An inducible, conditional and targeted B cell ablation mouse model for studying B cell functionality in the pathogenesis of human diseases

Abstract

Primary objective of my MS thesis project is to characterize and develop a B cell ablation model for investigating the pathogenesis of human diseases such as hepatitis and systemic lupus erythematosus (SLE). Conditional and targeted cell ablation is a powerful approach for studying cellular functions in vivo. However, currently available cell ablation models still have some limitations and therefore limit their broader application in biomedical research. For example, two of the most common cell ablation methods currently employed utilize the herpes simplex virus 1 thymidine kinase (HSVtk) or diphtheria toxin (DT) receptor combined with their respective transgenic strategies. The ablation using HSVtk transgenic mice eliminates dividing cells, but does not affect non-dividing cells. In addition, because of its extremely high potency (a single molecule of DT-A, the active cleavage product of DT is sufficient to induce apoptosis), dose dependent responses are difficult to achieve and off-target effects are frequently observed. These facts highlight the unmet need to develop alternative methods of targeted cell ablation, which our model very successfully addresses. Our recently established approach using intermedilysin (ILY)-mediated cell ablation that is specific for human CD59 (hCD59) expressing cells, obviates these problems and provides an excellent and significantly improved alternative approach to the existing cell ablation methodologies. Intermedilysin (ILY), a toxin secreted by Streptococcus intermedius, exclusively binds to the human cell membrane protein CD59 (hCD59) but not to CD59 of any other species. Once bound, ILY rapidly and potently lyses the cells. Using genetic engineering, animal models can be created that express hCD59 in a spatially regulated manner. Administration of ILY will then selectively ablate only those cells in the animals that express hCD59 without any non-specific effect. To expand and facilitate the application of this newly generated model, we recently generated a Cre-inducible floxedSTOP-hCD59 transgenic mouse line (ihCD59), where specific hCD59 expression occurs following Cre-mediated recombination. By crossing ihCD59 mice with specific immune cell (T cells or macrophage) Cre transgenic lines, we obtained double transgenic mice expressing hCD59. ILY administration mediated specific cell ablation in these target cell populations in a dose dependent manner. Based on these results, I wanted to establish a new B cell ablation model for further studying B cell functionality in the pathogenesis of human diseases. CD19-Cre mice expressing the Cre-recombinase in B cell population were crossed with ihCD59 mice to generate the double positive transgenic mice (ihCD59+/-/CD19-Cre+/-). In Aim 1, I have demonstrated that hCD59 is specifically expressed in the B cell populations. In Aim 2, I have documented that 1) ILY has a large pharmacological window, and 2) ILY injection to ihCD59+/-/CD19-Cre+/- mice resulted in a rapid cell ablation of the B cell cells with off-target effect. Further, I have demonstrated that the specific ablation of B cells did not prevent the immune (Con A)-mediated hepatitis. In the future, I will apply this conditional B cell ablation model for investigating the functionality of B cells in the pathogenesis of human disease such as SLE.