Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy
Victoria Klepsch, Maria Pommermayr, Dominik Humer, Natascha Brigo, Natascha Hermann‐Kleiter, Gottfried Baier
Abstract
Abstract Background 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. Methods 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. Results Analyzing these Nr2f6 CRISPR/Cas9 knockout 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. Conclusions These findings indicate that Nr2f6 CRISPR/Cas9 knockout 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. Graphical abstract