PF462 SPECIAL AT‐RICH SEQUENCE BINDING PROTEIN 1 (SATB1) IS MARKEDLY EXPRESSED IN THE DARK ZONE GERMINAL CENTER B CELLS ON MURINE SPLEEN

Abstract

Background:Special AT‐rich Sequence Binding Protein 1 (SATB1) is known as a protein that binds to the AT‐rich region near the enhancer of various genes. SATB1 is expressed predominantly in the thymus, and controls T lineage‐specific gene transcription by organizing chromatin configurations in the cell nucleus. In addition, we previously reported that SATB1 plays important roles in the early differentiation of hematopoietic stem cells (HSCs) (Immunity, 2013; Cell Reports, 2018). Indeed, exogenous induction of SATB1 in HSCs enhances both T and B lymphopoietic potentials, whereas SATB1 deficiency skews HSC differentiation toward myeloid lineage at the expense of lymphocyte production. Furthermore, SATB1 also associates with the enhancer of immunoglobulin heavy chain gene. Thus, SATB1 plays a pivotal role in various aspects of lymphopoiesis, but its significance in the B lineage development has yet to be determined.Aims:We performed this experiment to know the role of SATB1 in the B lineage development.Methods:We used SATB1/Tomato reporter mice to monitor the precise SATB1 expression during B cell maturation in vivo. We also generated B cell‐specific SATB1 conditional knock out mice using Cre‐loxP‐mediated inactivation of SATB1. We crossed SATB1‐flox mice with mice expressing Cre recombinase under the control of the Mb1 gene promotor.Results:At first, we analyzed the B lineage development of SATB1/Tomato reporter mice. In bone marrow (BM), while markedly increasing with early lymphoid differentiation, SATB1/Tomato expression decreased in pro‐B and pre‐B stages and almost diminished in surface IgM+ immature B cells. However, SATB1/Tomato expression was revived after immature B cells migrated to the spleen. The expression of SATB1/Tomato increased with the maturation from T1 to T3 transitional splenic B cells. The up‐regulation of SATB1/Tomato in developing B cells positively correlated with IgD expression. Indeed, while transcripts for SATB1 gene were barely detected in the splenic IgM+IgD− stage, they were actively induced in IgM+IgD+ cells and became abundant in IgM−IgD+ B cells. When we incubated murine splenic B cells with an anti‐IgM antibody in vitro, SATB1/Tomato+ IgD+ population was significantly induced. Incubation of murine splenic B cells with anti‐IgM and anti‐CD40 also induced the same population. These results suggest that B cell receptor signaling induces SATB1 expression along with the transition of IgM to IgD. The acquisition of antigens by naive B cells triggers the formation of germinal centers (GCs). The mature GC is divided into two compartments, called the light zone (LZ) and the dark zone (DZ). The naïve B cells located in LZ receive the positive selection of higher‐affinity B cells by CD4+ helper T cells and migrate to DZ by expressing the DZ‐homing receptor CXCR4. The B cells in DZ are highly proliferative. The analysis of SATB1/Tomato reporter mice showed that SATB1+ IgD+ splenic B cells had a significantly higher expression of CXCR4 than the SATB1− IgD+ cells. In addition, CD83, the activation marker of LZ B cells, decreased in IgD+ B cells with increased expression of SATB1. The above results imply that the SATB1 protein might be involved in the modulation of Ig heavy chain and the formation of DZ in GC during splenic B cell maturation. To test this hypothesis, we analyzed Mb1‐Cre:SATB1 Flox/Flox mice. The histological analysis showed that the follicles of the spleen in Mb1‐Cre: SATB1 Flox/Flox mice are enlarged compared to those in control SATB1 Flox/Flox mice.Summary/Conclusion:With these results, we conclude that SATB1 plays some role in the formation of GCs.