To explore inhibition of NR2F6, a signaling regulator downstream of the TCR, Klepsch et al. first demonstrated that germline NR2F6 KO mice had improved tumor rejection and enhanced survival (60% KO vs. 25% WT) in response to αCTLA-4 in the B16-OVA model. To generate NR2F6-ablated T cells, Klepsch et al. transfected autologous CD3+ T cells from Cas9 transgenic mice with sgRNAs targeting NR2F6. In an MC38 tumor model, adoptive transfer of NR2F6-ablated T cells synergized with either αCTLA4 or αPD-L1 treatments to slow tumor growth and promote long term survival (67% vs. 33% and 38% vs. 0%, respectively, compared to controls).
Contributed by Samuel Goldman
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. Video abstract.
Author Info: (1) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. Victoria.Klepsch@i-med.ac.at. (2) Division of Translat
Author Info: (1) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. Victoria.Klepsch@i-med.ac.at. (2) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. (3) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. (4) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. Present address: Department of Internal Medicine II, Medical University of Innsbruck, Anichstrasse 35, A-6020, Innsbruck, Austria. (5) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. (6) Division of Translational Cell Genetics, Medical University of Innsbruck, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria. Gottfried.Baier@i-med.ac.at.
