Transcriptional analysis of the B cell germinal center reaction

Abstract

The germinal center (GC) reaction is crucial for T cell-dependent immune responses and is targeted by B cell lymphomagenesis. Here we analyzed the transcriptional changes that occur in B cells during GC transit (naive B cells --> centroblasts --> centrocytes --> memory B cells) by gene expression profiling. Naive B cells, characterized by the expression of cell cycle-inhibitory and antiapoptotic genes, become centroblasts by inducing an atypical proliferation program lacking c-Myc expression, switching to a proapoptotic program, and down-regulating cytokine, chemokine, and adhesion receptors. The transition from GC to memory cells is characterized by a return to a phenotype similar to that of naive cells except for an apoptotic program primed for both death and survival and for changes in the expression of cell surface receptors including IL-2 receptor beta. These results provide insights into the dynamics of the GC reaction and represent the basis for the analysis of B cell malignancies.

Figure 1

Isolation of tonsillar B cell subpopulations by magnetic cell separation. Tonsillar naïve B cells (IgD⁺, CD27⁻, CD38^low), GC CB (CD38^high, CD77⁺), GC CC (CD38^high, CD77⁻), and memory B cells (CD27⁺, CD38^low), were isolated by magnetic cell separation (see Methods). Flow cytometric analyses of representative isolations, tonsillar MCs before separation, and purified fractions are shown stained for CD38 and IgD, CD77, and CD27. Separation steps are summarized on the right.

Figure 2

Supervised analysis of changes in gene expression during the GC transit of B cell subpopulations. The genes identified in the four individual transitions (Fig. 6) by supervised pattern discovery using GENES@WORK were merged to visualize their expression changes during GC transit. Color changes within a row indicate expression levels relative to the average of the sample population. Values are quantified by the scale bar that visualizes the difference in the z_ge score (expression difference/standard deviation) relative to the mean (0). Genes are ranked according to their z_g score (mean expression difference of the respective gene between phenotype and control group/standard deviation). Shown are only those gene segments that differ 3-fold or more in their z_g score. The gene expression changes among the subsets are shown according to functional or operational categories; for additional categories see Fig. 7.

Figure 3

Analysis of c-Myc expression in normal and malignant B cells. (A) c-Myc mRNA expression in normal B cells and Burkitt lymphoma (BL), diffuse large B cell lymphoma (DLCL), and Epstein–Barr virus-transformed lymphoblastoid cell lines (LCL). c-Myc was represented by two probe sets on the U96A chip. Bcl2 and Bcl6, as well as the proliferation-associated genes PCNA and Ki67, are shown along with c-Myc as markers for the identity of the purified cell populations. The matrix is as in Fig. 2. (B) Immunohistochemical analysis of a tonsillar section for c-Myc (blue) and the B cell marker CD20 (red). Tonsillar epithelium, “marginal zone,” and GC light zone (LZ) and dark zone (DZ) are indicated (magnification ×10). (Insets) A higher magnification (×40) of the epithelium and the GC DZ, respectively. Note the expression of c-Myc in the nuclei of epithelial cells and the lack of expression in DZ CBs.

Figure 4

Immunohistochemical analysis of tonsillar tissue sections for IL-2Rβ. GCs, GC dark zone (DZ) and light zone (LZ), mantle zone, T cell zone, and tonsillar subepithelium are indicated (magnification ×10). Pax-5 (red), a B cell marker, and IL-2Rβ (blue). (Insets) A higher magnification (×40) of the GC LZ and the subepithelium, respectively. Arrows indicate Pax-5/IL-2Rβ double-positive cells.