Prolonged effect of BAFF on chicken B cell development revealed by RCAS retroviral gene transfer in vivo

Abstract

Chickens provide an important model for developmental biology as well as phylogenetic studies of the immune system. In many species cytokines are important regulators of immune functions and recently we identified the chicken homologue of BAFF (B cell activating factor of the TNF family), shown to be an essential survival factor for B cells in mammals. Characterisation of BAFF function in the phylogenetically distant chicken requires in vivo studies, but despite considerable progress to date no efficient transgene or knockout technology for chickens is available. Thus the retroviral vector system RCAS (replication-competent ASLV long terminal repeat (LTR) with a Splice acceptor) was used to generate chickens stably expressing chBAFF or a soluble chBAFF neutralizing decoy receptor. Expression of RCAS based proteins was demonstrated in ovo and for the complete observation period of 2 month after hatch. In contrast to injections with recombinant proteins the prolonged RCAS based cytokine expression allowed assessment of BAFF function not only in newly hatched chicks but also in more mature birds. We demonstrate that RCAS based secreted products were biologically active, with chBAFF overexpression leading to a drastic and continuous increase in B cell numbers and neutralisation of chBAFF by expression of a soluble decoy receptor leading to severely reduced B cell numbers in transduced birds. This increase in B cell numbers was matched by increased immunoglobulin levels. Using RCASBP transduced birds we provide evidence that the RCAS system provides a fast and highly efficient gene transfer system for analysis of secreted protein function in the chicken and we show that in the chicken BAFF is necessary for B cell survival but in contrast to mammals, not only mature B cells but also immature B cells in the bursa of Fabricius are maintained by BAFF.