in vivo, especially immune cells, in immune phenotype and function, becomes an important way to understand CRS more deeply.
In addition, the CD123 molecule is an important target for AML treatment at present, but serious toxic and side effects such as hematopoietic system inhibition, endothelial Cell Leakage Syndrome (CLS), CRS and the like appear in CAR-T treatment and clinical trials of monoclonal antibody drug treatment. [8,9] it has been reported in the literature that CD123 is expressed in HUVEC cells by IFN-. gamma., TNF-. alpha.and the like [10,11 ]. Therefore, it is important to study the expression change of CD123 in various cells under the CRS state.
However, current means of studying CRS are limited and not mature enough. At present, two established CRS models are mainly used, one is established by SCID-beige mice, T cells and tumor cells are human and are murine when cells such as monocyte phagocytosis and the like are used, and the simulation degree of the CRS models is limited to a certain extent [5 ]. Another model uses genetically modified humanized NSG mice that produce human IL-3, SCF and GM-CSF, thereby promoting human T, B and monocyte development and better mimicking CRS [4 ]. However, the current modeling technology has higher difficulty and higher cost, no finished product is available in China, and the function of human endothelial cells in CRS cannot be reflected.
The current common method for studying CAR-T function in vitro is to co-incubate T cells with target cells and then detect target apoptosis and cytokine release. This approach can demonstrate the effects of direct T cell contact with target cells in vitro, but does not mimic the effects of CAR-T cells on other cells indirectly after activation due to cytokine secretion. The Transwell permeable support is a microporous membrane structure attached to a plastic cup-shaped nest, and is shown in FIG. 2, where 1 is the Transwell, 2 is the upper compartment, 3 is the microporous membrane, and 4 is the lower compartment. At present, the main applications include transport experiments, secretion experiments, drug tropism/invasion experiments and co-culture experiments. A pore size Transwell of 0.4 μm allows cytokines to pass freely but not cells. In order to simulate the influence of cytokine secretion on other cells after the CAR-T cells and target cells are incubated together in vitro, the method adopts Transwell to design indexes such as indirect target cell immunophenotype research under the condition of in vitro CRS simulation, obtains better experimental effect when applied in vitro, and has no relevant disclosure at home and abroad at present.
Disclosure of Invention
In recent years, cellular immunotherapy, in particular CAR-T cell therapy, has continued to make breakthroughs. However, with more clinical trials and applications of CAR-T cell therapy, prevention and mechanism studies of CRS are becoming more and more important. At present, in vitro experimental methods lack a model for effectively simulating the influence of CRS on other cells. In this model, CAR-T co-incubation with tumor cells in the upper compartment of the Transwell simulated the release of cytokines in large amounts upon activation and expansion of CAR-T in the high tumor burden state after reinfusion. And the indirect target cells in the lower compartment are in the same cytokine state as CAR-T and tumor, but do not directly contact with CAR-T and tumor cells, so the cell factor in CRS state has a good in vitro model for researching the influence of the cytokine on the indirect target cells.
The model has the following advantages when used for simulating CRS in vitro:
1. the model can be used for researching various cells, besides endothelial cells, CD3+, CD56+, CD14+, CD11c + cells and CD34+ G-PBMCs in PBMCs, and can also be used for various suspended or adherent cells such as CAR-T cells, tumor cells, smooth muscle cells and the like, and the indirect influence of released cytokines on indirect target cells is researched while the CAR-T is incubated with the tumor cells in vitro, so that the interference of direct contact influence is eliminated. For example, the effect of cytokine action alone on indirect target cells can be compared by adding effector cells and target cells in a Transwell and placing the same target cells in the lower layer, thereby testing the separation of direct contact and indirect effect.
After the CAR-T cells are returned to a human body, the target antigen positive cells are recognized in the vascular cavity or organs of the human body to release a large amount of cytokines, so that the concentration of blood or specific cytokines is increased. In this process, the effect on a large number of cells negative for the target antigen is often caused by indirect recognition. Compared with direct incubation, the model can simulate paracrine under the CRS state and influence on remote cells.
