Need earlier readouts for your CAR tonic signaling?

John Nguyen, D.Phil

John Nguyen, D.Phil

R&D Scientist

Early Detection of Tonic Signaling in Primary Human CAR-T Cells by Flow Cytometry


Chimeric Antigen Receptors (CARs) are intended to trigger T cell activation in response to binding to specific antigens. However, certain CAR designs and excessive levels of CAR expression have the tendency to constitutively activate T cells in the absence of ligands, which has been described as tonic signaling. This chronic spontaneous stimulation has been shown to accelerate T cell differentiation and exhaustion, ultimately leading to dysfunctional CAR-T cells with poor persistence and potency. Therefore, it is highly recommended to screen new CAR constructs for tonic signaling during pre-clinical development.

Conventional methods to probe for tonic signaling include detection of CD3ζ phosphorylation in the absence of ligands (e.g., by Western Blot), as well as observation of abnormal T cell expansion kinetics and phenotypes which may manifest after extended culture1. However, with the ongoing trend towards shorter processes for CAR-T cell production, earlier, simpler, and less ambiguous readouts are desirable.

Here, we propose assessment of CAR ligand-independent surface expression of CD137 as an indicator of tonic signaling. On T cells, CD137 (also known as 4-1BB and TNFRSF9) is a co-stimulatory receptor of the tumor necrosis factor (TNF) receptor superfamily, and its expression strictly correlates with T cell receptor signaling. This property has previously been utilized to identify and isolate antigen-specifically activated T cells in various clinical settings, such as virus-specific T cells in ongoing infectious diseases2, tumor-reactive T cells from tumor infiltrates3, and alloreactive T cells in transplantation4.

We show by flow cytometry analysis that this expression pattern also applies to primary human CAR-T cells with tonic signaling through their CAR’s CD3ζ endodomain. 72 hours after removal of polyclonal stimulants (e.g., anti-CD3 / anti-CD28 antibody-coated beads), CD137 surface expression remains elevated in T cells transduced with chronically activating CARs compared to untransduced T cells and CAR-T cells without tonic signaling, long before differences in other markers of T cell activation, exhaustion and differentiation (e.g., CD25, CD69, PD-1 and CCR7) become detectable. The kinetics and specificity of CD137 expression, coupled with the simplicity of including it in standard immunophenotyping panels for flow cytometry, make it a useful and attractive tool for pre-clinical characterization of CARs.

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