CN114457037B - Construction method and application of C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells - Google Patents
Construction method and application of C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells Download PDFInfo
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Abstract
The invention discloses a construction method and application of a C57BL/6J mouse-derived CD19 chimeric antigen receptor T cell, wherein the method uses C57BL/6J as a research background to explore whether IL-2 exists or not and CD3 under different stimulation conditions + T cell optimal stimulation activation and incubation time; respectively stimulating activated T cells by using different stimulators of the coated anti-CD3/CD28, the coated anti-CD3+Soluble anti-CD28 and the coated anti-CD3 3, and continuously culturing after the stimulation is finished; mCD19CAR-T cells were then prepared using retroviral infection. Compared with the prior art, the invention has the following advantages: (1) The method of the invention can better stimulate and activate T cells, thereby constructing and obtaining GFP + The percentage of the CAR-T cells reaches 22.4%, and the prepared CAR-T cells can meet the requirement of subsequent experiments; (2) The mCD19CAR-T cells constructed by the method have better cell proliferation capability later, and the cell curative effect is ensured.
Description
Technical Field
The invention belongs to the technical field of cell biology and relates to an optimization of C57BL/6J mouse CD3 + The T cell in vitro stimulation condition, optimal infection and culture time, in particular to a construction method and application of a C57BL/6J mouse-derived CD19 chimeric antigen receptor T cell.
Background
Chimeric antigen receptor (chimeric antigen receptor, CAR) refers to a recombinant transmembrane molecule consisting of an extracellular single chain antibody variable region, a hinge transmembrane region, and an intracellular signaling domain; chimeric antigen receptor T cells (CAR-T cells) are T cells that utilize vectors to transfer CAR genes into T cells, making them T cells with specific recognition and killing of tumor cells. CAR-T cell therapy is becoming more and more widely used clinically, playing an important role in anti-tumor immunity. FAD approved three CD19CAR-T cell products Kymriah, yescarta and Tecartus, respectively, for treatment of pediatric acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL) subtype, and relapsed/refractory Mantle Cell Lymphoma (MCL). Currently, CAR-T cell therapies are considered as effective solutions for the treatment of recurrent or refractory tumors, especially hematological malignancies.
Studies have reported that about 50% of patients with relapsed/refractory diffuse large B-cell lymphoma (R/RDLBCL) after CD19CAR-T cell treatment reach complete remission, but the rate of relapse after 1 year of follow-up is about 65%; there are also relevant clinical experiments showing that the initial complete remission rate of Kymriah for non-hodgkin lymphoma is 39.5%, the total survival rate of 6 months is 70.7%, stephen J et al report that the total effective rate is only 53.0% with 40.3 months of follow-up time, and about 61% of patients develop disease progression or recurrence. The data demonstrate that although the therapeutic efficacy of CD19CAR-T cells is still satisfactory, the long-term survival rate of patients is still not ideal, so that the recurrence after disease treatment is a difficult problem to be solved after CAR-T cell treatment, and further, the relevant mechanism of the recurrence of the disease after CD19CAR-T treatment is very important to be studied in a laboratory.
Since the overall structures of the mouse and human immune systems are very similar, mice are often the experimental tool of choice for most immunologists. The laboratory selects two strains of mice of BALB/C and C57BL/6J in the mechanical study, and the primary problem is the optimization of the in vitro stimulation activation system of the primary T cells of the mice. In the early stage, the in vitro BALB/c stimulation activation system is optimized, the CD19CAR-T cells of the BALB/c background are stably prepared, and the requirement of subsequent experiments can be met. However, at present, C57BL/6J is a common strain in oncology and immunology research, most transgenic mice are manufactured by taking C57BL/6J as a genetic background, so that optimizing an in-vitro stimulation activation system of C57BL/6J T cells and stably constructing CD19CAR-T cells are a precondition for the next mechanism research.
