CAR and TCR form individual signaling synapses and do not cross-activate, however, can co-operate in T cell activation

Abstract

In engineered T cells the CAR is co-expressed along with the physiological TCR/CD3 complex, both utilizing the same downstream signaling machinery for T cell activation. It is unresolved whether CAR-mediated T cell activation depends on the presence of the TCR and whether CAR and TCR mutually cross-activate upon engaging their respective antigen. Here we demonstrate that the CD3ζ CAR level was independent of the TCR associated CD3ζ and could not replace CD3ζ to rescue the TCR complex in CD3ζ KO T cells. Upon activation, the CAR did not induce phosphorylation of TCR associated CD3ζ and, vice versa, TCR activation did not induce CAR CD3ζ phosphorylation. Consequently, CAR and TCR did not cross-signal to trigger T cell effector functions. On the membrane level, TCR and CAR formed separate synapses upon antigen engagement as revealed by total internal reflection fluorescence (TIRF) and fast AiryScan microscopy. Upon engaging their respective antigen, however, CAR and TCR could co-operate in triggering effector functions through combinatorial signaling allowing logic “AND” gating in target recognition. Data also imply that tonic TCR signaling can support CAR-mediated T cell activation emphasizing the potential relevance of the endogenous TCR for maintaining T cell capacities in the long-term.

Integration of ζ-deficient CARs into theCD3-zetagene conveys potent cytotoxicity in T and NK cells

“…Like TRAC -, CD3 ζ - editing causes TCR-ablation. Furthermore, the CAR’s CD3ζ-domain cannot rescue TCR/CD3-expression in CD3 ζ-KO T cells 66 . Together, this avoids the risk of alloreactivity in TCRα/β + T cells.…”

Endogenous Signaling Molecule Activating (ESMA) CARs: A Novel CAR Design Showing a Favorable Risk to Potency Ratio for the Treatment of Triple Negative Breast Cancer

“…Whereas CAR and TCR activation are usually not interconnected but instead induce additive effector functions [ 65 ], ESMA CARs bridge the gap and employ synergistic mechanisms of both CAR costimulation and (TCR) endogenous stimulatory signaling. This way, redundant signaling of multiple CAR ITAMs that could induce early T cell anergy and imposes different safety risks could be avoided.…”

Integration of ζ-deficient CARs into the CD3 ζ gene conveys potent cytotoxicity in T and NK cells

“… 17 , 27 Like in the TRAC approach, CD3ζ- editing causes TCR-ablation, because the CAR’s CD3ζ-domain cannot rescue TCR/CD3 expression in CD3ζ -KO T cells. 53 Together, this circumvents alloreactivity in T cells and should minimize the risk for GvHD if residual TCR + T cells are efficiently depleted before allogeneic application of CD3ζ -edited CAR-T cells. 54 , 55 Therefore, the CD3ζ- approach may be preferentially suited for allogeneic applications.…”

“…The CD3ζ-locus is a CAR-integration site which limits necessary transgene size and shares features and advantages with the TRAC knock-in in T-cells 17,27 . Like TRAC-, CD3ζ-editing causes TCR-ablation, because the CAR's CD3ζ-domain cannot rescue TCR/CD3-expression in CD3ζ-KO T-cells 52 . Together, this circumvents alloreactivity in T-cells and should minimize the risk for GvHD if residual TCR + T cells are efficiently depleted prior to allogeneic application of CD3ζ-edited CAR-T-cells 53,54 .…”