T cell-intrinsic PKD3 fine-tunes differentiation into CD8+ central memory T cells and CD8 single positive thymocyte development.

Members of the Protein kinases D (PKD) family are described as regulators of T cell responses. From the two T cell-expressed isoforms PKD2 and PKD3, so far mainly the former was thoroughly investigated and is well understood. Recently, we have investigated also PKD3 using conventional as well as conditional T cell-specific knockout models. These studies suggested PKD3 to be a T cell-extrinsic regulator of the cells' fate. However, these former model systems did not take into account possible redundancies with the highly homologous PKD2. To overcome this issue and thus properly unravel PKD3's T cell-intrinsic functions, here we additionally used a mouse model overexpressing a constitutively active isoform of PKD3 specifically in the T cell compartment. These transgenic mice showed a slightly higher proportion of central memory T cells in secondary lymphoid organs and blood. This effect could not be explained via differences upon polyclonal stimulation in vitro, however, may be connected to the observed developmental aberrances in the CD8 single positive compartment during thymic development. Lastly, the observed alterations in the CD8+ T cell compartment did not impact proper immune response upon immunization with ovalbumin or in a subcutaneous tumour model suggesting only a small to absent biological relevance. Taking together the knowledge of all our published studies on PKD3 in the T cell compartment, we now conclude that T cell-intrinsic PKD3 is a fine-tuner of central memory T cell as well as CD8 single positive thymocyte development.

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

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 Nr2f6CRISPR/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 Nr2f6CRISPR/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.