CAR-T cell therapy has now become an important treatment regimen for non-hodgkin lymphomas, particularly for relapsed/refractory patients. However, current data indicate that maintaining long-term remission remains a significant hurdle to its use despite the high rate of induction remission following CAR-T cell therapy. The factors that influence the efficacy of CAR-T cells and the mechanisms of primary disease recurrence are of great concern, but the mechanisms involved are not completely understood. Therefore, the mouse model is applied in a laboratory to further reveal the recurrence mechanism of the CAR-T cells after treatment, reduce disease recurrence and have important significance for improving the long-term survival rate of patients.
The prior studies show that the efficacy of CAR-T cells is directly related to the activity, expansion and infection efficiency of T cells, while IL-2 is a T cell growth factor necessary for T cell proliferation, and C57BL/6J is used as a common animal strain in laboratories. Studies show that two different strains of BALB/C and C57BL/6J mice have significant differences in innate immune system function, the former shows more Th2 and M2 dominant immune responses, while C57BL/6 mice show Th1 and M1 dominant immune responses, and the experiment uses a previously optimized BALB/C in vitro stimulation optimization system to prepare C57BL/6J mCD19CAR-T cells with poor efficiency and reduced cell activity.
In vitro T cell stimulation requires TCR-CD3 to provide an initial activation signal and CD28 to provide a co-stimulatory signal, studies have reported that free antibodies generally do not provide sufficient receptor cross-linking to activate intracellular signaling events, T cells can be effectively expanded using immobilized anti-CD3 and anti-CD28 antibodies, and stimulation of T cells with anti-CD3 antibodies alone may render them unreactive, unable to produce IL-2 or fulfill their function. In addition, death occurs soon after T-cell activation by in vitro stimulation, and related studies have reported that IL-2 can prevent death of activated T-cells in vitro and can stimulate proliferation of T-cells.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the defects in the prior art, the invention aims to search whether the IL-2 exists or not and search the CD3 under different stimulation conditions by taking the C57BL/6J as a research background + T cell optimal stimulation activation and incubation time; respectively stimulating activated T cells by using different stimulators of the coated anti-CD3/CD28, the coated anti-CD3+Soluble anti-CD28 and the coated anti-CD3 3, and continuously culturing after the stimulation is finished; mCD19CAR-T cells were then prepared using retroviral infection. In view of this, the present invention provides a C57BL/6J mouse-derived CD19 chimeric antigen receptorT cell construction method and application.
The technical scheme is as follows: a method for constructing a C57BL/6J mouse-derived CD19 chimeric antigen receptor T cell, wherein the method comprises the steps of stimulating activated T cells in an environment containing IL-2 under one of the following 3 conditions, and then preparing mCD19CAR-T cells by retroviral infection;
condition 1: culturing by using anti-CD3 and anti-CD28 antibody coated culture plates;
condition 2: culturing by using an anti-CD3 antibody coated culture plate, and directly adding an anti-CD28 antibody after inoculating cells;
condition 3: only anti-CD3 antibodies were used to coat the plates.
Preferably, the IL-2 final concentration is 5U/mL-300U/mL. Low concentrations of IL-2 are more favorable for early memory T cell production, but effector T cells are less produced, and high doses of IL-2 can cause T cells to fail; 100U/mL is considered to be the standard for T cell production commonly used today, so the final concentration of IL-2 most preferred in the present invention is 100U/mL.
Preferably, the final concentration of anti-CD3 and anti-CD28 antibodies is 1 μg/mL-5 μg/mL. In the early experiments of the invention, the final concentration of 2 mug/mL and 5 mug/mL are used for T cell stimulation and activation, and after 36 hours, the surface activation marker CD25 of the T cells is detected, and two groups of indiscriminate cells are found (the result is shown in figure 5), so that the final concentration of the anti-CD3 and anti-CD28 antibodies is 2 mug/mL. Further, the anti-CD28 antibody is soluble.
