Stromal Notch Ligands Drive Notch2-Dependent Transdifferentiation of Follicular B Cells into Marginal Zone-like B Cells in Lymphopenic Environments
Authors: Gómez Atria Daniela , Gaudette Brian T. , Perkey Eric , Londregan Jennifer , Kelly Samantha , Ludewig Burkhard , Siebel Chris , Ryan Russell J. H. , Pear Warren S. , Allman David , Maillard Ivan 
Published: February 2021

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting homeostatic proliferation of mature lymphocytes. Although this process operates in multiple clinically relevant settings, including after bone marrow transplantation and chemotherapy as well as in aging individuals, little is known about its underlying molecular mechanisms and functional consequences. In mice, mature naïve B cells include mostly follicular B (FoB) cells in spleen and lymph nodes, as well as specialized marginal zone B (MZB) cells in the spleen with innate-like functions and a capacity for rapid plasma cell differentiation. To identify mechanisms controlling the size and composition of the peripheral B cell pool, we developed a mouse model in which highly purified CTV-labelled B6-CD45.1 FoB cells were adoptively transferred intravenously to lymphopenic B6-CD45.2 Rag2-/- mice, lacking mature B and T cells, or control B6-CD45.2 lymphoid-replete recipients. Within two days after transfer of CD19+ CD93neg CD21int CD23high B6-CD45.1 FoB cells (106, &gt;99% purity), CD45.1+ donor-derived B cells had upregulated expression of surface IgM (sIgM) and CD21 in the spleen of Rag2-/- recipients, but not in wild type control recipients. At day 4 and day 8, the majority of transferred B cells remained sIgMhi and CD21hi in Rag2-/- recipients and gradually showed decreased CD23 and sIgD as well as increased CD1d expression, consistent with loss of their FoB phenotype and acquisition of a characteristic MZB cell phenotype (sIgMhisIgDlow CD21hi CD1dhi CD23low). These phenotypic changes were followed by a burst of proliferation (CTV dilution) between day 4 and day 8 after transfer. Immunofluorescence microscopy of host Rag2-/- spleen sections at day 2 post-transfer identified clusters of transferred B cells localized around CD169+ macrophages at the white pulp/red pulp interface close to the marginal sinus, with subsequent proliferation in this area. We next investigated if Notch signaling regulates this transdifferentiation process, by analogy to its role in normal MZB cell homeostasis. Treatment of Rag2-/- recipients with blocking antibodies against Delta-like-1 Notch ligands (anti-DLL1) or Notch2 receptors (anti-NRR2) completely inhibited lymphopenia-induced FoB to MZB cell conversion and proliferation. To identify the cellular source of DLL1 Notch ligands, we studied transferred B cells in Rag2-/-;Ccl19-Cre+;Dll1f/f recipients, lacking Dll1 expression in all Ccl19-Cre+ fibroblastic reticular cells and their progeny. In these mice as compared to Rag2-/- recipients, FoB to MZB transdifferentiation was almost completely abrogated and transferred cells no longer clustered with marginal sinus-associated macrophages. Thus, stromal DLL1 Notch ligands are critical to regulate the size and composition of the splenic peripheral B cell pool in lymphopenic mice through DLL1/Notch2 interactions. Furthermore, FoB cells are not locked in their FoB cell fate, as commonly assumed, but are instead endowed with plastic transdifferentiation potential in response to DLL1/Notch2-mediated signals that function as a sensor of B cell lymphopenia. We speculate that these adaptive physiological functions of the Notch signaling pathway play an important role in the homeostasis of mature B cells within their stromal microenvironment, and that they can be hijacked during malignant transfomation in Notch-dependent mature B cell lymphomas such as CLL and marginal zone lymphoma.

Disclosures
Siebel: Genentech: Current Employment. Maillard:Allogene: Consultancy; Regeneron: Consultancy; Genentech: Consultancy.