NFkB Crel Protein Amount Determines the Selection of Germinal Center B Cells

Most B cell lymphomas originate during B cell differentiation and selection in the germinal center (GC). A key event in the GC is the selection of GC B cells to produce high-affinity antibody-producing plasma cells and memory B cells. GCs are transient structures formed in response to immunization and infection. GCs are the site of B cell proliferation coupled with somatic hypermutation of the B cell receptors (BCRs) to produce high-affinity antibodies. GC B cells circulate between two distinct zones: the light zone (LZ) and the dark zone (DZ). Proliferation and somatic hypermutation occur in the DZ and transit to the LZ to test for BCR affinity. GC B cells are selected based on their BCR affinity to differentiate into plasma cells and memory B cells in LZ. Following selection in the LZ, GC B cells can also reenter the DZ for additional rounds of division and somatic hypermutation. A higher expression of the cell cycle regulator cMyc characterizes the positively selected GC B cells. High cMyc-expressing LZ GC B cells show enhanced expression of the NFkB gene signatures, and importantly, cMyc is a cRel target gene. It has been demonstrated that the transcription factor NFkB cRel is required for B cell proliferation and GC B cell maintenance. cRel-deleted GC B cells show reduced somatic hypermutation and affinity maturation. Surprisingly, transgenic overexpression of cRel in GC B cells does not affect somatic hypermutation and reduce, if any, affinity maturation. Hence, the function of cRel in regulating GC B cell selection remains unknown.

To understand the function of cRel in GC B cell selection, we have generated cRel fluorescence reporter mice (mTFP1-cRel) and measured the kinetics of cRel expression at the protein level in GC B cells. We found that cRel expression is higher in GC B cells compared to non-GC B cells, including memory B cells, and interestingly, cRel expression increases from early to late GC B cells. Analysis of cRel expression in DZ and LZ B cells showed that about 10% of LZ B cells increased cRel expression compared to DZ B cells. We discovered that high cRel-expressing LZ B cells have higher expression of Myc (cRel target gene), and cell cycle regulator CyclinD2. Surprisingly, cRel expression at the mRNA level remains similar in low and high cRel protein-expressing GC B cells. Higher somatic hypermutation leads to higher affinity maturation. We examined somatic hypermutation in the low and high cRel expressing LZ B cells by sequencing IgH J_H4-intronic enhancer downstream of the rearranged V_J558DJ_H4 element. We found that high cRel-expressing LZ B cells have a higher somatic hypermutation than low cRel-expressing LZ B cells. Single cell study reveals high cRel LZ B cells are enriched for precursor plasma cells and precursor memory B cells. Overall, our results suggest that cRel protein but not transcript levels regulate GC B cell selection. Our studies resolve conflicting findings about the function of cRel in GC B cells somatic hypermutation and affinity maturation.

No relevant conflicts of interest to declare.