Team:SYSU-CHINA/Measurement

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Measurement

Measurement

This year, SYSU-CHINA developed a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of Human Herpesvirus 6. Since our project relies heavily on the regulatory function of the tet-ON promoter, a major part of our project is to characterize the properties of it. Its response to different concentration of doxycycline, a small molecule drug approved by FDA, and the kinetics of expression upon induction can be vital to further develop effective treatment for the adverse effects of CAR T therapies. In order to achieve such goal, we employed different methods to measure the promoter's dose-dependent response and its expression kinetics under optimal drug concentration, including fluorescence intensity measurement using microscopy, western blots and flow cytometry. We also characterized our circuits in two different chassis, HEK-293T cell lines and Jurkat leukemia cell lines. Some of our data was shown below.

Figure 1. Optimal concentration of Dox for treating cells
A: Fluorescence images of HEK293T cells transfected with pEF1a-GFP-T2A-U24 or transfected with ptetON-GFP-T2A-U24 and treated with different concentration of Dox, ranging from 0 ng/ml to 600 ng/ml.
B: Relative fluorescence intensity of the images in A(3 replicates, data not show) after analyzed by ImageJ.
C: Western blot result of the cells harvested in A.
D: Normalized protein level of the images in C after analyzed by ImageJ.



Figure 2. Expression time course of U24 in HEK293T cells
A: Western blot result of the HEK293T cells (transfected with of ptetON-GFP-T2A-U24 and treated with 100 ng/ml Dox) harvested at different time points: 0h, 2h, 4h, 8h, 12h, 24h.
B: Normalized protein level of the images in A after analyzed by ImageJ.



Figure3 . Protein half-Life of U24
A: Western blot result of the HEK293T cells (transfected with of ptetON-GFP-T2A-U24, treated with 100 ng/ml Dox and 200μg/ml CHX) harvested at different time points: 0h, 2h, 4h, 6h, 8h.
B: Normalized protein level of the images in A after analyzed by ImageJ.

Unlike measurement using bacteria, measurement experiments done with mammalian cells are more likely to have a broader variation and sometimes inconsistent results. Several factors may contribute to the variation, including varying transfection efficiency from sample to sample and intrinsic cell-to-cell variation. As shown in our flow cytometry result, only a small fraction of cells exhibited elevated GFP fluorescence intensity, indicating a low transfection efficiency. A large number of untransfected cells may therefore mask the actual results. Thus we recommend using single cell anaylsis for mammalian cells measurements, or establish a stable cell lines, preferably from a single colony.

Figure 4. Measurement of single cell fluorescence of HEK293T cells transfected with ptetON-GFP-T2A-U24 with or without doxycycline
This figure shows the distribution of fluorescence intensity of GFP, which reflect the expression level of U24. The red line is the sample001, stands for untransfected HEK293T cells(unstained), as control. The blue one is the sample010, stands for HEK293T cells transfected with pEF1a-CAR and ptetON-GFP-T2A-U24. The orange one is the sample011, stands for HEK293T cells transfected with pEF1a-CAR and ptetON-GFP-T2A-U24, treated with Dox. 10,000 cells are analyzed.

In addition, we tested a new universal method to detect surface chimeric antigen receptors (CARs) expression. The method utilizes the binding property of protein L to light kappa chain in immunoglobulins, which also presents in the scFv domain of CARs. By staining cells with biotin-protein L and R-phycoerythrin(PE)-conjugated streptavidin, we can detect surface CAR expression.

In summary, our work provided data from first-hand experience for one of the most widely-used regulatory promoters in mammalian cell lines, which can bring benefits to teams in the future who are eager to use this promoter. In addition, we performed measurement experiments with mammalian cells using different methods. The experience may provide insights for teams in the future to better conduct measurement on mammalian cells.