3. Is convenient for collecting and detecting indirect target cells. Due to the Transwell separation, the cells in the lower compartment can be collected for detection directly, and the difficulty of separating the cells from the upper compartment is avoided.
4. The effects of direct contact of CAR-T cells with indirect target cells can be excluded and the effect of cellular secretions (predominantly cytokines) on indirect target cells can be studied separately. Due to the limitation of sample material selection, the effector cells and indirect target cells are not always from the same donor. If effector cells are directly co-incubated with indirect target cells, allogeneic rejection may occur due to MHC mismatch, thereby interfering with the results of the experiment. By adopting the model for research, effector cells from different donors and indirect target cells can be incubated together without considering the interference of rejection reaction caused by MHC mismatching on the experiment.
Drawings
FIG. 1 CRS Induction schematic in the prior art
FIG. 2 schematic diagram of CRS model in vitro according to the present invention
FIG. 3CD123-CAR-41BBZ-EGFR retroviral vector Structure schematic SP: a signal peptide; VL: a variable region of a light chain; and Lk: a hinge region; VH: the variable region of the heavy chain; h: a CD8a hinge; TM: the CD8 transmembrane region; 2A: P2A peptide. tEGFR: truncated human epidermal growth factor receptor
FIG. 4 flow assay cell phenotype results A Positive Rate of CD3 after sorting and CAR expression efficiency after CAR-T transfection B efficiency of expression of CD123 by different tumor cells
FIG. 5 uses the model of the invention to study the effect of CD 123-targeting CAR-T incubation on the CD123 expression level of HUVEC on the changes in CD123 expression levels in endothelial cells following incubation with AML tumor cells, data are presented as
Figure BDA0002378460690000041
The (n ═ 6 or 4) Control group represents the untreated group. Comparing the two samples by t test<0.05,**P<0.01,***P<0.001 and P<0.0001。
FIG. 6 supernatant cytokine detection from cytokine ELISA after incubation of CD 123-targeted CAR-T and AML tumor cells using the model of the invention, data are shown as
Figure BDA0002378460690000042
(n-3) identification represents cells co-incubated with HUVEC. The Control group represents the unprocessed group. Comparing the two samples by t test<0.05,**P<0.01,***P<0.001 and P<0.0001。
FIG. 7A is a graph showing the detection of the positive rate of CD34, CD38 and CD123 in G-PBMCs-derived CD34+ cells before incubation; panel B is a Control group representing untreated groups using this model to study the effect of CD 123-targeting CAR-T incubation on CD34+ cell CD123 expression levels after incubation with AML tumor cells. The comparison between the two samples was performed by t-test, P <0.05, P <0.01, P <0.001 and P < 0.0001.
FIG. 8A Control group representing untreated groups was investigated using the model of the invention to investigate the effect of CD 123-targeting CAR-T incubation on CD3+ cell CD123 expression levels in PBMCs after incubation with AML tumor cells. The comparison between the two samples was performed by t-test, P <0.05, P <0.01, P <0.001 and P < 0.0001.
FIG. 9A Control group representing the untreated group was investigated using the model of the invention to investigate the effect of CD 123-targeting CAR-T incubation on CD56+ cell CD123 expression levels in PBMCs after incubation with AML tumor cells. The comparison between the two samples was performed by t-test, P <0.05, P <0.01, P <0.001 and P < 0.0001.
FIG. 10-C-CD 11c + cells CD123 expression Control group representing the effect of CD 123-targeting CAR-T incubation with AML tumor cells on CD123 expression levels of CD11c + cells in PBMCs after incubation with the inventive model. The comparison between the two samples was performed by t-test, P <0.05, P <0.01, P <0.001 and P < 0.0001.
