Real-time monitor system with function of evaluating specific CTL immunostimulatory effect of cytokines on EBV-LMP2

A technology of EBV-LMP2 and cytokines, applied in the direction of cell culture active agents, animal cells, vertebrate cells, etc., can solve the problems that the immune enhancement effect needs to be further clarified, the optimal concentration has not been systematically established, etc., and it is easy to be widely used , simple operation and low technical requirements

Inactive Publication Date: 2020-09-11
BEIJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the optimal concentrations of IL-2 and IL-21 have not been systematically established, and the immunoenhancing effects of IL-2 and IL-21 on EBV-LMP2-specific CD8+ CTL-mediated cytotoxicity still need to be further elucidated

Method used

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  • Real-time monitor system with function of evaluating specific CTL immunostimulatory effect of cytokines on EBV-LMP2
  • Real-time monitor system with function of evaluating specific CTL immunostimulatory effect of cytokines on EBV-LMP2
  • Real-time monitor system with function of evaluating specific CTL immunostimulatory effect of cytokines on EBV-LMP2

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0090] Experimental example 1. rAd-LMP2 vaccine immunized mice to induce LMP2-specific CD8+ CTL

[0091] Female C57BL / 6 mice aged 4-6 weeks were selected and randomly divided into two groups: PBS group and rAd-LMP2 group, 5 mice in each group. Dilute the rAd-LMP2 vaccine to 2x109VP / mL and inject 100uL each intramuscularly. PBS intramuscular injection of 100uL each. They were immunized three times at 0, 2, and 4 weeks, and at the 5th week, the spleen was taken to separate lymphocytes. ELISpot results such as figure 1 It shows that the number of SFC / 106spleen lymphocytes (spleen lymphocytes) induced by rAd-LMP2 immunization in C57BL / 6 mice is 478, and SFC / 106spleenlymphocytes (spleen lymphocytes) are EBV-LMP2-specific CD8+ T cells. The number of cells secreting IFN-γ was detected to measure the level of vaccine-induced LMP2-specific CTL responses.

experiment example 2

[0092] Experimental example 2, RTCA detection of the optimal seeding density of C3-LMP2-GFP cells

[0093] Collect C3-LMP2-GFP cells at 2.5×10 per well 3 ,5×10 3 ,1×10 4 ,2×10 4 Cells were inoculated onto E-Plate 96 plates, and the proliferation curve of C3-LMP2-GFP was detected by RTCA within 24 to 96 hours, and the optimal inoculation density was selected. According to the proliferation curve and CI value (Cell Index) of C3-LMP2-GFP cells within 96 hours, select 5×10 3 / well is the optimal inoculation density, the result is as follows figure 2 shown.

experiment example 3

[0094] Experimental example 3. Real-time monitoring of the killing efficiency of LMP2-specific CD8+ CTL on C3-LMP2-GFP target cells by RTCA

[0095] According to the optimal seeding density of C3-LMP-GFP, C3-LMP2-GFP cells were divided into 5 × 10 3 The cell density per well was inoculated to E-Plate 96 plates, and then the E-Plate 96 plates were placed in the iCELLIgence system and proliferated for 24 hours. At the same time, the LMP2-specific CD8+ T cells in the SPL of the immunized mice were stimulated and activated with EBV-LMP2 polypeptide for 24 hours, and the unstimulated and activated SPL was used as a control. Then inoculate the activated SPL into the corresponding wells of the E-Plate 96 plate according to the effect-to-target ratio of 10:1, 25:1, 50:1, and 100:1. The plate was then inserted into the iCELLigence system and the adherence rate of the target cells continued to be monitored for up to 72 hours; the data was then exported for analysis. The result is as ...

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Abstract

The invention provides a real-time monitor system with a function of evaluating the specific CTL immunostimulatory effect of cytokines on EBV-LMP2 and belongs to the field of cell culture in vitro. The real-time monitor system can be used for continuously and dynamically monitoring the immunological enhancement effect of IL-2 and IL-21 on T cell mediated cytotoxicity in real-time. The method detection results indicate that the highest killing efficiency enhancing effect on the EBV-LMP2 specific CD8+T cell is achieved when the IL-2 concentration is 60U / mL and the IL-21 concentration is 10ng / mL;and a remarkable immunological enhancing effect can be obtained by individually adding IL-21 with the final concentration of 10ng / mL. Results indicate that RTCA can be used for monitoring the immunological enhancing effect of the IL-2 and IL-21 on T cell functions in real time.

Description

technical field [0001] The present invention relates to the field of in vitro culture of interleukin (Interleukin, IL) and T cells, in particular to the real-time monitoring of IL-2 and IL-21 on EBV-LMP2-specific CD8+ cells using real-time cell analysis technology. Immunoenhancing effect of T cell killing efficiency. The invention also relates to the establishment and application of the detection method. Background technique [0002] The occurrence of NPC is closely related to EBV infection. EBV only expresses sub-immune dominant EBNA1, LMP1 and LMP2 in NPC cells, among which LMP2 is non-carcinogenic and expressed in all NPC cells, which can induce effective cellular immune responses, which is recognized It is an ideal target for the development of NPC immunotherapy strategies. At present, many reports at home and abroad have shown that the adoptive transfer of EBV-LMP2-specific T cells to treat NPC patients can significantly enhance the LMP2-specific CD8+CTL response in p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12Q1/02
CPCC12N5/0638C12N2501/2302C12N2501/2307C12N2501/2321C12N2503/00G01N33/5011G01N33/505
Inventor 曾毅葛玉洋周志祥滕智平周玉柏
Owner BEIJING UNIV OF TECH
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