-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

2549 Stroma-Driven Notch2 Signaling Controls Naïve B Cell Fate By Regulating Microenvironmental Positioning within the Spleen

Program: Oral and Poster Abstracts
Session: 203. Lymphocytes and Acquired or Congenital Immunodeficiency Disorders: Poster II
Hematology Disease Topics & Pathways:
Fundamental Science, Research, non-Hodgkin lymphoma, Lymphomas, B Cell lymphoma, Diseases, immune mechanism, immunology, Lymphoid Malignancies, Biological Processes
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Anneka Allman1*, Brian Gaudette, PhD1*, Samantha Kelly1*, Nagham Alouche, PharmD, PhD2*, Leolene Carrington, PhD1*, Eric Perkey, MD, PhD3*, Riley Outen4*, Ashley Vanderbeck1*, Katlyn Lederer1*, Tanner F. Robertson, PhD5*, Janis K. Burkhardt5*, Anastasia N. Tikhonova, PhD6, Iannis Aifantis, PhD7, Ute Koch, PhD8*, Freddy Radtke, PhD9*, Burkhard Ludewig, DVM10*, Lena Tveriakhina, PhD11*, Achim Gossler, PhD12*, Christian W. Siebel, PhD13*, Daniela Gomez Atria, PhD1*, Sanjiv Luther, PhD2*, David Allman, PhD1* and Ivan Maillard14

1University of Pennsylvania, Philadelphia, PA
2University of Lausanne, Epalinges, Switzerland
3University of Michigan, Ann Arbor, MI
4University of Pennsylvania, Philadelphia
5Children's Hospital of Philadelphia, Philadelphia, PA
6Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada
7Department of Pathology, NYU Grossman School of Medicine, New York, NY
8EPFL SV ISREC, Lausanne, Switzerland
9Ecole Polytechnique Fédlrale De Lausanne (EPFL), Lausanne, Switzerland
10Cantonal Hospital St.Gallen, St Gallen, Switzerland
11Harvard Medical School, Boston, MA
12Hannover Medical School, Hannover, Germany
13Genentech, South San Francisco, CA
14Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania, Philadelphia, PA

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;ROSA26YFP 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 CD1dhigh 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.

Disclosures: Carrington: Interius BioTherapeutics: Current Employment. Ludewig: Stromal Therapeutics: Membership on an entity's Board of Directors or advisory committees. Siebel: Genentech: Current Employment. Gomez Atria: GSK: Current Employment. Maillard: Regeneron: Research Funding; Genentech: Research Funding; Garuda Therapeutics: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH