(425) Development of an AICDA-Cre^ERT2 Ferret Model to Study Adaptive Humoral Immunity

Background: Immunity and inflammation are key components of nearly all inherited and acquired diseases. In the context of cystic fibrosis (CF), altered and hyperactive lung inflammation has been attributed to viscous mucus secretions in the airways and CFTR expression in lymphocytes. Humoral and cellular responses may also be a significant barrier to viral-mediated gene therapies for CF. Much of our understanding of how immunity impacts disease progression is based on genetic studies in congenic mice. Thus, there is an increased need for genetic models to study immune responses in outbred species that also develop CF disease similar to humans. High-affinity, isotype-switched antibody responses are produced by B cells following effective T cell-dependent B cell activation. This involves the processes of somatic hypermutation (SHM) and class-switch recombination (CSR). Both SHM and CSR are initiated by the expression of activation induced cytidine deaminase or AID (AICDA gene). A deeper understanding of the contextual activation of SHM and CSR is needed to improve our understanding of human disease and improve immunologic therapies, including therapies for CF. For example, whereas effective anti-viral or anti-bacterial vaccines must elicit induction of high affinity antibodies for durable protection, an optimal gene therapy vector must avoid significant induction of high affinity antibodies to allow for repeat dosing. Further, in CF lung transplant recipients, chronic lung allograft dysfunction (CLAD) is often marked by the emergence of graft- and auto-reactive B cells. Thus, a better understanding of the context-specific physiology of SHM and CSR is needed to develop targeted therapies for CLAD. Transgenic AICDA-Cre^ERT2 mice have been used to trace affinity-selected B cells, but current rodent models fail to adequately phenocopy human CLAD and CF pathology. Ferrets share similar airway anatomy and cell biology with humans and are naturally susceptible to many of the same pathogens. Moreover, ferret lung transplants faithfully model the full human spectrum of CLAD histopathology, including the formation of tertiary lymphoid organs. Here, we report the generation and characterization of AICDA-Cre^ERT2 ferrets.

Methods: Unlike the current AICDA-Cre^ERT2 knock-in mouse model, which disables one AICDA allele and reduces the efficiency of SHM and CSR, we inserted IRES-Cre^ERT2 into the 3’-UTR of the ferret AICDA gene.

Results: Endogenous levels of AICDA mRNA in the spleen, thymus, and lymph nodes did not differ between transgene positive and negative littermates. In combination with Cre reporter ferrets (ROSA-TG), we have demonstrated functionality of the model in vitro. We efficiently expanded ferret CD79b+ B cells—isolated from peripheral blood mononuclear cells—in commercially available B cell expansion media. Reporter conversion was seen in cultures treated with 4-hydroxytamoxifen and not in untreated cultures.

Conclusions: These animals will facilitate future experiments to lineage trace antigen experienced B cells in contexts that are highly relevant to human disease. We envision that that AICDA-Cre^ERT2::ROSA-TG ferrets will have utility in studying vaccines (viral and bacterial), lung transplant rejection, and humoral immunity to gene therapy vectors. Once bred onto a CF background, CFTR^G551D:AICDA-Cre^ERT2::ROSA-TG ferrets will be an excellent model for studying humoral responses in the CF disease state.

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