Regulation of IgD Expression and Its Role in B Cell Transformation

A developing B lymphocyte first acquires expression of immunoglobulin M (IgM) at the immature stage, and along maturation into the transitional stage it starts to co-express another isotype–IgD. IgM and IgD are generated by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus, so that they share the N-terminal antigen-binding variable region, but differ in the C-terminal constant region, encoded by Cμ or Cδ exons, respectively. Maturation into follicular (naïve) B cells, IgD levels surpass those of IgM; while upon antigen-stimulation and entering germinal center reaction, follicular B cells turn off IgD expression. Trans-acting factors have been proposed to control IgM and IgD expression since several decades ago. However, the first of such factors, Zfp318, was just reported last year (Enders et al. Proc Natl Acad Sci USA 2014; Pioli et al. J Immunol 2014). Still, other factors and signaling cascades regulating IgD expression remain to be identified.

In this context, we accidentally discovered that expression of the Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1) in B cells specifically abolishes IgD but not IgM expression; LMP1 signaling suppresses IgD expression at the mRNA level. Using microarray we screened differentially expressed genes in LMP1-expressing versus normal mouse B cells, and then looked up these genes in ImmuGen database (http://www.immgen.org) to identify those whose expression patterns correlate with that of IgD at various B cell developmental stages. These analyses returned us two new candidates Rapgef4 and CD55, in addition to Zfp318. By CRISPR technology, we further demonstrated that mutation of Rapgef4 or CD55 in mature B cells results in downregulation of IgD but not IgM. To define their role in regulating IgD expression in vivo, mice carrying conditional null alleles Rapgef4^F/F and CD55^F/F, are being generated.

The facts that IgD is an evolutionarily conserved Ig isotype and its expression is precisely regulated during B cell developmental and functional stages suggest an important role in B cell biology/function. Indeed, a recent study suggested that IgD, by sequestering CD19 coreceptor from IgM, might play a negative role in B cell receptor (BCR) signaling (Klasener et al. eLife 2014). Interestingly, flow cytometric analysis of 12 diffuse large B cell lymphoma (DLBCL)-like malignancies arising in our mouse model revealed that 10 cases are low/negative for IgD and the other 2 cases express intermediate levels of IgD, while they all express surface IgM or IgG (Zhang et al. Cell Rep 2015; data not shown); human DLBCLs often retain surface IgM expression (Wright et al. Proc Natl Acad Sci USA 2003; IgD expression data not available). Basing on aforementioned observations, we hypothesize that downregulation of IgD may be important for LMP1-induced B cell transformation and for DLBCL pathogenesis in general. Accordingly, in this work, we will reconstitute IgD expression in LMP1⁺ lymphoma cell lines (Zhang et al. Cell 2012), and determine if it suppresses lymphoma cell growth in vitro and in vivo. Similarly, we will reconstitute IgD expression in a few IgD^-mouse DLBCL lines and monitor its impact on lymphoma growth. In addition, as we have 2 DLBCL lines expressing intermediate levels of IgD, we will use CRISPR technology to delete/mutate Cδ coding sequence in these lymphoma lines, and see if the mutated cells (losing IgD expression) outcompete the non-mutated cells.

Overall, this work will lead to molecular insights into IgD regulation. It may also elucidate the functional significance of IgD downregulation in B cell lymphoma pathogenesis/maintenance, and guide therapy development targeting BCR signaling.