Preferably, the stimulation activation time of the conditions 1, 2 and 3 is 12-36h, and the continuous culture time in the IL-2 environment is 24-72h.
Preferably, the method comprises the following specific steps:
(1) CD3 of C57BL/6J after separation and purification + T cells are inoculated in a culture plate treated in the condition 1, the condition 2 or the condition 3, and the culture stimulation is carried out for 12-36 hours;
(2) Transferring the cells subjected to the stimulated culture in the step (1) to a new culture plate, adding IL-2, and continuing to culture for 24-72 hours;
(3) Transferring the cells after the activation culture in the step (2) to a new culture plate, adding mCD19 retrovirus and virus infection enhancing liquid, centrifuging, continuing to culture for 6-8 hours, replacing a fresh culture medium, adding IL-2, continuing to culture, and collecting the cells.
Preferably, the culture plate treated by the condition 2 is used for culturing and stimulating for 24 hours in the step (1), and IL-2 is added in the step (2) for further culturing for 48 hours.
Preferably, the cells collected in step (3) are subjected to flow detection, GFP + The CAR-T cell fraction reached 22.4%.
The C57BL/6J mouse-derived CD19 chimeric antigen receptor T cell obtained by constructing any one of the methods.
The use of the above-described C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells for the treatment of non-hodgkin's lymphoma.
The construction method of the invention has the principle that:
(1) Activation of T cells
Double signaling is required for T cell activation in vivo. The first signal is from the TCR recognizing MHC/antigen peptide complex, delivering an antigen specific recognition signal; the second signal is provided by the costimulatory molecule of the APC (CD 28 or 4-1 BB), which is a nonspecific costimulatory signal. Thus in vitro CD3 + T cells bind to solid phase coated anti-CD3 and anti-CD28, providing the first and co-stimulatory signals required for T cell activation and expansion.
(2) Preparation of CAR-T cells
Co-culturing a plasmid with anti-CD 19, a pro-transfection reagent and packaging cell Plat-E to obtain Retrovirus; the retrovirus is infected with activated T cells, and the mCD19CAR-T cells are obtained by genetic modification.
The schematic of CAR-T cell constitution is shown in FIG. 6.
The beneficial effects are that: (1) The method of the invention can better stimulate and activate T cells, thereby constructing and obtaining GFP + The percentage of the CAR-T cells reaches 22.4%, and the prepared CAR-T cells can meet the requirement of subsequent experiments; (2) The mCD19CAR-T cells constructed by the method have better cell proliferation capability later, and the cell curative effect is ensured.
Drawings
FIG. 1 is a CAR of example 1T cell preparation and percentage detection results, wherein A is CD3 of BALB/C and C57BL/6J + Bright field and fluorescent photographs (100X) of T cell infected mCD19CAR-T retrovirus; b is a CAR-T cell detection flow gate setting scheme; c is the percentage of GFP+ CAR-T cells; wherein the FSC forward scatter signal is related to cell relative volume and size; SSC side scatter signals, representing fine structural changes inside cells, are more sensitive to changes in the envelope, cytoplasm, and nuclear membrane; a represents the area, representing the relationship between the signal intensity and the cell size; w represents the width, representing the time it takes for a cell to pass through the laser beam; h represents the height and represents the intensity of the signal. GFP is a green fluorescent protein in CAR-T cells; B. the abscissa in graph C does not represent the order of magnitude relationship, and the negative and positive group cells can be distinguished only by setting a gate through a control group.
FIG. 2 is a graph showing the results of proliferation conditions of C57BL/6J CD3+ T cells, wherein A is C57BL/6J mouse CD3 + T cells were continued to culture for 24h, 48h and 72h after stimulation with Plated-CD3/CD28, plated-CD3+Soluble-CD28, and Plated-CD3, respectively, for 12h, and CD3 was recorded under a microscope + T cell clone number and cell clone number graph; after 24h of stimulation, the culture was continued for 24h, 48h and 72h, and CD3 was recorded under a microscope + T cell clone number and cell clone number graph.
