Expression pattern of DNMTs in tonsillar B-cell subsets. (A) Cartoon illustrating the stages of B-cell development and T-dependent B-cell immune response. B cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow and released into the periphery upon maturation. Naive B cells encounter antigen-primed T cells in secondary lymphoid organs and enter the germinal center (GC) reaction followed by differentiation into memory B cells or antibody-secreting plasma cells (PCs). (B) Purification of B-cell subsets. CD20, IgD, and CD38 cell surface expression were used to distinguish the cell populations: Naive B cells (CD20+IgD+CD38lo), GC B cells (CD20+IgD−CD38+), memory B cells (CD20+IgD-CD38−), and PCs (CD20lo/+IgD−CD38hi). The percentage of each subset within tonsillar B cells is shown in the FACS plot in the middle panel. Naive, GC, memory, and PC populations were purified from eight individuals for gene expression and DNA methylation profiling on microarrays. The purity of each cell population (>90%) was confirmed by flow cytometry analysis of post-sorted cells (right panel). (C) Transcript abundance of DNMTs in B-cell subsets by quantitative RT-PCR. DNMT expression levels are normalized to actin expression level in each cell type. The expression level in one biological replicate of naive B cells is arbitrarily set to one. Error bars denote standard deviation of three biological replicates. (D) Expression patterns of DNMT1 and DNMT3A in tonsils. (Upper left, upper right) Immunohistochemical staining of DNMT1 at 200× and 400× magnifications, respectively. (Lower left, lower right) Immunohistochemical staining of DNMT3A at 200× and 400× magnifications, respectively. (E) Up-regulation of DNMT3B in GC B cells. Tonsil sections were costained with anti-DNMT3B (red) and anti-MTA3 (green). Nuclei were labeled with DAPI (blue). DNMT3B expression colocalized with MTA3, which is a marker for GC B cells (Fujita et al. 2004). Scale bar, 50 μm.
