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RBPJ-dependent Notch signaling initiates the T cell program in a subset of thymus-seeding progenitors

Nature Immunology volume 20, pages 1456–1468 (2019)Cite this article

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Abstract

T cell specification and commitment require Notch signaling. Although the requirement for Notch signaling during intrathymic T cell development is known, it is still unclear whether the onset of T cell priming can occur in a prethymic niche and whether RBPJ-dependent Notch signaling has a role during this event. Here, we established an Rbpj-inducible system that allowed temporal and tissue-specific control of the responsiveness to Notch in all hematopoietic cells. Using this system, we found that Notch signaling was required before the early T cell progenitor stage in the thymus. Lymphoid-primed multipotent progenitors in the bone marrow underwent Notch signaling with Rbpj induction, which inhibited development towards the myeloid lineage in thymus-seeding progenitors. Thus, our results indicated that the onset of T cell differentiation occurred in a prethymic setting, and that Notch played an important role during this event.

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Fig. 1: RBPJind mice allow for controlled T cell development.
Fig. 2: Regulation of T lymphopoiesis in RBPJind mice induces thymic architectural changes.
Fig. 3: The appearance of DN2 cells in the RBPJind mouse thymus is delayed upon induction of Notch responsiveness.
Fig. 4: The appearance of RBPJind DN2 cells in wild-type thymus is delayed upon induction of Notch responsiveness.
Fig. 5: Notch signaling is required for the thymic appearance of T lineage-competent ETPs.
Fig. 6: RBPJind-noDox thymic DN1a/b cells are myeloid biased.
Fig. 7: BM LMPPs undergo Notch signaling.
Fig. 8: Notch signaling in BM inhibits myeloid skewing in TSPs.

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Data availability

The data that support the findings of this study are available from the corresponding author on request. Raw and processed RNA-Seq and single-cell RNA-Seq data are available from the Gene Expression Omnibus database under accession number GSE128964.

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Acknowledgements

We are grateful to G. Awong and C. McIntosh for expertise and assistance in flow cytometry and cell sorting, and to C. Lee and L. Wells for assistance with animal care. We are thankful to R. Dickson and T. McGaha for assistance with the single-cell RNA-Seq analysis, and M. K. Anderson for helpful discussions and critical review of the manuscript. This work was supported by grants from the Canadian Institutes of Health Research (MOP-119538 and FND-154332), National Institutes of Health (NIH-1P01AI102853-01) and Krembil Foundation. E.L.Y.C. was supported by an Ontario Graduate Scholarship. P.K.T. was supported by a Banting and Best Doctoral Research Award. J.C.Z.-P. is supported by a Canada Research Chair in Developmental Immunology.

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  1. These authors contributed equally: Edward L. Y. Chen, Patrycja K. Thompson.

Authors and Affiliations

  1. Sunnybrook Research Institute, Toronto, Ontario, Canada

    Edward L. Y. Chen, Patrycja K. Thompson & Juan Carlos Zúñiga-Pflücker

  2. Department of Immunology, University of Toronto, Toronto, Ontario, Canada

    Edward L. Y. Chen, Patrycja K. Thompson & Juan Carlos Zúñiga-Pflücker

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E.L.Y.C. and P.K.T. designed and performed the experiments, analyzed the data, and wrote the manuscript. J.C.Z.-P. designed the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Juan Carlos Zúñiga-Pflücker.

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J.C.Z.-P. is a co-founder of Notch Therapeutics.

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Integrated supplementary information

Supplementary Figure 1 Generation of the RBPJind mouse and regulation of Notch responsiveness in vitro.

(a) Schematic of the RBPJind mouse in which in vivo expression of RBPJ-HA in hematopoietic cells, and thus Notch responsiveness, is dependent on Dox treatment. (b) Western blot analysis detecting HA in TetOS-RBPJ-HA tail fibroblasts that were either untransfected or rtTA transfected, and untreated, treated with Dox for 72 hours, or treated with Dox for 72 hours and Dox removed for 24 hours in vitro as indicated. The black horizontal lines denote cropping of the gels. (c) Flow cytometry analysis for T lineage and B lineage outcomes from RBPJCtr and RBPJind-noDox BM LSKs cultured on OP9-DL1 for 12 days in the presence (+ Dox) or absence (- Dox) of Dox, or for 8 days in the presence of Dox followed by 4 days in the absence of Dox (+ Dox - Dox) as indicated. (d) Flow cytometry analysis for T lineage outcomes from RBPJCtr and RBPJind-noDox BM LSKs cultured on OP9-DL4 or OP9 for 8 days in the presence (+ Dox) or absence (- Dox) of Dox as indicated. Data are representative of three independent experiments (n=3 mice per group).

