3.2 Future research
3.2.1 The influence on helper T cell differentiation caused by the mutations p.H962R and p.R1154C on DOCK8
We seek to study whether these two mutations would affect the potential of naïve T cells to differentiate into different helper T cell population. We plan to isolate the naïve T cells from the patients’ T cell blast via fluorescence-activated cell sorting (FACS), then respectively treat with different inducing cytokines, including IFNγ, IL-4 and IL-6/21, followed by assessing the expression of effector cytokines after the stimulation of αCD3/CD28.
3.2.2 Jurkat cell electroporation/transfection protocol modification
We plan to adapt the Jurkat cell electroporation in the following aspects. First, play around with the parameter settings. It has been demonstrated that the transfection efficiency of 15-kb plasmid into cancer cells could be significantly elevated by adjusting to a single high voltage pulse (1000V/cm, 100us) followed by a lower voltage (between 25~100V/cm) and long pulse duration (e.g. 10ms). We would first start from this setting as the adjustment. Second, we need to enhance the transportation condition as much as possible, including processing the assay when the outdoor temperature is proper and using insulated container for carry.
If the adjustment of electroporation fails to obtain stable and usable transfection efficiency and cell survival, another potential solution is switching to lentiviral transduction. We have purchased
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the lentiviral vector inserted with DOCK8 cDNA. Currently, we are in the process of generating the two missense point mutations on the lentiviral DOCK8 plasmid.
3.2.3 Molecular experiments in Jurkat cells
First, all the molecular assays processed in HEK293 cells should be repeated in Jurkat cells after 3.2.2 is done, including DOCK8-STAT3 nuclear translocation and the interaction between them tested by immunoprecipitation.
Second, DOCK8-transfected Jurkat cells enable us to study the pathways stimulated upon TCR signaling, including the cytoskeleton rearrangement. After it is doable to stably and effectively transfect Jurkat cells, phalloidin staining should be processed to observe whether/how the actin polarization triggered by TCR ligation is disrupted with the presence of mutant DOCK8 protein.
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