Disruption of the Bcl6 gene results in an impaired germinal center formation

Abstract

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6(-/-)RM). Lymphogenesis in primary lymphoid tissues of Bcl6(-/-)RM is normal, and Bcl6(-/-)RM produced control levels of primary IgG1 antibodies specific to T cell-dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.

Figure 1

Bcl6 expression in spleen from immunized C57BL/6. Spleen sections from C57BL/6 mice on day 14 (A and B) or day 7 (C and D) after immunization were stained with anti-Bcl6 Ab (A and B) or anti-Bcl6 and anti-λ light chain Abs (C and D). Brown signals in nuclei and membranous blue signals indicate Bcl6 protein and λ light chain, respectively. Sections were counterstained with hematoxylin (A and B). Arrows and arrowheads indicate centroblasts and centrocytes respectively.

Figure 2

Normal development of B and T cells in Bcl6^−/−RM. Representative flow cytometric analysis of BM cells (A), thymocytes (B), and splenocytes (C and D) from Bcl6^+/+RM (left column) and Bcl6^−/−RM (right column). Numbers indicate relative percentages of positive cells within a quadrant.

Figure 3

Control levels of primary Abs in sera from immunized Bcl6^−/−RM. Bcl6^−/−RM (closed circles) and Bcl6^+/+RM (open circles) were immunized with DNP–OVA. IgM and IgG1 anti-DNP titers in the sera were measured by ELISA.

Figure 4

GC formation is impaired in spleen from Bcl6^−/−RM. Splenocytes from Bcl6^+/+RM (A) and Bcl6^−/−RM (B) immunized with DNP–OVA on day 14 after immunization were stained with PNA and anti-B220 Ab. The numbers in the figures indicate percentages of PNA^high B220⁺ cells relative to total B220⁺ cells. Splenic sections from the immunized Bcl6^+/+RM (C and E) and Bcl6^−/−RM (D and F) were stained with PNA (C and D) or anti-B220 Ab (E and F) (brown). Hematoxylin counterstain. G, germinal center; T, T cell–rich PALS; M, marginal zone; F, primary follicle.

Figure 4

GC formation is impaired in spleen from Bcl6^−/−RM. Splenocytes from Bcl6^+/+RM (A) and Bcl6^−/−RM (B) immunized with DNP–OVA on day 14 after immunization were stained with PNA and anti-B220 Ab. The numbers in the figures indicate percentages of PNA^high B220⁺ cells relative to total B220⁺ cells. Splenic sections from the immunized Bcl6^+/+RM (C and E) and Bcl6^−/−RM (D and F) were stained with PNA (C and D) or anti-B220 Ab (E and F) (brown). Hematoxylin counterstain. G, germinal center; T, T cell–rich PALS; M, marginal zone; F, primary follicle.

Figure 5

PALS-associated focus formation is not impaired in spleen from Bcl6^−/−RM. Splenic sections from Bcl6^+/+RM (A) and Bcl6^−/−RM (B) immunized with NP–CG on day 7 after immunization were stained with anti-λ light chain Abs (blue) and PNA (brown).

Figure 6

B cells are responsible for the defect of GC formation in Bcl6^−/− RM. Spleen sections from the DNP–OVA immunized RAG1^−/− reconstituted with B cells from Bcl6^+/+ RM and T cells from Bcl6^−/− RM (A), or with B cells from Bcl6^−/−RM and T cells from Bcl6^+/+RM (B) were stained with PNA (brown). Hematoxylin counterstain.

Figure 7

Nascent GC is absent in spleen from Bcl6^−/−RM. Spleen sections from Bcl6^+/+RM (A) and Bcl6^−/−RM (B) on day 3 after immunization were stained with anti-BrdU Ab and PNA. Strong blue signals in the nuclei and membranous weak blue signals indicate BrdU uptake and cell surface Igs, respectively. Brown signals in follicle of Bcl6^+/+RM indicate PNA-reactive GC cells.

Figure 8

Proliferation capacity of splenic B cells from Bcl6^−/−RM. Proliferative responses of splenic B cells from Bcl6^+/+RM (shaded bars) and Bcl6^−/−RM (closed bars) to indicated stimuli. Results represent means and variations (SD) from triplicate cultures. The data presented are representative of three independent experiments.