Supplementary Figure 2 Maintenance of MZB cells requires Notch signaling in RBPJind mice.

(a) Flow cytometry analysis of the splenic phenotype of RBPJCtr-Dox, RBPJind-noDox, RBPJind-Dox6wk and RBPJind-Dox3wk-noDox3wk mice. Left to right: analysis of CD4 and CD8 αβ T cells, γδ T cells (DN gated) and marginal zone B cells (B220+IgM+ gated). DN gated: gated on CD4-CD8-. Data are representative of three independent experiments (n=3 mice per group). (b) Total splenic cellularity of RBPJCtr-Dox, RBPJind-noDox, RBPJind-Dox6wk and RBPJind-Dox3wk-noDox3wk mice showing mean + standard deviation (n=3 mice per group).

Supplementary Figure 3 Kinetics of DN2 differentiation from RBPJCtr ETPs and LMPPs.

(a) Flow cytometry analysis of DN2 kinetics of RBPJCtr thymic ETPs and BM LMPPs cultured on OP9-DL4 with Dox for 1, 2 and 3 days. (b) Flow cytometry analysis of kinetics of RBPJ-HA expression by RBPJind-Dox BM cells and thymocytes following 4, 8 and 24 hours of Dox treatment in mice. Cells from uninduced (RBPJind-noDox) mice and cells from induced (RBPJind-Dox) mice, but that were unstained or stained with secondary antibody only, served as negative controls. Data are representative of three independent experiments (n=2 mice pooled for each experiment in a and n=3 mice per group in b).

Supplementary Figure 4 Gating strategy and purity assessment for isolation of thymic DN1a/b cells.

Thymic DN1a/b cells are defined and were sorted as Lin-CD44+CD25-c-KithiCD24-/lo.

Supplementary Figure 5 RBPJind-noDox thymic DN1a/b cells display strong myeloid potential.

(a) Flow cytometry analysis for myeloid lineage and DC lineage outcomes of RBPJCtr, RBPJind-noDox and RBPJind-Dox6d thymic DN1a/b cells and BM LSKs cultured on OP9-DL1lo with Dox for 8 days. (b) Flow cytometry analysis for plasmacytoid DC (left), and lymphoid and myeloid DC (right) development from thymic DN1a/b cells and BM LSKs from RBPJCtr, RBPJind-noDox and RBPJind-Dox6d mice cultured on OP9-DL1lo with Dox for 8 days. Data are representative of three independent experiments (n=2 mice pooled for RBPJCtr and n=8 mice pooled for RBPJind-noDox and RBPJind-Dox6d for each experiment).

Supplementary Figure 6 Gating strategy for identification of BM progenitors and T lineage differentiation from previously Notch unresponsive LMPPs.

(a) HSCs are defined as Lin-Sca-1+c-Kit+Flt-3-, MPPs as Lin-Sca-1+c-Kit+Flt-3lo, LMPPs as Lin-Sca-1+c-Kit+Flt-3hi and CLPs as Lin-Sca-1loc-KitloFlt-3hiCD127+. (b) Flow cytometry analysis for T cell potential of RBPJCtr, RBPJind-noDox and RBPJind-Dox6d LMPPs cultured on OP9-DL4 with Dox for 8 days, and B cell potential of RBPJCtr, RBPJind-noDox and RBPJind-Dox6d LMPPs cultured on OP9 for 14 days. (c) Flow cytometry analysis for DN2 development from RBPJCtr, RBPJind-noDox and RBPJind-Dox6d LMPPs and CLPs cultured on OP9-DL4 with Dox for 3 days. Data are representative of three independent experiments (n=2 mice pooled for each group for each experiment in b and n=3 mice pooled for each group for each experiment in c).

