Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.

To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.     

Early Growth Response Genes 2 and 3 Regulate the Expression of Bcl6 and Differentiation of T Follicular Helper Cells

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GCs), and Tfh differentiation requires the function of B cell lymphoma 6 (BCL6). We have now discovered that early growth response gene 2 (EGR2) and EGR3 directly regulate the expression of Bcl6 in Tfh cells, which is required for their function in regulation of GC formation. In the absence of EGR2 and -3, the expression of BCL6 in Tfh cells is defective, leading to impaired differentiation of Tfh cells, resulting in a failure to form GCs following virus infection and defects in production of antiviral antibodies. Enforced expression of BCL6 in EGR2/3-deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of EGR2/3 that is important for Tfh cell development and Tfh cell-mediated B cell immune responses.     

EZH2 codon 641 mutations are common in BCL2-rearranged germinal center B cell lymphomas

Mutations at codon 641 of EZH2 are recurrent in germinal center B cell lymphomas, and the most common variants lead to altered EZH2 enzymatic activity and enhanced tri-methylation of histone H3 at lysine 27, a repressive chromatin modification. As an initial step toward screening patients for cancer genotype-directed therapy, we developed a screening assay for EZH2 codon 641 mutations amenable for testing formalin-fixed clinical specimens, based on the sensitive SNaPshot single nucleotide extension technology. We detected EZH2 mutations in 12/55 (22%) follicular lymphomas (FL), 5/35 (14%) diffuse large B cell lymphomas with a germinal center immunophenotype (GCB-DLBCL), and 2/11 (18%) high grade B cell lymphomas with concurrent rearrangements of BCL2 and MYC. No EZH2 mutations were detected in cases of Burkitt lymphoma (0/23). EZH2 mutations were frequently associated with the presence of BCL2 rearrangement (BCL2-R) in both the FL (28% of BCL-R cases versus 0% of BCL2-WT cases, p<0.05) and GCB-DLBCL groups (33% of BCL2-R cases versus 4% of BCL2-WT cases, p<0.04), and across all lymphoma types excluding BL (27% of BCL2-R cases versus 3% of BCL2-WT cases, p<0.003). We confirmed gain-of-function activity for all previously reported EZH2 codon 641 mutation variants. Our findings suggest that EZH2 mutations constitute an additional genetic "hit" in many BCL2-rearranged germinal center B cell lymphomas. Our work may be helpful in the selection of lymphoma patients for future trials of pharmacologic agents targeting EZH2 and EZH2-regulated pathways.