FIG. 11A Control group representing the untreated group was investigated using the model of the invention to investigate the effect of CD 123-targeting CAR-T incubation on CD14+ cell CD123 expression levels in PBMCs after incubation with AML tumor cells. The comparison between the two samples was performed by t-test, P <0.05, P <0.01, P <0.001 and P < 0.0001.
Detailed Description
As shown in FIG. 2, the CRS model in vitro used in the present invention comprises an upper compartment 2, a microporous membrane 3 and a lower compartment 4, wherein A cells and B cells are cultured in the upper compartment and target cells are cultured in the lower compartment. The specific model building method will be described in detail below.
First, preparing the material
1. Collecting a specimen: collecting 10mL of peripheral blood specimen of AML patient or healthy donor by using EDTA-K2 anticoagulant tube, and obtaining Peripheral Blood Mononuclear Cells (PBMCs) and primary AML tumor cells of healthy people by using a Ficoll density gradient centrifugation method. K562, MOLM-13 cells were purchased from ATCC cell banks. Human primary Umbilical Vein Endothelial Cells (HUVEC) were purchased from Sciencell, USA.
2. Reagents for experiments: transwell (12mm bottom area, 0.4 μm pore size; Corning, USA); RPMI1640 medium (Gibco, USA); fetal bovine serum (FBS, french, Biowest); phosphate buffered saline (PBS, Gibco, USA); penicillin/streptomycin double-resistant solution (Gibco, usa); XVIVO-15 serum-free immune cell culture medium (Lonza, USA); lymphocyte separation (Ficoll, GE, usa); CD3/CD28 sorting and activating magnetic beads (Thermo Fisher, USA); endothelial cell culture medium (ECM, Sciencell, usa); EDTA-K2 anticoagulation tube (INSEPACK, China); magnetic particle concentration instrument (MPC, Thermo Fisher Co., USA); ethylenediaminetetraacetic acid buffer (EDTA, MACS, germany); CD34 MicroBead Kit UltraPure (MACS, Germany); flow through antibodies (Biolegend, usa); anti-human CD123-APC or PE, anti-human CD11c-FITC, anti-human CD56-PE, anti-human CD3-PercP, anti-human CD19-FITC, anti-human CD14-PE, anti-human CD66b-PercP, anti-human CD38-FITC flow antibodies are purchased from American BioLegend, human TNF-alpha, IFN-gamma, IL-2, IL-3, IL-4, IL-6 factor ELISA detection kit (China Unico Bio Inc)
Second, method
(one) Experimental groups
In experiments with HUVEC cells as indirect target cells, the experiments were divided into the following 12 groups according to different a cells and B cells: control group, NT group, CART123 group, MOLM-13 group, K562 group, AML-2 group, NT + MOLM-13 group, CART123+ MOLM-13 group, NT + K562 group, CART123+ K562 group, NT + AML-2 group, CART123+ AML-2 group. In CD34+ cell and PBMCs experiments, an IFN-gamma 1000IU/ml treatment group, a TNF-alpha 1000IU/ml treatment group, an IFN-gamma + TNF-alpha 1000IU/ml treatment group and an IL-4100 ng/ml treatment group are added on the basis.
(II) cell preparation
Preparation of a cells: including CART123 cells as well as NT (untransfected group) cells. The CD 123-targeting CAR structure was anti-CD 123 scFv-41BB-CD3 ζ -tEGFR (see FIG. 2). In FIG. 3, 10ml of peripheral blood was collected from healthy volunteers and PBMCs were isolated by Ficoll. Mixing CD3/CD28 magnetic beads of Thermo Fisher company in a ratio of CD3+ cells to magnetic beads of 1:1 in a 15ml centrifuge tube, gently shaking at room temperature for 40 minutes, placing on a magnetic frame, gently blotting adherent cells after 2 minutes, adding PBS to resuspend cells, centrifuging 400g of cells, removing supernatant, adding complete medium (XVIVVO-15 + 10% by volume FBS + IL-250 IU/ml), and mixing at 5 × 105Cells were seeded per ml cell number. Infection of cells 48 hours later with a retrovirus reverse-bearing the CAR target Gene. Adding or replacing fresh culture medium every 1-2 days, and controlling cell density at 5 × 105-2×106And/ml. CAR expression efficiency was measured 5 days after transfection, see figure 4, panel a. A graph shows that the positive rate of CD3 after sorting and the expression efficiency after CAR-T transfection are detected, the positive rate of CAR can reach 81.7%, and the positive rate of CD3 reaches 92.5%.