Supplementary Figure 7 Limiting dilution analysis of T cell potential from RBPJind BM progenitors.

(a) Representative flow plots of DN2/DN3 positive single-cell wells from BM CD62L+ LMPPs and CLPs from RBPJCtr and RBPJind-noDox mice. Cells were placed on OP9-DL4 with Dox and analyzed 10 days after. (b) Calculation of the T cell progenitor frequency (with the 95% upper and lower confidence limit) in BM CD62L+ LMPPs and CLPs from RBPJCtr and RBPJind-noDox mice. Data are from one independent experiment (n=2 mice pooled for each group).

Supplementary Figure 8 BM LMPP subsets expressing thymus-homing genes undergo Notch signaling.

(a) Dot plot analysis of Hes1 expression in total LMPPs, Sell+ LMPPs, Selplg+ LMPPs, Ccr9+ LMPPs and Ccr7+ LMPPs from RBPJCtr mice (n=2614, 1038, 1727, 158 and 100 cells, respectively), RBPJind-noDox mice (n=2729, 760, 1541, 160 and 68 cells, respectively), RBPJind-Dox3d mice (n=1268, 412, 749, 63 and 38 cells, respectively) and RBPJind-Dox6d mice (n=2074, 708, 1280, 159 and 78 cells, respectively). For Hes1 gene expression fold-change values, see Supplementary Table 11. (b) t-SNE analysis showing cluster location of Sell+Selplg+Ccr9+Ccr7+ LMPPs from RBPJCtr mice (n=4 cells), RBPJind-Dox3d mice (n=2 cells) and RBPJind-Dox6d mice (n=4 cells). Data are from one independent experiment (n=3 mice pooled for each group).

Supplementary information

Supplementary Information

Supplementary Figs. 1–8.

Reporting Summary

Supplementary Table 1

Comparison of the genes enriched in RBPJCtr and RBPJind-noDox thymic DN1a/b cells.

Supplementary Table 2

Comparison of the genes enriched in RBPJCtr and RBPJind-Dox6d thymic DN1a/b cells.

Supplementary Table 3

Comparison of the genes enriched in RBPJind-noDox and RBPJind-Dox6d thymic DN1a/b cells.

Supplementary Table 4

Comparison of the biological pathways involving genes enriched in RBPJCtr and RBPJind-noDox thymic DN1a/b cells.

Supplementary Table 5

Genes enriched in BM LMPP cluster 3.

Supplementary Table 6

Genes enriched in BM LMPP cluster 4.

Supplementary Table 7

Genes enriched in BM LMPP cluster 5.

Supplementary Table 8

Genes enriched in BM LMPP cluster 6.

Supplementary Table 9

Genes enriched in BM LMPP cluster 7.

Supplementary Table 10

Genes enriched in BM LMPP cluster 8.

Supplementary Table 11

Hes1 expression in RBPJCtr, RBPJind-noDox and RBPJind-Dox6d BM LMPP subsets.

Supplementary Table 12

Cluster 5 distribution of RBPJCtr, RBPJind-noDox and RBPJind-Dox6d Sell+Selplg+Ccr9+ BM LMPPs.

Supplementary Table 13

Comparison of the genes enriched in RBPJCtr and RBPJind-noDox Sell+Selplg+Ccr9+ BM LMPPs.

Supplementary Table 14

Comparison of the genes enriched in RBPJCtr and RBPJind-Dox6d Sell+Selplg+Ccr9+ BM LMPPs.

Supplementary Table 15

Comparison of the genes enriched in RBPJind-noDox and RBPJind-Dox6d Sell+Selplg+Ccr9+ BM LMPPs.

Supplementary Table 16

Comparison of the biological pathways involving genes enriched in RBPJCtr and RBPJind-noDox Sell+Selplg+Ccr9+ BM LMPPs.

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Chen, E.L.Y., Thompson, P.K. & Zúñiga-Pflücker, J.C. RBPJ-dependent Notch signaling initiates the T cell program in a subset of thymus-seeding progenitors. Nat Immunol 20, 1456–1468 (2019). https://doi.org/10.1038/s41590-019-0518-7

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