CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition.

The objective of this experiment was to characterized the molecular mechanisms behind the different effects of PD-1 disruption in low affinity (LA) and high affinity (HA) HER2-28z CAR-T cells. The transcriptomic profile of mock and PD-1 KO CAR-T cells following antigen recognition was compared by using the nCounter® CAR-T Characterization Gene Expression Panel (Nanostring). RNA samples were isolated from CAR-T cells generated from three independent donors. CAR-T cells were cocultured with SKOV3 tumour cells (effector/target ratio of 1:3) for 48h. CD45+ cells were then flow sorted and total RNA was extracted using the RNeasy Mini kit (Qiagen). Samples were prepared according to the manufacturer’s protocols for the nCounter CAR-T Characterization Panel (Nanostring). Cartridges were run on the nCounter Sprint Profiler. Gene expression levels were normalized against the housekeeping genes and data analysis was conducted using the Rosalind Platform