B cell preparation: the target cells include target antigen negative: human erythroleukemia cell line K562(ATCC), and positive for target antigen: human acute myeloid leukemia MOLM-13(ATCC), and primary AML cells AML-2 and AML-3 derived from peripheral blood of patients. The primary AML cells select a peripheral blood specimen of an AML patient with high tumor load, PBMCs are obtained by separation, and the CD45/SSC is detected by a flow cytometer to set a gate, so that the juvenile cell population is higher (more than 80%). The CD123 expression ratio of each target cell is shown in B diagram of FIG. 4. Diluting the cord blood and PBS according to a ratio of 1:2, adding 20ml of Ficoll density gradient lymph separation liquid into a 50ml centrifuge tube, inclining the centrifuge tube, and slowly adding 30ml of diluted blood sample to the upper layer of the separation liquid. Centrifuging at 400 Xg for 30 min at 4 ℃, sucking the middle layer gray mononuclear cell layer into a 50ml centrifuge tube, adding 5 times volume of PBS, mixing uniformly, centrifuging at 400 Xg for 10 min, washing for 2 times, adding 300 mu l of EDTA buffer solution containing 0.5% BSA, resuspending and counting. The monocyte suspension is added with 100 mul of FcR Blocking reagent and CD34 MicroBeads UltraPure respectively and mixed evenly by adopting CD34 magnetic bead sorting technology of Germany and American danni company, and then is placed in a refrigerator at 4 ℃ for incubation for 30 minutes. After the incubation was completed, 10ml of MACS buffer 1700rpm was added and centrifuged for 10 minutes, and the supernatant was removed and 500. mu.l of MACS buffer was added for resuspension. The column was pre-wetted with 500. mu.l of MACS buffer, the suspension was passed through the column, washed 3 times with 500. mu.l of MACS buffer, the column was removed from the magnetic field, 2ml of MACS buffer was added, the solution was quickly pushed out into a 15ml centrifuge tube with a piston, and counted for future use. The target cell CD123 expression level was detected by flow cytometry.
C, cell preparation: HUVEC, human PBMCs and mobilized human PBMCs (G-PBMCs) from CD34+ cells. HUVEC were cultured in vitro in ECM medium to about 80-90% confluency, trypsinized until the cells floated, neutralized with equal amount of FBS, and centrifuged for 5 minutes at 400g in 5-fold volume of RPMI1640 medium. After centrifugation, the cells were removed from the supernatant, resuspended in ECM medium and passaged by inoculation at the appropriate density. If the cells are used for co-culture, the cells are re-suspended in RPMI1640+ 10% FBS medium before being seeded. In order to maintain the HUVEC character, 7 generations of the previous HUVEC cells are adopted in the experiment.
CD34+ cell preparation protocol: PBMCs are respectively added into 100 mu l of FcR Blocking reagent and CD34 MicroBeads UltraPure and mixed uniformly by adopting CD34 magnetic bead sorting technology of Germany and American danni company, and then the mixture is placed in a refrigerator at 4 ℃ for incubation for 30 minutes. After the incubation was completed, 10ml of MACS buffer 1700rpm was added and centrifuged for 10 minutes, and the supernatant was removed and 500. mu.l of MACS buffer was added for resuspension. The column was pre-wetted with 500. mu.l of MACS buffer, the suspension was passed through the column, washed 3 times with 500. mu.l of MACS buffer, the column was removed from the magnetic field, 2ml of MACS buffer was added, the solution was quickly pushed out into a 15ml centrifuge tube with a piston, and counted for future use.
