B Cell Development and the Splenic Microenvironment in Dlk-1 Deficient Mice

• Published in: Blood 2012-11, Vol.120 (21), p.1251-1251
• Main Authors: Degheidy, Heba A, Branchaw, Allison L, Bauer, Lucy C, Bauer, Steven R
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.1251.1251

Abstract 1251 DLK-1 is a transmembrane and secreted protein that plays a crucial role in normal B cell development and differentiation. In a previous study we showed that DLK-1 knockout mice (Dlk1−/− mice (KO)) have distinct differences in B cell fractions in the spleen and bone marrow compared to wild-type Dlk1+/+ (WT) mice. KO mice showed a decrease in follicular (FO) B cells and an increase in the size of the marginal zone (MZ) and number of MZ B cells in the spleen of 8 week old mice. Furthermore, there was an exaggerated primary T-dependent antigen-specific humoral immune response. The mechanisms underlying the changes in splenic B cell fractions between KO and WT mice are not yet clear. It has been suggested that stromal microenvironmental cells form distinct cellular niches that influence different stages of B cell development. Alterations in these stromal niches due to absence of DLK-1 may be an underlying cause of the observed splenic B cell fraction alterations. It was previously shown that Galectin-1 (GAL1) plays an important role in the bone marrow microenvironment and affects proliferation and differentiation of normal mouse pre-BII cells (Blood April 21, 2011). This study is designed to investigate the splenic stromal cells in DLK-1 deficient mice as a step toward understanding these B cell alterations, and to test whether the stromal cell derived-GAL1 influences splenic B-cell development. For detection of B cell fractions, a multicolor flow cytometry panel consisting of anti-IgD/IgM/CD23/CD93/CD45R/CD21/CD35 was used. B cell fractions were identified as follows: MZ (CD23neg-low, CD21high), FO (CD23high, CD21intermediate), Tr1 (CD23neg, CD21neg, AA4.1pos, B220pos), Tr2 (CD23pos, AA4.1pos, B220pos). For detection of stromal cell fractions, an anti-CD11c/Ter119/CD19/Gr-1/Tie-2/CD45/CD31/CD117/CD34 panel was used. Stromal cells were identified as follows: CD45neg, lineage− (Ter119, CD19, GR1, CD11c, and CD34), CD117neg, CD34neg, Tie2neg. Two stromal cell fractions were detected: CD31 +and CD31−. Galectin-1 expression was evaluated on CD31+ and CD31−stroma cell fractions. An eight color flow cytometric panel consisting of antibody directed to MHC II, CD11c, Gr-1, CD8a, B220, and CD11b was used to evaluate myeloid, lymphoid and plasmacytoid dendritic cell fractions. Our data showed an increase in MZ B-cells (p