Differential Expression and Non-Redundant Functions of NF-κB Transcription Factor Subunits in Germinal Center (GC) B Cell Subpopulations

Abstract 216

The majority of B cell-derived neoplasms, including Hodgkin and Non-Hodgkin lymphoma and multiple myeloma (MM), arise from antigen-specific B cells that have undergone the germinal center (GC) reaction of T-dependent immune responses. Recent work has demonstrated that GC-derived tumors frequently harbor genetic mutations in nuclear factor-κB (NF-κB) signaling pathway components, resulting in the constitutive activation of NF-κB signaling, thus identifying NF-κB as a critical player in GC-lymphomagenesis. Moreover, there is evidence for a preferential activation of particular NF-κB transcription factor subunits in tumor subtypes. Despite extensive knowledge about the biology of NF-κB, its potential function in the physiology and development of GC B cells, the presumptive tumor precursor cells, is largely unresolved.

The NF-κB signaling cascade comprises 5 different subunits, which occur as homo- and heterodimers and can be activated via two different routes, the canonical (classical) and the alternative (non-canonical/classical) NF-κB pathways. RELA, c-REL and p105/p50 represent the subunits of the canonical, while RELB and p100/p52 comprise those of the alternative pathway. It is known that there is no active NF-κB signaling in tonsillar GC centroblasts. Conversely, NF-κB activation was shown to occur in a subset of GC centrocytes. In this study, we demonstrate that each of the 5 NF-κB subunits exhibit nuclear translocation in centrocytes. Surprisingly, we observed that centrocytes expressing the plasma cell master regulator BLIMP1 showed strong immunofluorescence (IF) staining for the alternative NF-κB subunit p100/p52 and weak expression of the canonical subunits p105/p50 and c-REL compared to surrounding lymphocytes. Plasma cells localized in the tonsillar subepithelium showed the same pattern of expression. This observed differential expression of alternative vs. canonical NF-κB subunits in plasma cells and B cells, respectively, is supported by gene expression profiling data of human B cell subpopulations. Moreover, we observed that a mouse lymphoma cell line (M12) shows activation of the alternative NF-κB pathway upon induction of plasma cell differentiation. Also, Western and IF analysis of MM vs. diffuse large B cell lymphoma (DLBCL) cell lines revealed high protein levels and nuclear translocation of both p52 and RELB and low levels and cytosolic localization of c-REL in MM cell lines, while the opposite pattern was observed in the analyzed DLBCL lines. In summary, the elevated protein expression and presumed activity of the alternative over the canonical NF-κB pathway in plasma cells and their precursors suggests that activation of the alternative NF-κB pathway in centrocytes may contribute to plasma cell development and/or physiology.

To elucidate the in vivo function of individual NF-κB transcription factor subunits, we started by determining the extent to which deletion of c-REL specifically in GC B cells affects the biology and differentiation of GC and post-GC B cells. We generated and then crossed a conditional loxP-flanked rel (c-REL) allele to mice that express the Cre-recombinase in GC B cells instructed to undergo class switch recombination (Cγ1-Cre mice). Following immunization with a T-dependent antigen, PNA⁺CD95⁺ GC B cell numbers were markedly reduced in immunized rel^fl/flCγ1-Cre mice compared to rel^+/+Cγ1-Cre control mice. In addition, immunohistochemical analysis of spleen sections for BCL6 and IgG1 showed significantly smaller GCs, and a strong reduction in the numbers of GC-derived IgG1-secreting plasma cells, in rel^fl/flCγ1-Cre mice compared to controls. Consistent with these findings, we observed that rel^fl/flCγ1-Cre mice showed dramatically reduced numbers of nitrophenyl (NP) hapten-specific cells 14 days after immunization with NP-KLH compared to the control mice. Taken together, these findings suggest that c-REL may be required for the maintenance of GC B cells or for their selection into the post-GC compartment. Of note, the results demonstrate that deletion of a single NF-κB subunit in GC B cells can have drastic effects, suggesting a lack of general redundancy of the canonical subunits during the GC reaction. These findings imply that c-REL activation needs to be tightly controlled during GC B cell development, and raise the possibility that other NF-κB subunits may also exert unique functions in GC B cell differentiation.