Main Project

Transfection and Confirmation

Knockdown

Three finished knockdown plasmids were used to generate lentivirus for transduction of Jurkat and HEK293T cells. On transduced cells fluorescence-activated cell sorting (FACS) was performed to isolate CFP-positive cells (Figure 1). Western blotting followed by anti-HIF1A-antibody detection (Figure 2) shows the decrease of protein level confirming knockdown of HIF1A. These results proved replicable over multiple independent experiments. [here knockdown efficiency quantification will be inserted]

Knockout

Following cloning each Cas9 plasmid with one of the gRNAs was transiently transfected into Jurkat cells by electroporation. Transfected cells were sorted for GFP expression by FACS either directly into single cells to found clonal cultures or manual dilution series of sorted cells was performed to this end. Alternatively, the transfected cells were selected by puromycin and thereafter diluted to find single cells.

Unfortunately, these cultures often did not grow or grew more slowly than standard doubling time as mammalian cells in culture are dependent on growth factors secreted by their peers in order to survive and proliferate. Therefore, we unfortunately were unable to grow clonal knockout cultures to sizes sufficient for analysis. For knockout mutations were planned to be identified by T7E1 assay on genomic DNA (Figure 3).

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.

Characterization of the CRE Promoter

The cAMP response element-containing promoter (pCRE), which is pH responsive, was characterized in vitro in Jurkat and HEK293T cell lines. For this purpose, the pH of the test media had to be adjusted.

Promoter characterization

In order to characterize the CRE promoter, stably transduced HEK293T and Jurkat lines were created expressing eCFP under a minimal promoter with multiple CRE sites. Induction was performed with pH adjusted media. Constitutively expressed mCherry was used as transduction marker. For tests in HEK293T, PEI transfection of the pH receptor TDAG8, which is not expressed in these cells, was performed. (Ausländer et al., 2014). To generate a high expression by activating the signaling cascade downstream of the receptor, the stable cell lines were induced with forskolin and IBMX. Forskolin activates the cAMP-producing enzyme adenylyl cyclase and IBMX inhibits cAMP-hydrolyzing phosphodiesterases (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. a) Jurkat cells stably transduced with 4xCRE-pTal:eCFP were incubated 24 h in pH adjusted RPMI 1640. b) The experiment was repeated inducing with Forskolin (10 µM) and IBMX (10 µM). c) and d) HEK293T cells stably transduced with 4xCRE-pTal:eCFP with and without transient TDAG8 were induced similarly to a) and b). Results show the represent the amount of eCFP positive cells c) and the d) mean fluorescence intensity. All data points are mean values of triplicates, error bars represent standard deviation. Significant differences in a) and b) were determined using ANOVA, for c) and d) significant differences were determined using one-tailed student’s t-test (Excel 2017); * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant differences are not marked.

Jurkat 4xCRE-pTal:eCFP cells show a 1.5 fold increase of fluorescence at pH 6.5 compared to 7.7 even though the amount of eCFP positive cells remains low (Fig. 1a). The same trend was obtained via additional stimulation with forskolin and IBMX for which the percentage of eCFP positive cells is about 15 fold higher (Fig. 1b). It was shown that Jurkat 4xCRE-pTal:eCFP cells are pH responsive. As overall expression via CRE remains on a low level, further optimization of the construct has to be performed. High expression via induction with forskolin and IBMX indicates that higher expression rates are obtainable. This could be achieved by the use of another minimal promoter or different amount of enhancer elements. Furthermore, the density of TDAG8 on the membrane could be adapted in order to optimize the signaling of the cells.
In HEK293T containing 4xCRE-pTal:eCFP the amount of eCFP positive cells was pH and TDAG8 independent at about 90% (Fig. 1c). Against the expectations, HEK293T cells without TDAG8 induced with forskolin and IBMX show a higher amount of eCFP positive cells than cells with transiently introduced TDAG8. As the differences between the conditions could not be pointed out with percent positive cells, the mean fluorescence intensity (MFI) was evaluated (Fig. 1d). Fluorescence intensity of cells without TDAG8 increases towards lower pH showing a 1.3 fold increase from pH 7.7 to 6.5. As HEK293T are not supposed to have TDAG8 receptors, other pathways must be used to activate the CRE promoter. HEK293T containing transiently introduced TDAG8 show an at average 20% lower fluorescence intensity against the expectations. HEK293T induced additionally with forskolin and IBMX without TDAG8 show a 40% higher fluorescence intensity than the cells with TDAG8 leading to the conclusion that transiently transfected cells show lower expression caused by the stress of the PEI transfection. Overall, 4xCRE-pTal:CFP in HEK293T cells is highly leaky which would have to be optimized in further steps. Another cell line may be better suited for initial promoter tests. As Jurkat cells are closer to the intended application, 4xCRE-pTal:CFP is to be optimized in this cell line.

pH Adjustment

To simulate the tumor microenvironment, it was crucial to adjust the pH of the medium. RPMI 1640 medium is strongly buffered by bicarbonate buffer (buffer range: pH = 5.1 – 7.1, Fig. 2) and HEPES (buffer range: pH = 6.8 - 8.2) (Dawson et al., 1986). Furthermore, the pH in the medium is influenced by the 5% CO₂ atmosphere in the incubator and production of lactic acid by cells (Damaghi et al., 2013).

Figure 2: Bicarbonate buffer. At lower pH, the equilibrium is shifted to CO₂ and H₂O causing evaporation of CO₂.

