TLR2 activation potentiates P-glycoprotein-mediated methotrexate efflux and enhances cytotoxicity of human NK cells against acute lymphoblastic leukemia

Introduction: Acute Lymphoblastic Leukemia (ALL) remains the most prevalent childhood malignancy. While chemotherapy has improved survival rates, multidrug resistance (MDR) mediated by P-glycoprotein (P-gp/ABCB1) overexpression persists as a major cause of treatment failure and relapse. Natural Killer (NK) cells are pivotal for anti-leukemic surveillance but are often compromised during treatment due to their susceptibility to chemotherapy, a vulnerability intrinsically linked to their own expression of P-gp. Strategies to transiently enhance NK cell chemoresistance could therefore preserve immune function and improve therapeutic outcomes. We hypothesized that stimulating Toll-Like Receptor 2 (TLR2) on NK cells could modulate P-gp expression or activity, enhancing their resilience to cytotoxic drugs. Methods: In this study, we first characterized NK cells from pediatric ALL patients, confirming the constitutive expression of both the therapeutic target (TLR2) and the drug efflux pump (P-gp) across all major subpopulations. Using a healthy donor model, we then dissected the functional consequences of specific TLR2 heterodimer engagement. Results: While agonists for both TLR2/1 (PCSK) and TLR2/6 (LTA, MALP-2) induced functional activation, their effects on P-gp were divergent. In a PBMC context, stimulation with the TLR2/1 agonist PCSK significantly enhanced the efflux of the chemotherapeutic agent Methotrexate (MTX), but not Rhodamine 123. This functional enhancement occurred without increasing P-gp surface expression, suggesting a modulation of transporter kinetics. Crucially, mechanistic assays in purified NK cells revealed that this MTX efflux enhancement relies on the cellular microenvironment, whereas direct high-dose TLR2/1 stimulation paradoxically led to P-gp loss. Furthermore, we demonstrated that the immunomodulatory effects of PCSK extend beyond chemoresistance to directly potentiate anti-leukemic effector functions; PCSK stimulation significantly enhanced NK cell cytotoxicity against RS4;11 leukemic blasts without compromising effector viability. Discussion: These findings identify a novel immunomodulatory axis where TLR2 signaling differentially regulates P-gp function and effector capacity in NK cells depending on the specific heterodimer engaged and the cellular context. We propose that controlled TLR2/1 stimulation represents a potential dual-benefit strategy to protect NK cells from chemotherapy-induced suppression while boosting their anti-leukemic activity in ALL.