(III) Co-culture of cells
1ml of RPMI1640+ 10% FBS was added to a 12-well plate in which a Transwell was nested, and the mixture was allowed to stand for 30 minutes or longer to allow the culture medium to be balanced inside and outside the Transwell. In a 2mm bottom area, 0.4 μm pore size Transwell (nested in a 12-well plate), the corresponding cells were added in upper compartments in groups.
Control group: 1. blank control group: no cells were added; 2. separate groups of effector cells: adding 1X 105CART123 or NT cells; individual groups of target cells: adding 5X 105Target cells including K562, MOLM-13 and primary AML groups (AML-2 in HUVEC experiment, AML-3 in PBMCs and CD34+ experiment; effector cells and target cells co-incubation group, i.e. experimental group, target cells 1 × 105+ effector cells 5X 105The group is NT + MOLM-13 group, CART123+ MOLM-13 group, NT + K562 group, CART123+ K562 group, NT + AML-2 group and CART123+ AML-2 group. Each group had 3 secondary wells, with 6 more wells prepared for control in flow assays. Adding target cells to the lower layer, including
1. Target cells HUVEC: HUVEC cells (before passage 7) resuspended in RPMI1640+ 10% FBS were added to the lower layer to give a confluency of approximately 50% and a total volume of 1.5 ml. After culturing for about 36h, the Transwell was carefully removed, the lower layer medium was aspirated (-80 ℃ frozen for detection of cytokines), washed once with PBS, cells were trypsinized, digestion was stopped with an equal volume of FBS, PBS was added to centrifuge the cells, the supernatant was removed and ready for flow detection.
2. Target cells PBMCs or CD34+ cells: PBMCs resuspended in RPMI1640+ 10% FBS or sorted CD34+ cells were added to the lower layer in a total volume of 1.5ml per well. After 24-36h of culture, the Transwell was carefully removed, the lower cell suspension was aspirated, centrifuged to remove the supernatant and ready for flow assay.
The culture medium in the incubation experiment is RPMI1640+ 10% FBS.
(IV) flow assay
Using BD AccuriTMC6 flow detector. The CD34+ phenotype after sorting by magnetic beads was detected by FITC-labeled CD38, PE-labeled CD34, and APC-labeled CD 123. When the target cells are HUVEC or CD34+, the cells are adjusted to 100 μ l after each well is cultured, CD123 antibody is added, and a tube of CD123 negative control is made by using the cells of the control group; when the target cells are PBMCs, dividing the cell suspension cultured in each hole into two tubes, wherein 100ul of cell suspension in each tube is added in 1 tube, and FITC-labeled CD11c, PE-labeled CD56, Percp-labeled CD3 and APC-labeled CD123 are added in each tube; FITC labeled CD19, PE labeled CD14, Percp labeled CD66b and APC labeled CD123 were added to 2 tubes, and compensation adjustment and negative control were performed for each channel single marker group.
Vortex each tube, incubate in the dark at room temperature for 15min, add appropriate amount of PBS, 500 Xg horizontal centrifugation at room temperature for 5min, discard the supernatant, add PBS 100. mu.l on the computer to determine.
(V) cytokine detection
The frozen cytokines were assayed for levels of the cytokines TNF- α, IFN- γ, IL-2, IL-3, IL-4, IL-6 by ELISA.
Third, statistical analysis method
The statistical treatment is carried out by adopting Graphpad Prism 7 software, the data comparison between two groups adopts t test, and the difference with P <0.05 has statistical significance. The experimental results were replicated for three independent samples and expressed as mean ± SEM.
