Resting innate-like B cells leverage sustained Notch2/mTORC1 signaling to achieve rapid and mitosis-independent plasma cell differentiation

Abstract

Little is known about how cells regulate and integrate distinct biosynthetic pathways governing differentiation and cell division. For B-lineage cells it is widely accepted that activated cells must complete several rounds of mitosis before yielding antibody-secreting plasma cells. However, we report that marginal zone (MZ) B cells, innate-like naïve B cells known to generate plasma cells rapidly in response to blood-borne bacteria, generate functional plasma cells despite cell cycle arrest. Further, short-term Notch2 blockade in vivo reversed division-independent differentiation potential and decreased transcript abundance for numerous mTORC1-and Myc-regulated genes. Myc loss compromised plasma cell differentiation for MZ B cells, and reciprocally induced ectopic mTORC1 signaling in follicular B cells enabled division-independent differentiation and plasma cell-affiliated gene expression. We conclude that ongoing in situ Notch2/mTORC1 signaling in MZ B cells establishes a unique cellular state that enables rapid division-independent plasma cell differentiation.

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells

“…Next, we studied the ability of donor-derived B cells recovered from Rag2 -/recipients to rapidly produce antibodies when stimulated ex vivo with CpG only (a response mimicking T cell-independent differentiation and a key functional property of MZB but not FoB cells). Indeed, MZB cells are characterized by a preactivated state and by their capacity to produce plasma cells more rapidly than FoB cells, with reduced requirements for prior division and for exposure to cytokines (28,34). In ELISpot assays, CpG stimulation of CD45.1 + B cells recovered at day 8 from Rag2 -/recipients induced a high frequency of antibody-secreting cells, similar to that observed with normal MZB cells and much higher than with FoB cells (Figure 3E).…”

“…The effects of Notch signaling on MZB cells are exquisitely dosage-sensitive, as shown by haploinsufficiency of multiple critical genes in the pathway (22)(23)(24)(25)(26). Emerging evidence indicates that Notch signaling is not only required for the development of MZB cells, but that continuous Notch signals are essential for their maintenance and function (27,28). Furthermore, induced expression of a gain-of-function Notch allele in FoB cells drove their differentiation to a MZB cell fate, showing that plastic changes in differentiation can be driven by Notch, at least under artificial conditions (29).…”

“…We next assessed functionally whether FoB transdifferentiation into MZ-like B cells and their subsequent proliferation in lymphopenic environments required Notch signaling. We transferred FoB cells into B6 or Rag2 -/recipients treated either with an isotype control or an anti-Notch2 antibody, which efficiently blocks ligand-induced cleavage and activation of Notch2 receptors in vivo (28,36,37). In Rag2 -/hosts that received anti-Notch2 as compared with control antibodies, phenotypic changes associated with transdifferentiation of FoB cells into MZ-like B cells were completely abrogated (Figure 6, A-D).…”

“…We then investigated whether donor-derived B cells displayed evidence of Notch signaling, as well as the kinetics and extent of Notch-regulated gene expression in the presence or absence of lymphopenia (Figure 5, D and E, and Supplemental Figure 2C). Normal MZB cells depend on Notch2-mediated signals for their development as well as for their maintenance and function (22,27,28). We used a gene signature that we previously identified as downregulated in steady-state MZB cells after 24 hours of in vivo antibody-mediated Notch2 inhibition, in comparison with that of B6 FoB and MZB cells (28).…”

“…Normal MZB cells depend on Notch2-mediated signals for their development as well as for their maintenance and function (22,27,28). We used a gene signature that we previously identified as downregulated in steady-state MZB cells after 24 hours of in vivo antibody-mediated Notch2 inhibition, in comparison with that of B6 FoB and MZB cells (28). Donor-derived MZB cells sorted from Rag2 -/recipients displayed upregulation of Notch-regulated gene expression, and the degree of upregulation was even larger than observed in normal B6 MZB cells as compared with FoB cells (suggesting the occurrence of intense Notch signals in B cells exposed to lymphopenia).…”