FIG. 3 is a graph showing the results of optimal activation time of C57BL/6J CD3+ T cells in an IL-2 environment, wherein A is C57BL/6J mouse CD3 + T cells were continued to culture for 24h, 48h and 72h after stimulation with Plated-CD3/CD28+IL-2, plated-CD3+Soluble-CD28+IL-2 and Plated-CD3+IL-2, respectively, for 12h, and CD3 was recorded under a microscope + T cell clone number and cell clone number graph; after 24h of stimulation, the culture was continued for 24h, 48h and 72h, and CD3 was recorded under a microscope + T cell clone number and cell clone number graph; after stimulation for 36h, the culture was continued for 24h, 48h and 72h, and CD3 was recorded under a microscope + T cell clone number and cell clone number graph.
FIG. 4 is a graph of CAR-T cell preparation and percent assay results, wherein A is the bright field and fluorescence photograph (100X) of C57BL/6J CD3+ T cells infected after 24h stimulation with a coated-CD3+Soluble-CD28+IL-2, coated-CD3+IL-2, coated-CD 3/CD28+IL-2 stimulator, respectively; b is the percentage of gfp+ CAR-T cells under different stimulation conditions (48 h); wherein the FSC forward scatter signal is related to cell relative volume and size; a represents the area, representing the relationship between the signal intensity and the cell size; GFP is a green fluorescent protein in CAR-T cells; the abscissa in the B diagram does not represent the order of magnitude relationship, and the negative and positive group cells can be distinguished only by arranging a gate through a control group.
FIG. 5 is the effect of final anti-CD3 and anti-CD28 antibody concentrations of 2. Mu.g/mL, 5. Mu.g/mL on T cell stimulation activation in a preliminary experiment of the present invention; wherein FSC-A is forward angle scattered light, related to cell size and volume; CD25: surface markers of T cell activation.
FIG. 6 is a schematic of CAR-T cell composition.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Retroviral plasmid mCD19 carrying the anti-mouse CD19 antibody gene and Green Fluorescent Protein (GFP) and packaging Plat-E cells were kept by the laboratory; retroviral pro-transfection reagent X-tremgeNE 9DNA transfection Reagent was purchased from Roche company; both opti-MEM and FBS are available from Gibco corporation; DMEM high sugar medium and RPMI 1640 medium were both purchased from kemel biosystems; recombinant human interleukin 2 (IL-2) was purchased from PEPROTECH company; hiTransG P virus infection enhancement fluid was purchased from Ji Kai gene; female BALB/C and C57BL/6J mice for 6-8 weeks were purchased from Vetong Lihua corporation; the flow cytometer is BD LSR Fortessa; the fluorescence microscope was OLYMPUS DP72.
Example 1 preparation of different strains of mouse CAR-T cells and percent detection
Retroviral infection was performed using the previously optimized BALB/c T cell in vitro activation system to prepare CAR-T cells. Grouping: (1) activated CD3 of control group, i.e. uninfected virus + T cells; (2) BALB/c group; (3) c57BL/6J group. Fluorescent brightness was observed under a fluorescent microscope for 24h, 48h and 72h. After 72h of virus infection, cells were collected and flow cytometry was performed to detect GFP, respectively + Percentage of CAR-T cells.
