Stroma-Driven Notch2 Signaling Controls Naïve B Cell Fate By Regulating Microenvironmental Positioning within the Spleen

Abstract

Marginal zone (MZ) B cells are naïve innate-like B cells that function as a first line of defense in the spleen within the blood-rich marginal zone. Previous research revealed a critical role for Notch2 receptors in B cells and Delta-like1 (Dll1) ligands in Ccl19-Cre + fibroblastic stromal cells to support the development and maintenance of MZ B cells in mice. Yet, little is known about how MZ B cells integrate extracellular signals and Notch-regulated transcriptional programs to support their positioning and function. To understand the basis of Dll1/Notch2's specificity, we used monoclonal antibodies to acutely block Notch2 receptors vs. Dll1 or Dll4 Notch ligands in vivo. Dll1 and Notch2 blockade, but not Dll4 inhibition, rapidly decreased MZ B cell numbers and Notch2-regulated transcription in B cells. Analysis of fluorescent reporter alleles revealed co-expression of Ccl19-Cre;ROSA26 YFP and Dll1-mCherry or Dll4-mCherry within white pulp follicles, showing that both Delta-like ligands are available in fibroblastic stromal cells, although with differential abundance and spatial distribution. To determine if the reliance of MZ B cells on Dll1 was due to ligand availability in the appropriate stromal niche, we inactivated endogenous Dll1 and Dll4 via Ccl19-Cre-mediated recombination, and restored Dll1 or Dll4 expression selectively via separate Cre-inducible expression alleles in the Hprt locus. Dll4 could not sustain Notch2-dependent MZ B cells even when expressed in the correct stromal niche, suggesting that Dll1/Notch2 interactions have unique biochemical and functional properties. As MZ B cells are known to shuttle between the follicle and the MZ across the marginal sinus, we assessed where Dll1/Notch2-mediated signals are delivered to MZ B cells. High abundance of intracellular Notch2 and expression of the Notch target gene, Hes1 (as revealed via a Hes1-GFP reporter) was apparent in CD1d high MZ B cells across the marginal sinus, including prominent signaling within B cell follicles (and not only in the MZ) (Figure 1A). We next evaluated the transcriptional programs controlled by Notch in MZ B cells. Notch2/Dll1-regulated genes included both Myc-dependent and Myc-independent cohorts, with the latter highly enriched for integrin and chemotactic receptor genes including S1pr1, shown previously to guide B cells towards the MZ across the marginal sinus. Myc itself was dispensable for MZ B cell positioning and retention in the spleen. Without S1pr1, B cells experienced Notch signaling within B cell follicles even without entering the MZ. Unlike wild-type B cells, mislocalized S1pr1-deficient MZ-like B cells were retained in the spleen upon Notch deprivation (Figure 1B). These findings identify splenic B cell follicles and not the MZ as a central hub for stroma-driven Dll1/Notch2 signaling, with Notch empowering subsequent B cell migration and positioning to the MZ through a Myc-independent transcriptional program. As many Notch-regulated transcriptional targets are conserved in human Notch-driven B cell lymphomas, we speculate that principles of stroma-driven Notch signaling and its downstream effects in B cells have been conserved during evolution from mouse to human B cells - with the Notch signature tagging the B cell subsets that rely on conserved Notch programs and the B cell malignancies that hijack them.