Preprint Club
A cross-institutional Journal Club Initiative

Key words

• Chimeric antigen receptor (CAR) T cell

• Immunotherapy

• Tumor immunology

Main Findings

In this preprint, Yi et al. investigate the relationship between CAR-T cell persistence and the FAS/FAS-ligand (FAS-L) signaling pathway. The authors first constructed a single-cell atlas of immune and non-immune cells from healthy donors and patients with diverse cancer types, and highlight that while FAS is expressed by various immune and stromal cells, FASLG (the gene encoding FAS-L) is highly restricted to T and NK cells. In addition, using RNA in situ hybridization, the authors observed that CAR-T cells had higher levels of FASLG expression compared to endogenous T cells.

To evaluate the role of FAS-signaling in persistence of CAR-T cells, in vitro and in vivo competitive fitness assays were performed to compare human CAR-T cells with disabled FAS-signaling (ΔFAS) and those with signaling intact. In vitro, CAR-T cells expressing ΔFAS showed a progressive enrichment when compared to CAR-T cells with intact FAS-signaling following repeated antigen stimulation. In vivo findings mirrored this result, where CAR-T cells expressing ΔFAS were enriched across multiple tissues, and enabled improved tumor control following adoptive transfer to models of two B cell tumors, B-cell acute lymphoblastic leukemia (B-ALL), and B-cell non-Hodgkin’s lymphoma (B-NHL). These results were paralleled in experiments using CAR-Natural Killer (CAR-NK) cells, where competitive assays again demonstrated that disruption of FAS-signaling significantly enhances persistence and anti-tumor potency of CAR-NK cells in a mouse model of B-NHL. In contrast, knockout of FASLG reduced the persistence of CAR-lymphocytes, while still facilitating CAR-mediated tumor killing. From this, the authors conclude that disruption of FAS-signaling provides advantages CAR-T and CAR-NK cells with respect to persistence and antitumor efficacy, without impacting the capacity for CAR-directed tumor lysis.

Limitations & Next Steps

• While the use of competitive fitness assays in mouse models of B-cell malignancies demonstrates an advantage of CAR-modified lymphocytes, the definition of persistence should be clarified. Is detection of CAR-expressing cells until tumor clearance is achieved sufficient, or is lifelong persistence of CAR-modified lymphocytes the objective?

• Given that the FAS-signaling pathway is dispensable for CAR-mediated tumor killing it would be of interest to evaluate the other receptors in the Tumor Necrosis Factor (TNF) family of death receptors. Other receptors (i.e. TRAIL) may play various compensatory roles in these processes, and should be investigated.

• What are the effects of a combined model of CAR-T and CAR-NK cell therapy in combination? Would this provide enhanced tumor control, and/or impact the persistence of the two populations?

• The paper uses two B cell malignancies as disease models. Is it anticipated that the disruption of FAS signaling will enhance the efficacy of CAR-therapies in solid and CNS tumors where there have been more challenges employing CAR-lymphocyte-based therapies?

• Further exploration of the effects of disrupted FAS signaling on other immune cells is of interest. Does this impact the interaction of CAR-T cells and macrophages, dendritic cells or other immune mediators involved in tumor lysis?

Significance/Novelty

What is the novelty of the preprint for the field specific?

• Yi et al. highlight the ability of the FAS-signaling pathway to be manipulated to enhance CAR-T and CAR-NK persistence. This offers new hope for cancers where CAR-lymphocytes have been short-lived or demonstrated limited clinical efficacy. Furthermore, as the effects of FAS and FAS-L in regulating in NK cells and CAR-NK cells has not been previously explored, this is particularly important for future studies of NK-based immunotherapies.

How does the result of the preprint matter for general immunologists and/or patients?

• This article has several implications for general immunologists and the public. Yi et al. offer a new strategy for improving genetically engineered CAR-lymphocytes for use in patients. As the efficacy of these therapies are closely related to their persistence in vivo, exploring the relationship between FAS-signaling and persistence identifies a new strategy to improve these therapies. In addition, this article demonstrates that modifications which enhance persistence do not come at the cost of tumor-killing efficacy. Ultimately, this is an exciting and novel opportunity to enhance CAR-therapies for patients and expand the applicability of these treatments.

Credit

Reviewed by Robyn Loves (University of Toronto, Department of Immunology) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interest in relation to their involvement in the review.