CD3 of C57BL/6J was assayed using the previously obtained BALB/C mouse T cell in vitro optimal activation System + After 36h T cell stimulation, mCD19CAR-T retrovirus was infected, the number of positive cells and fluorescence intensity were observed under a fluorescence microscope for 24h, 48h and 72h, the infection efficiency was primarily evaluated, and GFP was detected by flow + Percentage of CAR-T cells. As shown in FIG. 1A, the BALB/c-derived activated T cells are large in cell clone and scattered after 24 hours of virus infection, the cells are continuously proliferated for 48 hours, and the cells which are not proliferated for 72 hours die, but the clone density is increased; the fluorescence intensity was seen to increase gradually over 24h, 48h and 72h with infection time under fluoroscopy. B6 mouse T cells can obviously proliferate in 24 hours, and the cloning and the cell number in 48 hours and 72 hours are obviously reduced; GFP 72h post infection + The T cell numbers were highest. The percentage of positive cells after 72h of subsequent flow detection was analyzed for BABL/c mouse GFP according to the gating strategy of FIG. 1B + CAR-T positive rate was as high as 41%, while B6 mice were GFP + The percentage of CAR-T cells was only 5.23% (fig. 1C) the above results suggest that although higher infection efficiency was obtained 72h after virus infection of T cells, the activation proliferation status and timing of infection of mouse T cells of different backgrounds had a significant impact on obtaining CAR-T cells with high efficiency.
Example 2 construction condition optimization
1、C57BL/6J CD3 + Optimization of proliferation conditions of T cells in vitro
CD3 of C57BL/6J after separation and purification + T cells, according to 2X 10 6 Concentration of individual cells (1 mL/well) were seeded in 24-well plates treated under different conditions at 37℃with 5% CO 2 Incubator culture. The grouping is as follows: (1) coated anti-CD3/CD28 groups, i.e.plates coated with anti-CD3 and anti-CD28 antibodies overnight at 4 ℃ (final concentration of 2. Mu.g/mL); (2) coated anti-CD3+ soluble anti-CD28 cells were inoculated by coating the culture plates with anti-CD3 antibody overnight at 4 ℃Then, anti-CD28 antibody (the final concentration is 2 mug/mL) is directly added; (3) the coated anti-CD3 group coated the plates with anti-CD3 antibody alone (final concentration 2. Mu.g/mL). After 12h and 24h of stimulation, the cells were transferred to 6-well plates for further culture for 24h, 48h and 72h, photographed under a microscope and the cell clone numbers were recorded. The anti-CD28 antibody is soluble.
2、C57BL/6J CD3 + Optimal activation time of T cells in IL-2 environment
Purified CD3 + T cells were inoculated into 24-well plates treated as described above at the above concentrations, stimulated for 12h, 24h and 36h in the same culture manner, transferred to 6-well plates, and cultured for 24h, 48h and 72h with IL-2 (100U/mL) added, and the cell clone numbers were recorded under a microscope. The grouping is as follows: (1) set of Plated anti-CD 3/CD28+IL-2; (2) coated anti-CD3+ soluble anti-CD28+ IL-2; (3) set of coated anti-CD3+ IL-2. The anti-CD28 antibody is soluble.
3. CAR-T cell preparation in C57BL/6J background
According to the above groups, C57BL/6J CAR-T cells were prepared, activated 24h T cells were transferred into 6-well plates, 2.5mL of corresponding mCD19 retrovirus was added to each well, 40 uL/well 1 XHiTransG P virus infection enhancement solution was added, 1500g (lifting speed was 3), 80min, centrifuged at 30℃and then placed into 37℃and 5% CO 2 After a further incubation time of 6-8h, fresh medium was changed and IL-2 (100U/mL) was added for further incubation. Fluorescent brightness was observed under a fluorescent microscope for 24h, 48h and 72h. After 48h of virus infection, cells were collected and flow cytometry was performed to detect GFP, respectively + Percentage of CAR-T cells.
4. Statistical analysis
Flow cytometry results were analyzed using flowjo_v10 software, and data were plotted and counted by Graphpad Prism 8.0. The average comparison of Two samples adopts t test, the average comparison of a plurality of samples adopts multi-factor analysis of variance (Two-Way ANOVA), and P < 0.05 is statistically significant for the difference.
