Antibody Mediated Depletion of iNKT Cells Protects Against Hypoxia-Induced Pulmonary Injury in a Murine Model of Sickle Cell Disease

Background: Painful vaso-occlusive crises (pVOC) are caused by the polymerization of sickle deoxyhemoglobin, red cell sickling and vaso-occlusion. Vaso-occlusion is exacerbated by an inflammatory cascade that is initiated by iNKT cell activation. Townes sickle cell mice have mouse globins replaced by human globins, including the mutant human sickle b-globin gene (SS). NKT-14 is a monoclonal antibody directed specifically to the mouse invariant T cell receptor (iTCR). The antibody is a chimeric mouse IgG2a that, when bound to the mouse iNKT cell, promotes a rapid, specific and long lasting (> 14 days) depletion. Aims: Our primary goal was to determine if selective iNKT cell depletion with NKT-14 reduces lung inflammation and injury in response to hypoxia-induced RBC sickling in Townes SS mice. Methods: Hypoxic pulmonary injury was induced by placing mice in 8% O 2 (Coy Lab products) followed by reoxygenation. Some mice were subject to a second period of hypoxia and continuously monitored to determine carotid Hb-saturation (Starr MouseOx). Total cell numbers and activation state of iNKT cells and neutrophils in lung were determined by FACS. Pulmonary vascular leak was quantified with Evans Blue dye. Results: Twelve hours of 8% hypoxia followed 4 hours of reoxygenation (H/R) was found to produce non-lethal lung inflammation and injury in SS mice. Compared to isotype Ab controls, injection of four mice with 100µg/mouse of NKT-14 Ab three days prior to HR was found to decrease the number of pulmonary iNKT cells by 91% (P Conclusions: Selective depletion of mouse iNKT cells by injection of NKT-14 Ab three days prior to H/R results in substantial reductions in pulmonary inflammation, vascular leak and injury as measured by Hb-Sat in SS mice. The results suggest that prophylactic Ab-mediated depletion of iNKT cells in humans with SCD may be useful for reducing the incidence and severity of pVOC and acute chest syndrome. Disclosures Schaub: NKT Therapeutics Inc: Employment, Equity Ownership. Scheuplein: NKT Therapeutics Inc: Employment, Equity Ownership. Mashal: NKT Therapeutics Inc: Employment, Equity Ownership.