Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity

Dufva, Olli; Koski, Jan; Maliniemi, Pilvi; Ianevski, Aleksandr; Klievink, Jay; Leitner, Judith; Pölönen, Petri; Hohtari, Helena; Saeed, Khalid; Hannunen, Tiina; Ellonen, Pekka; Schmitz, Peter; Kankainen, Matti; Aittokallio, Tero; Keränen, Mikko; Korhonen, Matti; Mustjoki, Satu

doi:10.1182/blood.2019002121

Cited by 168 publications

(148 citation statements)

References 64 publications

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“…The Jurkat leukemic T-cell line is a widely used model system for the study of TCR function, 21 and we previously developed a triple parameter TCR signalling reporter cell line (TPR) based on the Jurkat line E6.1. 22 These reporter cells have been proven to be highly suitable to evaluate costimulatory pathways and the function of chimeric antigen receptors, [23][24][25] but to date, their potential to evaluate transgenically expressed TCRs in a high-throughput manner that still reflects physiological T-cell biology as seen in primary human T cells had not been tested. To facilitate highly sensitive and unbiased TCR functional characterisation, we introduced two additional modifications in the TPR cell line.…”

Section: Introductionmentioning

confidence: 99%

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

et al. 2020

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Objective Transgenic re‐expression enables unbiased investigation of T‐cell receptor (TCR)‐intrinsic characteristics detached from its original cellular context. Recent advancements in TCR repertoire sequencing and development of protocols for direct cloning of full TCRαβ constructs now facilitate large‐scale transgenic TCR re‐expression. Together, this offers unprecedented opportunities for the screening of TCRs for basic research as well as clinical use. However, the functional characterisation of re‐expressed TCRs is still a complicated and laborious matter. Here, we propose a Jurkat‐based triple parameter TCR signalling reporter endogenous TCR knockout cellular platform (TPRKO) that offers an unbiased, easy read‐out of TCR functionality and facilitates high‐throughput screening approaches. Methods As a proof‐of‐concept, we transgenically re‐expressed 59 human cytomegalovirus‐specific TCRs and systematically investigated and compared TCR function in TPRKO cells versus primary human T cells. Results We demonstrate that the TPRKO cell line facilitates antigen‐HLA specificity screening via sensitive peptide‐MHC‐multimer staining, which was highly comparable to primary T cells. Also, TCR functional avidity in TPRKO cells was strongly correlating to primary T cells, especially in the absence of CD8αβ co‐receptor. Conclusion Overall, our data show that the TPRKO cell lines can serve as a surrogate of primary human T cells for standardised and high‐throughput investigation of TCR biology.

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Section: Introductionmentioning

confidence: 99%

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

et al. 2020

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“…In order to elucidate the possible combinatorial role of anti-cancer or other drugs in preventing resistance to CAR T-cell immunotherapy, i.e., immunomodulatory impacts of drugs on cytotoxic properties of effector T-cells, and to explore the mechanism of CAR T-cell cytotoxicity, a detailed in vitro loss-of-function screening by means of CRISPR has conducted recently. The results revealed that cancer genetics affects the output of CAR T-cell therapy and also introduced the key role of signaling by death receptors in cytotoxic mechanism of CAR T-cells [ 150 ].…”

Section: The Deluxe Zone Of Cancer Therapy Where Crispr Meets Immunomentioning

confidence: 99%

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

et al. 2020

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Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called “adoptive cell transfer”, or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.

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“…For example, even in the absence of tonic signaling, how continuous presence of an auto or alloantigen would affect a CAR-or TCRengineered Tregs will be an important consideration. The metabolic phenotype of exhausted Tregs suggests that, as for CD8 + T cells and CD4 + T convs, strategies to modulate signaling pathway activity for example by use of kinase inhibitors (Dufva et al, 2020;Mestermann et al, 2019;Weber et al, 2019;Weber et al, 2020b), or modulation of transcription factor expression Lynn et al, 2019), could be strategies to limit this risk.…”

Section: Ts-car Tregs Are Dysfunctional In Vivo But Not In Vitromentioning

confidence: 99%

Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion

Lamarche

Novakovsky

et al. 2020

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Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how two methods which induce conventional T cell exhaustion -repetitive stimulation or expression of a tonic-signaling chimeric antigen receptor (CAR) -affect human Tregs. With each repetitive polyclonal stimulation Tregs progressively acquired an exhausted phenotype, and became less suppressive in vitro. Tregs expressing a tonic-signaling CAR rapidly acquired an exhausted phenotype and had major changes in their transcriptome and metabolism. Although tonicsignaling CAR-Tregs remained stable and suppressive in vitro, they lost in vivo function, as tested in a model of xenogeneic graft-versus-host disease. The finding that human Tregs are susceptible to exhaustion has important implications for the design of Treg adoptive immunotherapy strategies.

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Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity

Cited by 168 publications

References 64 publications

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

Repeated stimulation or tonic-signaling chimeric antigen receptors drive regulatory T cell exhaustion

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