CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis

Abstract

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

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Fig. 1: Location of proliferating and differentiating pre-B cells in the BM.
Fig. 2: Small pre-B cells are in intimate contact with CXCL12+ stroma.
Fig. 3: Cxcr4 is required for the development of small pre-B cells.
Fig. 4: Pre-B cell differentiation is directly regulated by CXCR4 signaling.
Fig. 5: CXCR4 signaling determines small pre-B cell identity.
Fig. 6: CXCR4 signaling sets the small pre-B cell epigenetic landscape.
Fig. 7: CXCR4 signaling is necessary for Igk recombination.
Fig. 8: ERK signaling downstream of CXCR4 enables pre-B cell differentiation.

Data availability

The sequences, ATAC-Seq data for WT small pre-B cells, mb1-Cre+Cxcr4fl/fl (Cxcr4 knockout) small pre-B cells, +IL-7, −IL-7 and −IL-7+CXCL12 cultured WT pre-B cells, and −IL-7 and −IL-7+CXCL12 cultured Cxcr4−/− small pre-B cells, as well as all of the corresponding RNA-Seq data, including for +IL-7+CXCL12 and −IL-7+CXCL12+ERK-i cultured pre-B cells, are deposited in the GenBank database (accession number GSE129311).

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Acknowledgements

We thank M. Olson, R. Ladd and D. Leclerc for cell-sorting services, and the ImmGen Consortium for data assembly. We thank H. Singh (University of Pittsburgh) for providing the PAX5 and FOXO1 ChIP-Seq data, and M. Schlissel (University of Michigan) for providing the -YFP reporter mice. This work is supported by US National Institutes of Health grants AI120715, AI128785 and AI143778 (to M.R.C.), AI120715-02 and AI128785-01A1 (to M.M.), F32AI143120 (to D.E.K.), T32GM007281 (to K.C.M.), UL1TR002003 (to M.M.-C.) and T32HD007009 (to M.K.O.). Part of the bioinformatics analysis was performed by the UIC Research Informatics Core, supported in part by NCATS through grant UL1TR002003.

Author information

M.M. and M.R.C. designed the experiments. M.M. carried out and analyzed most of the experiments. M.M. and M.M.-C. analyzed the high-throughput sequencing data. M.K.O and D.E.K. assisted with the ATAC-Seq, RNA-Seq and ChIP-Seq experiments, data analyses and flow cytometry. M.V. and K.C.M. performed some sorting and real-time PCR. J.I. and N.K. assisted with the confocal microscopy. I.A. helped to design some of the experiments. F.G. provided the RaDR-GFP mice. M.M. and M.R.C. oversaw the entire project and wrote the final manuscript.

Correspondence to Malay Mandal or Marcus R. Clark.

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Peer review information: Laurie Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Mandal, M., Okoreeh, M.K., Kennedy, D.E. et al. CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis. Nat Immunol 20, 1393–1403 (2019). https://doi.org/10.1038/s41590-019-0468-0

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