Analysis of results:
analysis of B6 background CAR-T cells in example 1 for inefficiency may be due to B6 CD3 + T cell inWhen the pre-activation condition is overdriven or the stimulation time is too long, and the virus enters cells and is still not sufficiently proliferated, the T cells die after failure. Therefore, we adjusted the activation conditions of B6 mouse T cells, stimulated with coated anti-CD3/CD28, coated anti-CD3+Soluble anti-CD28 and coated anti-CD3 for 12h and 24h respectively, transferred to 6-well plates, cultured for 24h, 48h and 72h continuously, and recorded the cell clone numbers under the lens. The results of FIG. 2 show that the clonal growth of cells is seen in each group after the 12h culture is stimulated for 24h, and the number of clones in the microscopic view is counted randomly, so that the number of clones in the plant anti-CD3+ Soluble anti-CD28 group can reach 110 cells/view, which is obviously higher than that in the plant anti-CD3/CD28 group and the plant anti-CD3 group; after 48h of culture, the clone numbers of the Plated anti-CD3+Soluble anti-CD28 group are still higher than those of other groups, but the clone numbers of the three groups are all drastically reduced; after 72h of incubation, the number of clones of the plated anti-CD3 stimulated cells was minimized, and there was no difference in the number of clones of the coated or soluble CD28 stimulated group. After 24h of stimulation of T cells under three conditions, the clone number of the Plated anti-CD3/CD28 group reaches the highest 24 th after culture, and then continuously declines, which is similar to the change trend after 12h of stimulation; the number of clones of the Plated anti-CD3+Soluble anti-CD28 group and the Plated anti-CD3 group started to rise to 48h after 24h of culture to peak values of 75 and 25 per field respectively, and then the number of clones was reduced; although the two groups of cells were consistent in their change pattern, the number of cell clones obtained by adding soluble CD28 to the stimulation system was higher at each time point than in the non-added group (fig. 2B). The results show that compared with the stimulation for 12 hours, the T cells of the B6 mice are more fully activated and have stronger continuous proliferation capacity after being stimulated for 24 hours; the use of soluble CD28 has a significant effect on maintaining the proliferative state of cells. The optimized cell clone number can be obtained by culturing for 48 hours after 24 hours of stimulation of the coated anti-CD3+Soluble anti-CD28.
According to the results shown in FIG. 2, activated cells were found to have reduced ability to proliferate continuously in the late stage of culture and to undergo massive death, so we were about to add cytokine IL-2 for promoting activated proliferation of cells, and continued to investigate the activation proliferation of T cells after adding IL-2 in different stimulation environments by using the optimal stimulation and culture time obtained above. Under the stimulation conditions of the coated anti-CD3/CD28, the coated anti-CD3+Soluble anti-CD28 and the coated anti-CD3 respectively, CD3 + IL-2 (100U/mL) was added to the T cell culture system and the culture was continued for 24h, 48h and 72h. As shown in FIG. 3, after IL-2 is added into three activation systems for stimulation for 12 hours, three groups of cells are quickly activated and form cloned cell clusters, the number of clones is the highest after 24 hours of culture, the number of clones is reduced after 48 hours, and the number of the cloned clusters has a return trend, which is different from the change trend of cell activation proliferation when IL-2 is not added; wherein the number of clones was higher for the soluble CD28 plus IL-2 group than for the other two groups at each time point (FIG. 3A). After 24h stimulation, T cells of the plated anti-CD3+Soluble anti-CD28+IL-2 group were cultured for 24h, cell activation was sufficient, clones were uniformly formed, and after 48h proliferation clones could reach 125 per field significantly better than other groups, and the 72h number was reduced without difference from the other two groups (FIG. 3B). As seen from the results of the 36h stimulation subsequent culture, the clone numbers of the three groups of cells were all slowly increased from 24h to a peak value of 48h, and the clone numbers were slightly decreased, wherein the treated anti-CD3+IL-2 group was superior to the treated anti-CD3/CD28+IL-2 group and the treated anti-CD3+Soluble anti-CD28+IL-2 group under the conditions of the culture for 24h and 48h (FIG. 3C). The above results demonstrate that IL-2 addition does not affect the change in proliferation trend due to the difference in stimulation and incubation time of T cell first and second signaling pathway molecules, but IL-2 is effective in maintaining proliferation capacity of activated T cells. Consistent with the results of no IL-2 factor addition, the treated anti-CD3+Soluble anti-CD28 group was stimulated for 24h and cultured for 48h, and the cell proliferation status was best, the cloning number was highest, and significantly higher than that of the IL-2-free group of FIG. 2. The results after 36h of stimulation show that the coated anti-CD3+IL-2 group cells also obtain a better activated proliferation state, but the clinical results of the actual application of the CAR-T cells show that the effect of the second generation CAR-T cells co-stimulated by the serial CD3 and CD28 or 4-1BB signals is obviously better than that of the first generation CAR-T cells only activating CD3 molecules. In summary, activated anti-CD3+ solid anti-CD28 and IL-2 added stimulated 24h group of T cells, with the best state for subsequent preparation of CAR-T cells, and 48h cultured T cells may have the best potential for expression of CAR molecules.
