Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells
Daniela Gómez Atria, Brian T. Gaudette, Jennifer Londregan, Samantha Kelly, Eric Perkey, Anneka Allman, Bhaskar Srivastava, Ute Koch, Freddy Radtke, Burkhard Ludewig, Christian W. Siebel, Russell J.H. Ryan, Tanner F. Robertson, Janis K. Burkhardt, Warren S. Pear, David Allman, Ivan Maillard
Daniela Gómez Atria, Brian T. Gaudette, Jennifer Londregan, Samantha Kelly, Eric Perkey, Anneka Allman, Bhaskar Srivastava, Ute Koch, Freddy Radtke, Burkhard Ludewig, Christian W. Siebel, Russell J.H. Ryan, Tanner F. Robertson, Janis K. Burkhardt, Warren S. Pear, David Allman, Ivan Maillard
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Research Article Immunology

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

Abstract

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2–/– mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone–like B cells when transferred into Rag2–/– lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Authors

Daniela Gómez Atria, Brian T. Gaudette, Jennifer Londregan, Samantha Kelly, Eric Perkey, Anneka Allman, Bhaskar Srivastava, Ute Koch, Freddy Radtke, Burkhard Ludewig, Christian W. Siebel, Russell J.H. Ryan, Tanner F. Robertson, Janis K. Burkhardt, Warren S. Pear, David Allman, Ivan Maillard

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Figure 5

Follicular B cells adoptively transferred into a lymphopenic environment attain a full marginal zone B cell transcriptome and upregulate a broad Notch signature.

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Follicular B cells adoptively transferred into a lymphopenic environment...
Congenically marked B6-CD45.1 FoB cells were labeled with CTV and i.v. transferred to either B6 or Rag2–/– hosts. RNA-Seq was then performed on B cells twice-sorted from animals euthanized after 4 and 8 days, sorting first on CD45.1/2 and second on follicular or marginal zone B cell gates (d8) or intermediate gates (d4) (see Supplemental Figure 2). (A) Principal component analysis of all samples with colors indicating transfer groups and times and shapes indicating cell-type sort gates. (B) Expression of a MZB/FoB signature — defined as genes differentially expressed between host B6 MZB and FoB cells (adjusted P value < 0.01, log2 fold change > 2) — is shown for all samples as the z score across each row. Samples in columns are hierarchically clustered by Spearman’s correlation. (C) Venn diagrams displaying differential gene testing results of the indicated comparisons. Shown are the number of genes differential (adjusted P value < 0.01, log2 fold change > 2) without respect to direction. (D) Volcano plots indicating the magnitude and significance of gene expression changes between indicated groups for all (gray), and highlighted (black/red) empirically defined MZB cell Notch2-regulated genes (genes significantly downregulated in MZB cells after 24 hours of anti-N2 antibody blockade) (28). Red indicates significance (adjusted P < 0.05; log2 fold change > 1). (B–D) Differential expression was calculated by empirical Bayes method with Benjamini-Hochberg correction with indicated cutoffs. (E and F) Log2 transcripts per million (TPM) are shown for indicated genes as 2-gene correlation (E) and selected individual genes (F). Adoptive transfer groups are shown by color, and sort gate is indicated by shape.