Periodic Reporting for period 1 - ReverT (Reversing T cell dysfunction in cancer by multimodal genetic screening)
Período documentado: 2022-10-01 hasta 2025-03-31
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.
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.
We have developed a system that relies on the Switch-ON promoter, developed previously by Chylinski et al. [4], which presents a loxP-STOP-loxP (LSL) cassette within the sgRNA scaffold, leading to its premature termination. In the presence of Cre recombinase, the LSL cassette is excised and efficient sgRNA expression occurs. We cloned this system into a retroviral backbone that includes a capture sequence after the sgRNA cassette for 10x Genomics sequencing. The capture sequence allows to combine a conventional CRISPR screen with single cell RNA sequencing, in a methodology denominated PERTURB-seq [5]. This approach enables us to examine the phenotypic changes that occur in an exhausted T cell following the perturbation of a specific gene.
In addition, to selectively identify and analyze the cells which have undergone successful recombination and are expressing a sgRNA, a Cre-induced fluorescent reporter (Ametrine) was introduced in the vector. Due to the dependence of the newly developed inducible vector on CRE-mediated recombination, we generated a new mouse model, the OT-1 Cas9 Cre-ERT2. In this model, T cells express the OT-1 TCR, which strongly recognizes the OVA peptide, Cas9 and the inducible version of Cre (Cre-ERT2). This inducible recombinase system will allow the temporal control of Cre activity with subsequent sgRNA expression through tamoxifen administration [6].
In addition, to selectively identify and analyze the cells which have undergone successful recombination and are expressing a sgRNA, a Cre-induced fluorescent reporter (Ametrine) was introduced in the vector. Due to the dependence of the newly developed inducible vector on CRE-mediated recombination, we generated a new mouse model, the OT-1 Cas9 Cre-ERT2. In this model, T cells express the OT-1 TCR, which strongly recognizes the OVA peptide, Cas9 and the inducible version of Cre (Cre-ERT2). This inducible recombinase system will allow the temporal control of Cre activity with subsequent sgRNA expression through tamoxifen administration [6].
With these screens, we have been able to compile a large list of T cell dysfunction genes, many of which we have validated in functional assays. This provides a highly valuable resource for several other projects in the lab, aiming to develop improved T cell therapies. For example, we have recently been able to isolate heterotypic T cell clusters from human melanoma metastases. Ex vivo, CD8+ T cells expanded from clusters exhibited a nine-fold increase in melanoma killing and enhanced cytokine production. Similarly, in adoptive cell transfer (ACT) experiments using patient-derived xenografts in mice, these T cells showed superior tumor killing, infiltration, and activation. These findings highlight that tumor-reactive CD8+ T cells are enriched in functional clusters, which can be isolated and expanded from clinical samples. These clusters, typically overlooked in standard single-cell sorting, offer valuable insights into tumor-immune interactions and hold therapeutic potential (Ibanez-Molero, Veldman et al, man. in revision for Nature). We are interested to explore taking advantage of this finding to integrate with our T cell dysfunction hits. Specifically, this offers a possibility to not only enrich for tumor-reactive T cells straight from clinical samples, but also to inactive T cell dysfunction genes, aiming to make highly active tumor-reactive T cells.
1. Lin, C.P. et al., Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness. Cancer Cell, 2024. 42(4): p. 623-645.e10.
2. Zhou, P., et al., Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer. Nature, 2023. 624(7990): p. 154-163.
3. Chung, H.K. et al., Multi-Omics Atlas-Assisted Discovery of Transcription Factors for Selective T Cell State Programming. bioRxiv, 2024: p. 2023.01.03.522354.
4. Chylinski, K., et al., CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nat Commun, 2019. 10(1): p. 5454.
5. Dixit, A., et al., Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell, 2016. 167(7): p. 1853-1866.e17.
6. Schwenk, F., et al., Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acids Research, 1998. 26(6): p. 1427-1432.
1. Lin, C.P. et al., Multimodal stimulation screens reveal unique and shared genes limiting T cell fitness. Cancer Cell, 2024. 42(4): p. 623-645.e10.
2. Zhou, P., et al., Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer. Nature, 2023. 624(7990): p. 154-163.
3. Chung, H.K. et al., Multi-Omics Atlas-Assisted Discovery of Transcription Factors for Selective T Cell State Programming. bioRxiv, 2024: p. 2023.01.03.522354.
4. Chylinski, K., et al., CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci. Nat Commun, 2019. 10(1): p. 5454.
5. Dixit, A., et al., Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell, 2016. 167(7): p. 1853-1866.e17.
6. Schwenk, F., et al., Temporally and spatially regulated somatic mutagenesis in mice. Nucleic Acids Research, 1998. 26(6): p. 1427-1432.