Asymmetrical Expression of PD1 and CD25 in T-cells Post-Initial Activation

Abstract After TCR engagement, T cells induce expression of CD25, which forms the IL2 high affinity receptor complex, concurrently with PD1, which plays a down-regulatory role in activation. Cessation of TCR stimulation leads to eventual contraction of T cell responses. Chimeric antigen receptor (CAR) T cell therapy has seen increased use in the past decade. CAR T-cells are activated and transduced ex vivo and expanded with low amounts of cytokines prior to administration. The expression of CD25 and PD1 on CAR T cells at different stages of the process has been incompletely characterized. Purified T cells were activated in vitro for 3 days followed by expansion using low dose IL2. We demonstrate that both CD25 and PD-1 are strongly upregulated during initial activation, but by day 8, under expansion conditions, PD1 is downregulated to negligible levels. Despite absence of TCR signaling, CD25 expression remains moderate-to-high explaining the ability to maintain proliferation. This discrepancy was higher in transduced CAR T-cells compared to activated non-transduced (NT) T cells indicating the possibility of CAR tonic signaling. Furthermore, restimulation of both NT and CAR-T cells after this expansion phase resulted in further increased CD25 expression whereas PD-1 expression remained low. Our studies suggest that during initial activation, TCR engagement is necessary to maintain PD-1, but not CD25 expression. However, upon restimulation, TCR engagement is capable of further upregulating CD25, but not PD-1 expression. These results indicate that TCR signaling induces asymmetrical expression of PD-1 and CD25, a dynamic interplay that may regulate T-cell function and memory response and that PD1 may not be a limiting factor in the process.