In vitro optimization of B6 mouse T cells was clarifiedTo further verify the efficiency of CAR-T cell production after virus infection following suitable activation conditions, B6 background CD3 was used here + 24h after T cell stimulation, retroviral infection with mCD19CAR, observation of the fluorescence intensity of CAR expression after various incubation times, and detection of GFP at 48h + CAR-T cell percentage. The infection of mCD19CAR in FIG. 4A is consistent with the activation and proliferation state of T cells in the previous results, and shows that the treated anti-CD3+Soluble anti-CD28+IL-2 group has obvious large clone formation in 48h of culture, and the number of positive cells and fluorescence intensity are higher than those of the treated anti-CD3/CD28+IL-2 group and the treated anti-CD3+IL-2 group under a fluorescent screen. Flow detection of GFP after 48h of culture in three groups + The CAR-T cell percentages, 11.6%, 22.4% and 8.03%, respectively, were higher than the CAR-T cell infection efficiency under the conditions of fig. 1 (fig. 4B). The result shows that under the condition of 24h stimulation of the coated anti-CD3+Soluble anti-CD28+IL-2, the efficiency of preparing the CAR-T cells after the B6 mice are subjected to T fine culture for 48h is highest, and the high mCD19CAR-T cells are obtained, so that the subsequent in-vivo and in-vitro experiment requirements can be met.
To sum up: in the embodiment, the 3 different stimulation conditions are used for stimulating and activating T cells, IL-2 is subjected to subsequent culture after the stimulation is finished, and the result shows that the clone number of the cells cultured for 48 hours is higher and better than that of the cells cultured for the treated anti-CD3+ Soluble anti-CD28+ IL-2 group stimulated for 24 hours. To further verify that the coated anti-CD3+Soluble anti-CD28+IL-2 stimulated and activated T cells better, this example used coated anti-CD3/CD28+IL-2, coated anti-CD3+Soluble anti-CD28+IL-2 and coated anti-CD3+IL-2 stimulated T cells for 24h, and used retrovirus infection to continue culturing for 48h and then tested GFP + The results again suggest that coated anti-CD3+Soluble anti-CD28+IL-2 is the optimal stimulatory activation system and that mCD19CAR-T cells were prepared. Thus, the best C57BL/6J CD3 was obtained by screening using different stimulus activation systems in the presence of IL-2 + T cell culture conditions optimize the optimal time for retroviral infection of mouse primary T cells, successfully prepares mCD19CAR-T cells with C57BL/6J background, and can be used for subsequent further research.
