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

Area of Science:

• Immunology
• Cancer Biology
• Gene Editing

Background:

• NR2F6 is a proposed immune checkpoint within effector T cells.
• Assessing NR2F6's therapeutic potential requires methods for its acute depletion in vivo.
• Understanding NR2F6's role is crucial for developing novel cancer immunotherapies.

Purpose of the Study:

• To establish a CRISPR/Cas9-mediated acute knockout protocol for NR2F6 in primary mouse T cells.
• To evaluate the therapeutic potential of NR2F6-ablated T cells in a preclinical cancer model.
• To investigate NR2F6's function as a T cell-inhibitory immune checkpoint.

Main Methods:

• Developed a CRISPR/Cas9 system using chemically synthesized sgRNA for electroporation into primary T cells from Cas9-transgenic mice.
• Generated Nr2f6 knockout T cells (Nr2f6^{CRISPR/Cas9 knockout}).
• Administered Nr2f6-ablated T cells via adoptive cell therapy (ACT) in MC38 tumor-bearing mice, combined with PD-L1 or CTLA-4 blockade.

Main Results:

• Nr2f6^{CRISPR/Cas9 knockout} T cells exhibited a hyper-reactive effector phenotype in vitro, similar to germline Nr2f6^-/- T cells.
• ACT with Nr2f6-ablated T cells significantly delayed MC38 tumor progression.
• Combination therapy (Nr2f6 ablation + PD-L1/CTLA-4 blockade) induced superior survival in tumor-bearing mice.

Conclusions:

• CRISPR/Cas9-mediated acute NR2F6 gene ablation in primary T cells is feasible and comparable to germline knockout.
• This approach validates NR2F6's role as a T cell-inhibitory immune checkpoint.
• NR2F6 inhibition is a promising sensitizing target to potentiate PD-L1 and CTLA-4 blockade therapies for enhanced tumor regression.