Fourthly, the result
(I) CD 123-targeting CAR-T cell identification and functional verification
CD 123-targeting CAR-T cell construction schematic (FIG. 1),
the T cells after 48h of activation are sorted by packaging retrovirus-infected CD3/CD28 magnetic beads, the proportion of CD3+ cells detected after 5 days can be more than 98%, and the infection efficiency of the T cells can reach about 80%. (FIG. 4A) different cells were tested for expression of CD123 from the source. (FIG. 4B) the K562 cell line expressed little CD123, and the MOLM-13 cell line, primary cells AML-2 and AML-3 all expressed CD123 at a high level.
(II) comparison of expression of CD123 in HUVEC cells
CD123 expression in each group of HUVEC cells after 36h incubation in the CRS model. As can be seen from figure 5, the CD123 levels of HUVECs were significantly upregulated when CD 123-targeting CAR-T cells were incubated with different AML cells, even when CD 123-targeting CAR-T cells were incubated alone. FIG. 5 shows the expression of Mean Fluorescence Intensity (MFI) for CD 123. CD 123% and MFI were significantly upregulated in the CAR-T alone group, CAR-T with K562, MOLM-13 and AML-2 incubated groups.
(III) comparison of cytokine levels in ELISA assays
After incubation for 36 hours in a CRS model, the culture was incubated, and the expression of different cytokines in the culture supernatant was examined by ELISA. (FIG. 5) IFN-. gamma., TNF-. alpha., IL-2, IL-3, IL-4, and IL-6 were significantly upregulated in the CAR-T cell co-incubation group and tumor cell co-incubation group, and IL-6 was also significantly upregulated in the CAR-T single group.
(IV) comparison of CD123 expression in CD34+ cells
Before incubation, flow detection showed that the proportions of CD34+, CD38+, and CD123+ were 72.5%, 75.6%, and 56.9%, respectively. FIG. 7A is a graph showing the positive rate of CD34, CD38 and CD123 in G-PBMCs-derived CD34+ cells before incubation, and the expression of CD123 in each group of CD34+ cells after 36 hours of culture in the CRS model. FIG. 7 is a B-graph showing the effect of CD 123-targeting CAR-T incubation with AML tumor cells on CD34+ cell CD123 expression levels studied using this model, with significant increases in cell CD123 levels after sorting of the CAR-T cells with MOLM13, K562, AML-3 cell incubations, IFN-. gamma.and TNF-. alpha.groups of CD34+ magnetic beads.
(V) comparison of CD123 expression in PBMCs in cells of different phenotypes
FIG. 8 is a graph showing the effect of CD 123-targeting CAR-T incubation and AML tumor cell incubation on CD3+ cell CD123 expression levels in PBMCs using the model of the present invention, showing that there is no significant difference in CD3+ cell CD123 expression.
FIG. 9 is a graph showing the effect of CD 123-targeting CAR-T incubation and AML tumor cell incubation on CD56+ cell CD123 expression levels in PBMCs using this model, showing that there is no significant difference in CD56+ cell CD123 changes.
FIG. 10 is a graph of CD11c + cell CD123 expression using the model of the invention to study the effect of CD 123-targeting CAR-T incubation after incubation with AML tumor cells on CD123 expression levels in PBMCs on CD11c + cells. From FIG. 10, it can be seen that CD 123% was down-regulated in the AML-3 group, TNF- α, IL-6 treated group, up-regulated in the NT and K562 incubation group, IFN- γ treated group and IL-4 treated group, with significant statistical differences. CD123 MFI was statistically significantly different in the MOLM-13 group, NT and MOLM-13 incubation group, NT and K562 incubation group, CAR-T and AML-3 incubation group, and IL-4 treatment group, with the IL-4 treatment group being the most upregulated. There was a significant statistical difference in the IFN-. gamma.and TNF-. alpha.treatment groups with the downregulation of CD123 MFI.