In addition: the implementation isExamples this experiment selected CD3 antibodies and CD28 antibodies for CD3 + Stimulated activation of T cells, TCR-CD3 provides an initial activation signal, CD28 provides a secondary signal that stimulates activation, increasing cytokine secretion and thus promoting T cell activation. Studies have reported that soluble antibodies generally do not provide sufficient receptor cross-linking to activate cells and that immobilized anti-CD3 and CD28 antibodies can effectively expand T cells. The stimulation of T cells with anti-CD3 antibodies alone, i.e., receiving only the first signal of activation, will activate T cells but will not produce cytokines such as IL-2, rendering the cells unusable. Therefore, three different stimulation forms of the coated anti-CD3/CD28, the coated anti-CD3+Soluble anti-CD28 and the coated anti-CD3 are designed, and after different stimulation activation and culture time, T cell activation and proliferation effects are detected. The results show that the application of the Soluble CD28 has better effect on maintaining the proliferation state of the T cells of the B6 mouse, and the optimized cell clone number can be obtained after 24 hours of stimulation of the coated anti-CD3+Soluble anti-CD28. Taken together, it was suggested that both the first and second signals were essential for T cell activation in vitro, but that the administration pattern of CD28 signals was different when the optimal activation and proliferation state of T cells was obtained due to the different type and sensitivity of B6 and BALB/c mouse T cell immune responses.
Studies have reported that T cells undergo post-activation failure death following in vitro stimulated activation. The experiment also shows that although B6 mouse T cells are fully activated and cloned under the condition of the coated anti-CD3+Soluble anti-CD28, the cloned cell mass in the subsequent culture can not be well maintained, and finally the CAR-T cell infection efficiency is low. Low doses of IL-2 are reported in the literature to be effective in preventing T cell death after activation in vitro, maintaining the proliferation state of T cells. Thus, the present invention was validated by adding IL-2 to the stimulated activation system. The results show that T cells of the 24h group are stimulated by activating the coated anti-CD3+Soluble anti-CD28 and adding IL-2, and the best state for preparing CAR-T cells subsequently exists. Culturing T cells for 48h under this condition produced CAR-T cells with significantly improved infection efficiency, confirming the important role of IL-2 in maintaining T cell activation.
Claims (5)
- A method for constructing CD19 chimeric antigen receptor T cells of c57bl/6J mouse origin, characterized in that the method comprises activating T cells in an IL-2-containing environment using the following conditions, and then preparing mCD19CAR-T cells by retroviral infection; wherein, the conditions for stimulating activated T cells are: culturing by using an anti-CD3 antibody coated culture plate, inoculating cells, and directly adding an anti-CD28 antibody, wherein the anti-CD28 antibody is soluble;the final concentration of the IL-2 is 100U/mL;the method comprises the following specific steps:(1) CD3 of C57BL/6J after separation and purification + T cells are inoculated in a culture plate treated by the conditions of stimulating and activating the T cells, and the culture stimulation is carried out for 12-36 hours;(2) Transferring the cells subjected to the stimulated culture in the step (1) to a new culture plate, adding IL-2, and continuing to culture for 24-72 hours;(3) Transferring the cells after the activation culture in the step (2) to a new culture plate, adding mCD19 retrovirus and virus infection enhancing liquid, centrifuging, continuing to culture for 6-8 hours, replacing a fresh culture medium, adding IL-2, continuing to culture, and collecting the cells.
- 2. The method of claim 1, wherein the final concentration of anti-CD3 and anti-CD28 antibodies is 1 μg/mL-5 μg/mL.
- 3. The method for constructing CD19 chimeric antigen receptor T cells derived from C57BL/6J mice according to claim 1, wherein the culture plate treated in the step (1) is stimulated for 24 hours, and IL-2 is added in the step (2) for further culturing for 48 hours.
- 4. The method of constructing C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells according to claim 1, wherein the cells collected in the step (3) are subjected to flow assay, GFP + The CAR-T cell fraction reached 22.4%.
- 5. Use of C57BL/6J mouse-derived CD19 chimeric antigen receptor T cells constructed by the method of any one of claims 1 to 4 in the preparation of a medicament for the treatment of non-hodgkin's lymphoma.
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