To overcome these obstacles, different acids were evaluated. As tumor cells secrete lactic acid it was tested to adjust the pH of the medium (Fig. 3). Bicarbonate buffering leads to instability of original or adjusted pH in RPMI 1640, making use of a titration curve inexpedient. Additionally, lactic acid is a weak acid with a pKa of 3.86 (Narendranath et al., 2001 [2]) and acts as a buffer itself. That makes setting up the pH even more difficult in this highly dynamic buffer system. As an alternative, hydrochloric acid, which completely dissociates with a pKa of -7 (Riedel et al., 2004), was tested. For that reason, the setup of the pH is more feasible with hydrochloric acid than with lactic acid.

Figure 3: Titration of lactic acid against RPMI 1640. Different concentrations of lactic acid in 2 ml RPMI 1640 have been measured in triplicates.

The influence of 5% CO₂ atmosphere on the pH of the medium was analyzed inside the incubator (Fig.4). The findings suggest that the carbonate buffer is equilibrated with the 5% CO₂ atmosphere after around 60 minutes showing a pH at about 0.3 below the pH outside the incubator. The strong increase at the beginning arises from evaporation of CO₂ before entering the incubator as the medium has a higher surface to volume ratio in a well compared to the bottle. The same effect is also responsible for the inconsistent original pH of stock RPMI 1640. The slow decrease of the pH after equilibration of the buffer results from the secretion of metabolites by Jurkat cells (Masters et al., 2007).
Based on these results, the pH of the induction media was set 0.3 above the final pH value, so the pH equilibrates to the desired value inside the incubator.

Figure 4: pH trend of RPMI 1640 containing Jurkat cells in incubator. 7.5 ml RPMI 1640 containing 250.000 cells per ml were incubated for 24h on a 6 well plate, pH was measured every 30 s.

Hypoxia Response Element Promoter

Construct and Conditions

Gene expression specific to low oxygen concentrations (hypoxia) as given in the tumor microenvironment was tested with promoters containing hypoxia response elements (HREs) (Schödel et al., 2011). However, it is difficult and expensive to cultivate mammalian cells under hypoxic conditions. The transcription factor hypoxia inducible factor 1 alpha (HIF1A) which binds to HREs is regulated by proline hydroxylase domain proteins (PHDs). Like in the absence of oxygen, modification of HIF1A by ΡΗDs can also be inhibited by cobalt dichloride (CoCl₂), which was therefore used to mimic hypoxia (Epstein et al., 2001).

Jurkat and HEK293T cell lines with stable integration of the TATA-like minimal promoter (pTal; Mahindhoratep et al., 2014), modified to contain quadruple HRE, expressing eCFP were generated. Transduced cells were sorted by fluorescence-activated cell sorting (FACS) for constitutively expressed CMV:mCherry selection marker. Alternatively, single enhancer element promoters were introduced into CHO-K1 by PEI transfection. For analysis, cells were incubated with CoCl₂ for 24 h prior to measurement by flow cytometry. Concentrations used vary between the cell lines due to different onset of toxicity.

Data and Discussion

Flow cytometry of Jurkat 4xHRE-pTal:eCFP cells shows increasing eCFP fluorescence correlating with moderate rising treatment (Fig. 1a). Promoter activity in this most relevant test cell line is strongest at 80 μM CoCl₂, decrease of signal at greater levels of induction may stem from toxicity of CoCl₂. Overall low expression is likely due to weak transcriptional activity inherent in this cell line (Michel et al., 2017).

Figure 1: Flow cytometry of hypoxia response element promoter tests. a) Jurkat, b) HEK293T cells stably transduced with 4xHRE-pTal:eCFP, and c) 1xHRE-pTal:eCFP or d) 4xHRE-pTal:eCFP PEI transfected into CHO-K1 cells. For analysis cells were incubated 24 h with indicated concentrations of CoCl₂. 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 and decreasing differences are not marked.

In HEK293T, 4xHRE-pTal:eCFP expression is already at very high level when untreated and decreases with addition of CoCl₂ (Fig. 1b). Less fluorescence with increasing treatment may also represent toxicity. Since it should show low expression without induction, the minimal promoter is leaky in this cell line. High basal expression can originate from high transcriptional activity in HEK293T (Thomas et al., 2005), which is consistent with findings for the CRE promoter. That construct is also based on pTal, therefore usage an alternative minimal promoter may improve results. As these cells were a testing system, and Jurkat cells are closer to the intended application of the CARTEL^TM AND gate, promoter expression should be optimized in Jurkat cells.

As HEK293T proved to be an inadequate promoter activation test system due to leakiness, the constructs 1xHRE-pTal:eCFP and 4xHRE-pTal:eCFP were transiently measured in CHO-K1 cells by flow cytometry (Fig. 1c, d). Rising promoter activity with increased treatment was observed with both constructs, with roughly double amount of cells activated when possessing four HREs rather than single HRE. This indicates that the direction for further improvement of this expression construct, also in T cells, could be addition of even more enhancer elements. Subsequent to optimization of the HRE promoter in Jurkat, the target primary T cells should be tested.

Hypoxia pathway activation by CoCl₂ in accordance with literature (Moroz et al., 2009) was confirmed via Western Blot assay of HIF1A stabilization on samples analyzed in the flow cytometer (Fig. 2). Detection of HIF1A excludes the possibility of failure to induce hypoxia.

Fig. 2: WB HRE lines from FACS experiments

Team:Freiburg/Results