PI3 Kinase Pathway Regulates CAR T Cell Trogocytosis

Chimeric antigen receptor (CAR) T cell mediated trogocytosis (CMT) is a phenomenon wherein the CAR shears off its cognate antigen from the surface of tumor cells and presents the antigen on its own surface. This leads to the low antigen density on the surviving tumor cell enabling its escape from CAR T cell killing. As the antigen is now expressed on CAR T cells, it leads to CAR T cells killing each other (CAR T cell fratricide). Preclinical and clinical studies (Ye li et al. Nat Med 2022) have demonstrated that CMT is a potential mechanism for CAR T resistance. However, the molecular mechanism underlying CMT is unknown.

We first determined whether specific CAR-antigen contact is necessary for CMT. We generated CD19 (FMC63) CAR T cell mutants (ΔscFv) that lack binding scFv and co-cultured them with CD19 expressing ALL cell line NALM6 and determined trogocytosis by flow cytometry. ALL cell lines only lost CD19 in the presence of CD19 CAR T cells but there was no CD19 loss when co-cultured with ΔscFv CAR T mutant. We next determined whether in addition to cell-cell contact, CAR signaling is essential for trogocytosis. We generated CD19 CAR T cells that lack CD3ζ signaling domain (Δsig) and co-cultured with NALM6 cells. Trogocytosis was observed with CD19 CAR T cells but not with the signaling mutant demonstrating that CAR signaling is important for CMT.

Previous studies have shown that T cell receptor (TCR) mediated trogocytosis is dependent on CD3ζ and involves the recruitment of phosphoinositide 3-kinase p110δ (PI3K p110δ) to the TCR (Martínez et al. Immunity 2011). As the CD3ζ domain is common between the CAR and TCR we hypothesized that the PI3K p110δ pathway is an important mediator of trogocytosis and blocking the pathway would mitigate membrane transfer.

To determine whether PI3K p110δ plays a role in CMT, we generated CD19 CAR Jurkat cells with p110δ knockdown (p110δ KD) using short hairpin RNA (shRNA) and co-cultured with NALM6 cells. There was a greater reduction of CD19 on the NALM6 cells co-cultured with CD19 CAR Jurkat cells compared to CD19 p110δ KD CAR Jurkat cells, suggesting that PI3K was an important mediator of trogocytosis. We next determined if p110δ inhibition in CD19 CAR T cells will have the same effect on trogocytosis. CD19 CART cells were treated with p110δ inhibitor ME401at 0.5 µM concentration overnight and co-cultured with NALM6 cells for one hour and trogocytosis determined by flow cytometry. We found reduction in CMT upon ME401 treatment compared to mock treated CART cells (median fluorescence intensity (MFI) % reduction of CD19 on NALM6 ; 41.8 % vs 76.9%).

We next determined whether p110δ interacted with the CD3ζ domain of the CAR. We co-cultured CD19 CAR T cells and NALM6 cells and then immunoprecipitated the CAR and probed for p110δ binding. We found that p110δ bound to the CAR domain, only upon stimulation with NALM6 cells with 4.5 fold higher intensity when compared to unstimulated CAR (p=0.028 ; N=3). This binding of p110δ to the CAR was inhibited in CAR T cells co-cultured with NALM6 in the presence of ME401. Furthermore, ME401 treatment of CAR T cells led to a reduction of AKT phosphorylation, a downstream effector of PI3K in the CAR T cells.

Rac1 GTPase is an important mediator of actin polymerization and trogocytosis and we determined whether p110δ inhibition with ME401 would affect Rac1 activation. We co-cultured CD19 CAR T cells with NALM6 cells and pulled down activated GTPase bound Rac1 using PAK1 beads. Rac1 was activated only when CAR T cells were stimulated with NALM6 cells and p110δ inhibitor ME401 was able to inhibit Rac1 activation by 2.9 folds as determined by the relative intensity of active Rac1 band.

We next determined whether p110δ inhibition of CAR T cells with ME401 adversely affected their cytotoxic ability. We treated CD19 CAR T cells with or without 0.5 µM ME401 and co-cultured with luciferase expressing NALM6 cells at different effector: target ratio and determined cytotoxicity using a luminescence-based assay. CD19 CAR T cells with or without ME401 treatment achieved 99% cytotoxicity in overnight assay suggesting that ME401 at 0.5µM does not affect T cell cytotoxicity. Moreover, ME401 treated CAR T cells exhibited a lower expression of PD1 (MFI 778 vs 1930) (p=0.002, N=3) and & LAG3 (10414 vs 20180) (p=0.1, N=3) compared to control CAR T cells.

We have shown that PI3K p110δ pathway is an important regulator of CAR mediated trogocytosis and targeting this pathway could be of potential clinical benefit.