A Toolbox for Binary and Rheostat-like Modulation of TOX Expression via Genome and Epigenome Editing in Primary Human T cells

T cells, key players in the immune response, can become exhausted due to persistent antigen stimulation. This exhaustion limits their effectiveness, promoting the expression of inhibitory receptors and diminishing their cytotoxic capabilities. TOX, a gene linked to T cell exhaustion, is a master regulator that influences chromatin accessibility in murine models.

Reduced TOX levels improve tumor control in mice, and high TOX expression in human lymphocytes is associated with worse disease outcomes. However, the precise mechanisms and significance of TOX in human T cell exhaustion remain unclear. In this study, researchers developed a method to genetically knock out or phenocopy the knockout of TOX in primary human T cells outside the body.

Both approaches led to almost total elimination of TOX mRNA. Utilizing enhancer prediction data, they identified specific human cis-regulatory regions that, when targeted with CRISPRoff, can silence TOX expression. Surprisingly, TOX deficiency did not affect survival or exhaustion markers in human CD8+ T cells in vitro. Furthermore, expression profiling showed that TOX knockouts only impacted TOX itself, with no downstream effects.

These findings demonstrate that completely eliminating or silencing TOX does not impact exhaustion marker expression or the transcriptome in primary human T cells stimulated by anti-CD3/anti-CD28. The researchers have created a powerful toolkit of genome and epigenome editing strategies to modify the expression of genes of interest in primary human T cells, aiming to gain insights into T cell function and develop improved T cell immunotherapies.