FIG. 11 is a graph of the effect of CD 123-targeting CAR-T incubation with AML tumor cells on CD14+ cell CD123 expression levels in PBMCs studied using the model of the invention. As can be seen from FIG. 11, CD 123% did not change significantly, and CD123 MFI was significantly up-regulated in the CAR-T group, NT group incubated with MOLM-13, CAR-T group incubated with MOLM-13, CT group incubated with K562, NT group incubated with AML-3, CAR-T group incubated with AML-3, and IL-4 group treated.
Reference documents:
1.Hay KA,Hanafi LA,Li D,Gust J,Liles WC,Wurfel MM,Lopez JA,Chen J, Chung D,Harju-Baker S et al:Kinetics and Biomarkers of Severe Cytokine Release Syndrome after CD19 Chimeric Antigen Receptor- modified T Cell Therapy.Blood 2017.
2.Shimabukuro-Vornhagen A,
Figure BDA0002378460690000101
P,Subklewe M,Stemmler HJ,
Figure BDA0002378460690000102
HA,Schlaak M,Kochanek M,
Figure BDA0002378460690000103
B,von Bergwelt-Baildon MS:Cytokine release syndrome.Journal for immunotherapy of cancer 2018,6(1):56.
3.Neelapu SS,Tummala S,Kebriaei P,Wierda W,Gutierrez C,Locke FL, Komanduri KV,Lin Y,Jain N,Daver N et al:Chimeric antigen receptor T- cell therapy-assessment and management of toxicities.Nat Rev Clin Oncol 2017.
4.Norelli M,Camisa B,Barbiera G,Falcone L,Purevdorj A,Genua M,Sanvito F,Ponzoni M,Doglioni C,Cristofori P et al:Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.Nature Medicine 2018.
5.Giavridis T,van der Stegen SJC,Eyquem J,Hamieh M,Piersigilli A, Sadelain M:CAR T cell–induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade.Nature Medicine 2018.
6.Obstfeld AE,Frey NV,Mansfield K,Lacey SF,June CH,Porter DL, Melenhorst JJ,Wasik MA:Cytokine release syndrome associated with chimeric-antigen receptor T-cell therapy:clinicopathological insights. Blood 2017 130(23):2569-2572.
7.Gust J,Hay KA,Hanafi LA,Li D,Myerson D,Gonzalez-Cuyar LF,Yeung C,Liles WC,Wurfel M,Lopez JA et al:Endothelial Activation and Blood- Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.Cancer Discov 2017, 7(12):1404-1419.
8.Gill S,Tasian SK,Ruella M,Shestova O,Li Y,Porter DL,Carroll M,Danet- Desnoyers G,Scholler J,Grupp SA et al:Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells.Blood 2014,123(15):2343-2354.
9.https://www.ashclinicalnews.org/news/third-patient-death-reported- sl-401-drug-trial-patients-bpdcn-aml/.
10.Korpelainen EI,Gamble JR,Vadas MA,Lopez AF:IL-3 receptor expression,regulation and function in cells of the vasculature. Immunology and cell biology 1996 74(1):1-7.
11.Korpelainen EI,Gamble JR,Smith WB,Goodall GJ,Qiyu S,Woodcock JM, Dottore M,Vadas MA,Lopez AF:The receptor for interleukin 3 is selectively induced in human endothelial cells by tumor necrosis factor alpha and potentiates interleukin 8 secretion and neutrophil transmigration.Proceedings of the National Academy of Sciences of the United States of America 1993,90(23):11137-11141。

Claims (14)

1.a system for simulating CRS influence on other cells in vitro is provided with an upper compartment and a lower compartment, wherein the upper compartment and the lower compartment are separated by a microporous membrane, and is characterized in that A cells and B cells in the upper compartment are co-cultured, and C cells are placed in the lower compartment.
2. The system of claim 1, wherein:
the A cells are selected from CAR-T cells targeting one of CD123 CD19, CD20, CD22, BCMA, MUC-1.
