Transcription factor Pax5 (BSAP) transactivates the RAG-mediated V(H)-to-DJ(H) rearrangement of immunoglobulin genes.

Immunoglobulin rearrangement from variable heavy chain (V(H)) to diversity (D)-joining heavy chain (J(H)), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage-specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of D(H)-proximal V(H)7183 genes. In exploring the mechanism for Pax5 regulation of V(H)-to-DJ(H) recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse V(H) gene segments. Pax5 bound to those sites in vitro and occupied V(H) genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)-RAG2 complex to enhance RAG-mediated V(H) recombination signal sequence cleavage and recombination of a V(H) gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin V(H)-to-DJ(H) recombination.

Analysis of H2-O influence on antigen presentation by B cells.

HLA-DM (DM; in mouse H2-DM) promotes the exchange of MHC class II-associated peptides, resulting in the accumulation of stable MHC class II-peptide complexes. In naive (but not germinal center) B cells, a large part of DM is tightly associated with HLA-DO (DO; in mouse H2-O), but the functional consequence of this association for Ag presentation is debated. Here, we have extended previous studies by examining the presentation of multiple epitopes after Ag internalization by fluid phase endocytosis or receptor-mediated uptake by membrane Ig (mIg) receptors. We find that the effects of H2-O are more complex than previously appreciated; thus, while only minor influences on Ag presentation could be detected after fluid phase uptake, many epitopes were substantially affected after mIg-mediated uptake. Unexpectedly, the presentation of different epitopes was found to be enhanced, diminished, or unaffected in the absence of H2-O, depending on the specificity of the mIg used for Ag internalization. Interestingly, epitopes from the same Ag did not necessarily show the same H2-O dependency. This finding suggests that H2-O may control the repertoire of peptides presented by B cells depending on the mIg-Ag interaction. The absence of DO/H2-O from germinal center B cells suggests that this control may be released during B cell maturation.

H2-O influence on antigen presentation in H2-E-expressing mice.

MHC class II molecules sample peptides generated in the endosomal/lysosomal system for cell surface presentation to CD4+ T cells. Peptide loading requires the endosomal/lysosomal resident HLA-DM (DM; H2-DM, mouse), but in B cells, DM is tightly associated with HLA-DO (DO; H2-O, mouse). We have previously shown that H2-O differentially modulates the processing and presentation of different antigenic epitopes on H2-Ab molecules. Using H2-Ead-transgenic mice, we here show that presentation of different epitopes by H2-Ed/b molecules is similarly influenced by H2-O after membrane immunoglobulin-mediated uptake of antigen. In addition, B cells from H2-Ead-transgenic mice (which co-express H2-Ab and H2-Ed/b molecules) show an altered pattern of presentation of H2-Ab-restricted epitopes. In H2-Ead-transgenic, H2-O-deficient mice, further changes in the peptide repertoire were observed. Thus, H2-Ed/b expression influences the epitopes presented by H2-Ab, and this effect is further altered by expression of H2-O.