Team:Freiburg/Results
Main Project
Characterization of the CTLA4 promoter
The CTLA4 promoter (pCTLA4), which is VEGF-A responsive, was characterized using Jurkat and HEK293T cells. Stable cell lines were generated containing pCTLA4 with quadruple NFAT binding sites (NFATbs) and the TATA like minimal promoter pTal (Mahindhoratep et al., 2014) expressing eCFP as reporter gene. Constitutively expressed mCherry was used as transduction marker.
The VEGF receptor 2 (VEGFR-2) was added by PEI transfection for HEK293T cells, which do not express this gene (Liu et al. 2014).
In order to characterize pCTLA4, transfected cells were induced with different concentrations of VEGF-A for 24 h.
To generate a high expression by activating the signaling cascade downstream of the receptor, the stable cell lines were induced with ionomycin causing influx of Ca2+ into the cells (Bittinger et al. 2004). Fluorescence was measured by flow cytometry after 24 h of treatment (Fig. 1).
Figure 1: Flow cytometry of hypoxia response element promoter tests.
Jurkat cells stably transduced with 4xNFATbs-pTal:eCFP were incubated 24 h with indicated concentrations of a) VEGF-A and b) VEGF combinated with ionomycin (5 µM). c) HEK293T cells stably transduced with 4xNFATbs-pTal:eCFP containing transient VEGFR-2 were incubated 24 h with indicated concentrations of VEGF. Data points are mean values of triplicates, error bars represent standard deviation. Significant differences were determined using one-tailed student’s t-test (Excel 2017) followed by Bonferroni-Hoch correction; * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant differences are not marked.
In Jurkat cells, 4xNFATbs-pTal:eCFP expression decreases with increasing VEGF-A concentration (Fig. 1a). Addition of ionomycin (5 µM) inverts the response on increasing VEGF-A concentration to the expected trend (Fig. 1b).
Generally low expression in Jurkat cells may arise due to transcriptional inactivity in these cells. As the promoter was expected to produce lower expression without induction, pCTLA4 seems to be leaky in HEK293T. The influence of ionomycin on the expression of 4xNFATbs-pTal:eCFP combined with VEGF-A can not be evaluated as the results do not allow conclusions without further experiments. Results indicate that VEGF-A is not an optimal input for our AND gate. Further experiments have to be performed as the CD4+ Jurkat cell line is not identical to primary T cells.
In HEK293T cells, 4xNFATbs-pTal:eCFP expression is already on a high level of 70 % positive cells without any treatment (Fig. 1c). Addition of VEGF-A (50 ng/ml) has no significant effect on the amount of eCFP positive cells. HEK293T cells with transiently induced VEGFR-2 show an average 7 % higher expression in general but no response on VEGF-A (50 ng/ml) as well.
High basal expression can originate from high transcriptional activity in HEK293T (Thomas et al., 2005). Using another minimal promoter could improve the results. Optimization of promoter expression should be done in Jurkat cells as this cell line is closer to the intended application in primary T cells.