3. The system of claim 1 or 2, wherein: the B cell is a target cell corresponding to the A cell, is a target antigen positive cell or a target antigen negative cell, and is selected from one of the following cells or cell lines: leukemia, lymphoma, multiple myeloma, liver cancer, lung cancer, kidney cancer, or human normal tissue.
4. The system of claim 3, wherein: the B cell is a positive control group or an experimental group and is selected from human leukemia cell line MOLM-13, primary AML cell, primary AML-2 and primary AML-3.
5. The system of claim 1, wherein: the C cells are selected from monocytes, macrophages, T cells, B cells, smooth muscle cells, stromal cells, tumor cells involved in or affected by the cytokine release syndrome.
6. The system of claim 5, wherein: the C cells are indirect target cells and are selected from HUVEC, PBMCs and CD34+ G-PBMCs.
7. The system of claim 1, wherein: the pore diameter of the microporous membrane is less than 3 mu m.
8. Method for using a system for modelling the effect of CRS on other cells according to one of claims 1 to 7, characterized in that it comprises the following steps:
1) co-incubating a cells with B cells in the upper compartment;
2) adding C cells to be researched into a lower compartment;
3) after incubating for a certain time, collecting the lower layer C cells for detecting phenotype, collecting the culture medium for detecting cell factors or freezing for later use.
9. Use according to claim 7, characterized in that:
the A cells are T cells, PBMCs are obtained by collecting human peripheral blood and separated by lymphocyte separating medium, and the PBMCs are activated by CD3/CD28 magnetic beads and cultured under the conditions of X-VIVO-15+ 10% FBS and IL-250 IU/ml.
10. Use according to claim 7, characterized in that:
the A cells are selected from CAR-T cells targeting one of CD123 CD19, CD20, CD22, BCMA, MUC-1.
11. Use according to claim 7, characterized in that: the B cell is a target cell corresponding to the A cell, is a target antigen positive cell or a target antigen negative cell, and is selected from one of the following cells or cell lines: leukemia, lymphoma, multiple myeloma, liver cancer, lung cancer, kidney cancer, or human normal tissue.
12. Use according to claim 11, characterized in that: the B cell is a positive control group or an experimental group and is selected from human leukemia cell line MOLM-13, primary AML cell, primary AML-2 and primary AML-3.
13. Use according to claim 7, characterized in that: the C cells are selected from monocytes, macrophages, T cells, B cells, smooth muscle cells, stromal cells, tumor cells involved in or affected by the cytokine release syndrome.
14. Use according to claim 13, characterized in that: the C cells are indirect target cells and are selected from HUVEC, PBMCs and CD34+ G-PBMCs.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115433709A (en) * 2022-06-13 2022-12-06 南京艾尔普再生医学科技有限公司 In-vitro experimental model for predicting myocardial cell transplantation immune rejection

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735500A (en) * 2019-01-25 2019-05-10 苏州茂行生物科技有限公司 A kind of CAR-T cell and its preparation method and application of secreting type targeting CD133
CN110684739A (en) * 2019-11-11 2020-01-14 深圳市体内生物医药科技有限公司 Chimeric antigen receptor T cell and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735500A (en) * 2019-01-25 2019-05-10 苏州茂行生物科技有限公司 A kind of CAR-T cell and its preparation method and application of secreting type targeting CD133
CN110684739A (en) * 2019-11-11 2020-01-14 深圳市体内生物医药科技有限公司 Chimeric antigen receptor T cell and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SUNYAO ET AL: "IFN-γ and TNF-α aggravate endothelial damage", 《ONCOTARGETS AND THERAPY》 *
孙耀: "靶向CD123的CAR-T治疗AML的临床及机制研究", 《中国优秀博硕士学位论文全文数据库(博士) 医药卫生科技辑(月刊)》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115433709A (en) * 2022-06-13 2022-12-06 南京艾尔普再生医学科技有限公司 In-vitro experimental model for predicting myocardial cell transplantation immune rejection

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