We conducted a systematic analysis to determine the effect of specific AP-1 family members on distinct CD8⁺ and CD4⁺ CAR-T cell functions. Herein, the identity of specific overexpressed transcription factors (TFs) are blinded as TF-A, TF-B, etc. and will be disclosed at the time of the presentation.

We identify TF-C as a lead candidate for CD8⁺ CAR-T cell modification providing improved specific proliferation, reduced activation-induced cell death, diminished exhaustion-marker expression, and a memory-like phenotype. TF-C attenuated IL-2 secretory capacity compared to conventional CAR-T cells. Conversely, TF-A/CAR-T cells consistently exhibited the highest levels of IL-2 and IFN-γ secretion accompanied with the best Agˡᵒʷ tumor control. Importantly, none of the tested TFs permitted antigen-independent proliferation. Transcriptional analysis of CD8⁺ TF-C/CAR-T cells revealed a signature of lower activation, reduced exhaustion, and a unique chemokine receptor profile compared to conventional CAR-T cells. The characteristics induced by TF-C significantly minimised lung sequestration compared to conventional CAR-T cells due to increased LFA-1 recycling via βII-spectrin in vivo.

TF-C-modification proved detrimental to the potential of CD4⁺ CAR-T cells to elicit effector functions, highlighting distinct preferences of T cell subsets for TF-modification. However, CD8⁺ TF-C/CAR-T cell proliferation was synergistically enhanced in the presence of CD4⁺ TF-A/CAR-T cells both in vitro and in vivo.

To conclude, overexpression of TF-C endows CD8⁺ CAR-T cells with favourable attributes facilitating engraftment, persistence, homing and exhaustion resistance. Our data pinpoint a blend of CD4⁺ TF-A/CAR and CD8⁺ TF-C/CAR-T cells augmenting the intrinsic properties of CAR-T cells conferring superior engraftment and augmented antitumor efficacy in vivo.