CCR5 edited CD4+ T cells maintain potency
Transfer of mRNA to T cells via electroporation can potentially affect cell viability and/or functionality. We addressed this point by evaluating viabilities 24 h post-treatment. CD4+ T cells electroporated with TALEN mRNA (both arms, or left arm only), GFP mRNA, or mock pulsed, showed a decrease in viabilities of ~30% when compared to untreated samples (Fig. 3A), implying that the electric pulse but not the RNA transfer had an impact on T cell viabilities. On the other hand, the temperature shift did not affect viability.
We next wanted to investigate if CCR5 edited cells retain their proliferative potential. To this end, the edited CD4+ T cells and control samples were cultivated under proliferation activating conditions. The total number of cells at days 7, 14 and 21 was comparable for all tested conditions (Fig. 3B-C). A time-series analysis [31] confirmed that the samples, independent of cultivation temperature or treatment, did not show significant differences in proliferation. Hence, we conclude that editing CCR5 did not affected the expansion capabilities of CD4+ T cells.
To explore if editing CCR5 has an impact on CD4+ T cell function in terms of cytokine release upon stimulation, we stimulated the cells with either phorbol 12-myristate 13-acetate (PMA), ionomycin or CD3/CD28/CD2 to trigger the release of TNF-α, IFN-γ or IL2, respectively. Supernatants of electroporated T cells were harvested 8–48 h after stimulation and analyzed by cytometric bead array. While we observed variations in the amounts of secreted cytokines among the four donors, the values did not significantly (two-sided t -test) differ when comparing the various treatment regimens (Fig. 3D-F). Unstimulated cells did not release measurable amounts of cytokines. In sum, these results demonstrate that editing CCR5 did not impair the ability of the engineered CD4+ T cells to respond to the different stimuli.