The key objective of this proposal is to identify new key targets that prevent or revert, in vivo T cell dysfunction in cancer.
We had a very successful start of this ERC project: we have already been able to establish a successful foundation in a publication in Cancer Cell, 2024 [1]. In this paper we describe how we can use multimodal stimulation screens - the key goal of this application - to uncover unique and shared genes limiting T cell fitness and dysfunction. We uncovered and compared genes either exclusively, or commonly, contributing to T cell fitness under different modes of TCR stimulation, namely chronic stimulation [1]. Specifically, we performed three genome-wide CRISPR-Cas9 knockout functional screens in primary mouse CD8+ T cells upon different stimulations: intense, acute, and chronic, covering key aspects of effector biology, namely: survival, proliferation, and persistence.
Importantly, even though these screens were performed in an in vitro setting, we were able to uncover, validate and characterize several regulators not previously reported that also improve T cell cytotoxic capacity and persistence in vivo. For example, one of the identified hits was DAP5, a key regulator of mRNA translation. We demonstrated that DAP5 inactivation promoted the expression of cell cycle-regulating genes, such as Ccnb1, Mki67, Ccne2, and Cenpe, whereas activation-induced immunosuppressive genes, such as Nr4a1, Pdcd1, Fas, and Tnfsf4 were suppressed. These observations may explain the phenotype induced by Dap5 inactivation: an activated cell cycle program at baseline which allows cells to achieve their effector status, resulting in attenuated activation and protection from dysfunction. We conclude therefore, that DAP5 serves as a nodal factor in T cell dysfunction and fitness, one of our key aims: it connects different aspects of T cell dysfunction, namely stress caused by acute and intense stimulation and by chronic stimulation. It may therefore serve as a valuable therapeutic target for different T cell therapies, including CAR-T, TCR and TIL, as it may reduce their dysfunction in patients upon treatment, a possibility we are interesting to explore.
As highlighted above, critical advances have been made by us but also others in identifying key molecules that limit T cell exhaustion in vivo [1-3]. However, to our knowledge no one has interrogated the reversibility (rather than prevention) of a truly exhausted state through CRISPR screens, which constitutes a key objective of this proposal, mainly due to the lack of appropriate tools. Within the first period of this grant, we have also developed such tools, aiming to ask this more challenging question. Specifically, we developed a Cre-mediated inducible sgRNA retroviral vector, which allows the timely induction of a genetic perturbation, which will be described below in detail. This approach will allow us to understand how an exhausted T cell phenotypically changes upon a specific gene perturbation. We also developed a second screening system allowing for temporal control of Cre, also described in detail below.
In full accordance with the proposal, we are now planning on performing an in vivo CRISPR screen using these newly developed tools to address the reversibility of an exhausted state, by inducing a genetic perturbation specifically when T cells present such state. To finalize the setup of this screen, we are now characterizing the T cell state of adoptively transferred OT-1 T cells throughout disease progression of our mouse syngeneic melanoma model (B16-OVA) to identify the best timepoint to induce sgRNA expression.
