Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy

NR2F6 has been proposed as an alternative cancer immune checkpoint in the effector T cell compartment. However, a realistic assessment of the in vivo therapeutic potential of NR2F6 requires acute depletion. Employing primary T cells isolated from Cas9-transgenic mice for electroporation of chemically synthesized sgRNA, we established a CRISPR/Cas9-mediated acute knockout protocol of Nr2f6 in primary mouse T cells. Analyzing these Nr2f6 T cells, we reproducibly observed a hyper-reactive effector phenotype upon CD3/CD28 stimulation in vitro, highly reminiscent to Nr2f6 T cells. Importantly, CRISPR/Cas9-mediated Nr2f6 ablation prior to adoptive cell therapy (ACT) of autologous polyclonal T cells into wild-type tumor-bearing recipient mice in combination with PD-L1 or CTLA-4 tumor immune checkpoint blockade significantly delayed MC38 tumor progression and induced superior survival, thus further validating a T cell-inhibitory function of NR2F6 during tumor progression. These findings indicate that Nr2f6 T cells are comparable to germline Nr2f6 T cells, a result providing an independent confirmation of the immune checkpoint function of lymphatic NR2F6. Taken together, CRISPR/Cas9-mediated acute Nr2f6 gene ablation in primary mouse T cells prior to ACT appeared feasible for potentiating established PD-L1 and CTLA-4 blockade therapies, thereby pioneering NR2F6 inhibition as a sensitizing target for augmented tumor regression. Video abstract.