Combined PD-1 and TIGIT blockade via siRNA enhances anti-leukemic T-cell function and promotes AML cell apoptosis

This study introduces a novel siRNA-based dual checkpoint blockade approach using readily available leukoreduction filter-derived T-cells, exploring dual PD-1/TIGIT siRNA-mediated knockdown to enhance anti-leukemic activity against Acute myeloid leukemia (AML) cell lines with distinct molecular characteristics (KG-1, representing minimally differentiated AML-M0; THP-1, representing monocytic AML-M5). T-cells were isolated from leukoreduction filters (98.1% purity), activated with CD3/CD28 Dynabeads and IL-2 to upregulate PD-1 (40.5%) and TIGIT (46.4%), and transfected with siRNA (70.8% efficiency), reducing PD-1 to 11.9% and TIGIT to 19.4%. Transfected T-cells were co-cultured with AML cells at E: T ratios of 1:1, 2:1, and 5:1. Viability, cytotoxicity, IFN-γ and caspase-3 levels, and Bax/Bcl-2 mRNA expression were assessed. Dual knockdown reduced THP-1 viability to 30% and KG-1 to 40% at 5:1, with cytotoxicity increasing to 44.3% (THP-1) and 48.8% (KG-1) (P < 0.01), outperforming single knockdowns (P < 0.01). IFN-γ peaked at 80 pg/mL (THP-1, 5:1, P < 0.0001), caspase-3 rose by 70% (P < 0.0001), Bax increased 3.0-fold, and Bcl-2 decreased 70% (P < 0.001). CD8⁺ T-cells dominated (~ 60% post-transfection). Statistical significance was confirmed for differences between KG-1 and THP-1 responses (P < 0.05), with THP-1 demonstrating greater susceptibility to dual checkpoint blockade. Dual PD-1/TIGIT knockdown additively enhances cytotoxicity, apoptosis, and T-cell activation, with stable CD4⁺/CD8⁺ ratios ensuring effector function. While this approach provides a cost-effective foundation for AML immunotherapy research, clinical translation requires validation in primary AML samples and in vivo models to address the complex bone marrow microenvironment and optimize delivery systems.