B Cell Development and the Splenic Microenvironment in Dlk-1 Deficient Mice
Authors: Degheidy Heba A , Branchaw Allison L , Bauer Lucy C , Bauer Steven R 
Published: October 2019

Abstract
Abstract 1251

Background:
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.

Methods:
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.

Results:
Our data showed an increase in MZ B-cells (p<0.005) and a decrease in FO B cells (p<0.005) in the KO mice. When looking at the splenic stromal cells, we found an increase in the CD31+ fraction and a decrease in the CD31- fraction in KO mice compared to WT mice (p<0.005). We observed that the CD31 + stromal cell expressed high galectin-1 (GAL1) levels compared to CD31− stroma cells. Also there was an increased percentage of myeloid dendritic cells in KO compared to WT (p< 0.05).

Conclusion:
We have shown that absence of DLK-1 leads to alterations in splenic stromal populations and that these may play an important role in altered B cell populations and function observed in DLK-1-deficient mice. We are investigating whether the CD31+ GAL1 +stroma cell is a key stroma cell that plays a critical role in splenic B cell development and is responsible for the B-lineage differences seen in DLK-1-deficient mice. Our observation that there is an increase in the myeloid dendritic cell fraction may explain the exaggerated primary immune response seen in the KO mice. These data show that DLK-1-deficiency leads to several changes in the splenic cellular microenvironment and suggest that these changes contribute to alterations in B-cell development and function.

Disclosures:
No relevant conflicts of interest to declare.