Determination of the Effects of Nrf2 upon the Early Events of Jurkat T Cell Activation by Use of CRISPR‐CAS9 Mediated Mutation

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a transcription factor activated by cell stresses, such as reactive oxygen species and electrophilic stimuli. Upon activation Nrf2 accumulates within the nucleus, where it induces transcription of its target genes. In mice a growing number of studies indicate a role for Nrf2 in the development of immune mediated diseases, including inflammatory diseases such as allergy and autoimmunity. Recently, we have shown that the Nrf2 activator, tBHQ, modulates early events of T cell activation in human Jurkat T cells. This is evidenced by a marked inhibition of the cytokine IL‐2, as well as a reduction in the expression of CD25, the high‐affinity subunit of the IL‐2 receptor. The suppression of IL‐2 by tBHQ correlates with a concurrent suppression of the transcription factor NFκB, a transcription factor known to have multiple binding sites within the IL‐2 promoter. Although we have shown that the synthetic food additive tBHQ modulates the early events of Jurkat T cell activation, the role of Nrf2 in these effects remains unclear. Accordingly, we implemented CRISPR‐CAS9 technology to determine the Nrf2‐specific effects upon the early events of human Jurkat T cell activation. Toward this end, we have successfully targeted exon one of Nrf2 and have identified several Nrf2‐mutatnt clones. Sanger sequencing of each mutant indicates that at least two of the clones have base pair insertions, resulting in frameshift mutations. (This work is funded by NIH grants ES018885 and GM092715).