Project Summary Granulocyte transfusion (aka. neutrophil transfusion or GTX) has been utilized as a therapeutic approach for the treatment of life-threatening bacterial and fungal infections in severe neutropenic patients, including hematopoietic cell transplantation (HCT) recipients. Clinical outcomes from GTX are disadvantaged by the short shelf life and compromised function of donor neutrophils. Spontaneous neutrophil death is heterogeneous and mediated by multiple pathways. Leveraging mechanistic knowledge and pharmacological screening, we identified a combined treatment, caspases–lysosomal membrane permeabilization–oxidant– necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which alters neutrophil fate by simultaneously targeting multiple cell death pathways. CLON-G treatment significantly prolongs neutrophil ex vivo survival, increasing their half-life from less than 1 day to greater than 5 days. Compared to freshly isolated neutrophils, the stored CLON-G-treated neutrophils display normal functions, including chemotaxis, ROS production, phagocytosis, and bacteria killing. These results led us to hypothesize that CLON-G treatment can significantly increase the ex vivo shelf life of granulocyte transfusion products without impairing the function of the neutrophils in the GTX recipients. Due to the prolonged intracellular retention of the drugs, CLON-G treatment may even improve the survival and increase the half-life of the transfused neutrophils in vivo and therefore further improve the efficacy of GTX. In this study, we will first test this hypothesis in a clinically relevant murine model of granulocyte transfusion in neutropenia-related pneumonia. We will determine whether the stored CLON-G-treated neutrophils can function normally and survive even longer in vivo compared to freshly isolated neutrophils (Aim 1A). Additionally, we will investigate the survival and function of transfused human neutrophils in vivo in neutrophil-depleted NSG mice (Aim 1B). Next, we will examine whether transfusion with stored CLON-G-treated neutrophils can ultimately enhance the inflammatory response and bactericidal capability of the recipients as effectively as, or even more effectively than, transfusion with untreated fresh neutrophils (Aim 2). Last, to identify novel CLON-G-inducible factors and pathways that play critical role in regulating neutrophil death and function, we will perform single-cell RNA sequencing (scRNA-seq) to reveal the molecular signature of CLON-G reprogrammed neutrophils (Aim 3) . Together, experiments proposed in the three aims will assist us to better understand the molecular and cellular mechanism leading to neutrophil death and to design novel clinical procedures for the long-term storage and application of neutrophils in transfusion medicine. This study also provides a new strategy for treatment of neutropenia-related pneumonia. The ability to utilize GTX through better understanding of granulocyte func...