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Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver

Nature Immunology volume 18, pages 1139–1149 (2017)Cite this article

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Abstract

The molecular events that initiate lymphoid-lineage specification remain unidentified because the stages of differentiation during which lineage commitment occurs are difficult to characterize. We isolated fetal liver progenitor cells undergoing restriction of their differentiation potential toward the T cell–innate lymphoid cell lineage or the B cell lineage. Transcripts that defined the molecular signatures of these two subsets were sequentially upregulated in lympho-myeloid precursor cells and in common lymphoid progenitor cells, respectively, and this preceded lineage restriction; this indicates that T cell–versus–B cell commitment is not a binary fate 'decision'. The T cell–bias and B cell–bias transcriptional programs were frequently co-expressed in common lymphoid progenitor cells and were segregated in subsets biased toward T cell differentiation or B cell differentiation, after interleukin 7 (IL-7) signaling that controlled the number of progenitor cells engaging in T cell differentiation versus B cell differentiation.

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Figure 1: HSA defines a T cell–NK cell–biased subset and a B cell–biased subset in the Flt3+α4β7− CLP compartment.
Figure 2: The molecular signatures of HSAlo CLPs, HSAhi CLPs and α4β7+ CLPs show enrichment for gene expressed specifically by ETPs, pro-B cells and ILCs, respectively.
Figure 3: T cell– and B cell–bias transcriptional signature are co-expressed in HSAint iCLPs before being segregated in HSAlo and HSAhi CLPs.
Figure 4: Expression of transcripts from the T cell–bias, B cell–bias and ILC signatures is regulated in CLPs with distinct differentiation potential.
Figure 5: Sequential expression of T cell– and B cell–bias signatures in distinct progenitor cells.
Figure 6: IL-7 signaling is not required for the expression of the B cell–bias signature but stabilizes Ebf1 expression in HSAint iCLPs.
Figure 7: IL-7 controls the number of CLPs that adopt the B cell fate or the T cell–ILC cell fate.

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Acknowledgements

We thank A. Bandeira and D. Guy-Grand for critical reading; S. Novault and S. Schmutz for support; H.-R. Rodewald (German Cancer Center DKFZ) for the IL-7Rα-Cre mouse line; and P. Chambon (IGBMC Strasbourg) for the TSLP-KO mice. This work benefited from data assembled by the ImmGen Consortium. Supported by the Pasteur Institute, Institut National de la Santé et de la Recherche Médicale (INSERM), the Ministère de la Recherche (C.B., C.R. and S.C.), Association pour la Recherche sur le Cancer (S.C. and R.G.), La Ligue Contre Le Cancer (C.B. and C.R.), Université Paris Diderot (C.B., R.G. and S.C.), Université Pierre et Marie Curie (C.R.), the Agence Nationale de la Recherche (project Myeloten (R.G.); program REVIVE (Investment for the Future) (A.C.); and project Twothyme (A.C.) and the Pasteur-Weizmann Foundation (A.C.).

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  1. Claire Berthault & Cyrille Ramond

    Present address: Research Center Growth and Signaling, INSERM U 1016, Institut Cochin, Paris, France

Authors and Affiliations

  1. Unit for Lymphopoiesis, Pasteur Institute. Immunology department, Paris, France

    Claire Berthault, Cyrille Ramond, Odile Burlen-Defranoux, Sylvestre Chea, Rachel Golub, Pablo Pereira, Paulo Vieira & Ana Cumano

  2. INSERM U1223, Paris, France

    Claire Berthault, Cyrille Ramond, Odile Burlen-Defranoux, Sylvestre Chea, Rachel Golub, Pablo Pereira, Paulo Vieira & Ana Cumano

  3. Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France

    Claire Berthault, Odile Burlen-Defranoux, Sylvestre Chea, Rachel Golub, Pablo Pereira, Paulo Vieira & Ana Cumano

  4. Université Pierre et Marie Curie, Paris, France

    Cyrille Ramond

  5. Département Génomes et Génétique, Plate-forme Transcriptome et Epigénome, Institut Pasteur, Paris, France

    Guillaume Soubigou

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Contributions

C.B. designed and performed most experiments, analyzed data and wrote the manuscript; C.R., O.B.-D. and S.C. performed experiments; G.S. performed the microarray; R.G. provided mouse lines and designed experiments; C.R., P.P. and P.V. contributed to the discussions and writing; and A.C. directed the research, designed experiments, analyzed data and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Kinetics for the expression of HSA and Ly6D in fetal and adult lymphoid progenitor cells.

(a) E12, E13, E15 and E17 FL cells were stained as described in Fig 1 and HSA expression was analyzed in the iCLP (Flt3+ α4β7-) compartment. (b) Absolute numbers of HSAlo, HSAint and HSAhi iCLPs per E13, E15 and E16 fetal livers. Data is representative of at least 3 independent experiments. (c) Plots of iCLP from E13, E15 and E17 FL showing the HSA and Ly6D expressions (high and intermediate levels of HSA are marked). (d) Plots show the expression of Ly6D in BM Flt3+ IL7Rα+ CLP. (e) Venn diagrams representing the number of overlapping upregulated (left) and downregulated (right) transcripts in the HSAhi, HSAlo and α4β7 CLP compared to HSAintiCLPs. (f) Quantitative RT-PCR gene expression analysis of the HSAint-signature gene Eltd1 in HSAlo, HSAint, HSAhi iCLPs and HSC (Lin- c-Kit+ Sca-1+ Flt3-). Histograms display relative quantity of expression normalized to Hprt. Data is representative of 3 independent experiments. (g) Histogram displays the frequency of Eltd1 expressing single cells in HSAint iCLPs expressing neither ETP nor proB specific genes (None), expressing Pax5 or Pira11, and HSAhi and HSAlo iCLPs. Data represents a pool of 5 independent experiments of 14 single cells each. (f, g) mean±SEM **p<0.01***p<0.001****p<0.0001 (Student’s t test).

Supplementary Figure 2 Deletion of RBPJ in IL-7rα-expressing cells does not affect the B cell potential or expression of the T cell–biased transcriptional signature of HSAlo iCLPs.

(a) E14 CLPs isolated from (IL7RαCre/+ ROSAYFP) RBPJfx/+ (top panel) and RBPJfx/fx(bottom panel) littermates embryos were stained as described in Fig 1. PIRA/B expression was analyzed in α4β7- HSAlo and HSAintCLPs, stained as described in Fig 1. Data is representative of more than 5 independent experiments. (b) Absolute numbers of HSAlo, HSAint and HSAhi iCLP per E14 FL of RBPJfx/+ and RBPJfx/fx. Dot plots displays absolute CLP numbers in 5 embryos from 3 independent experiments, each dot represents a single embryo. (c) B cell differentiation potential of single HSAlo, HSAint and HSAhi iCLPs from E14 RBPJfx/+ and RBPJfx/fx was assessed as described in Fig 1. Data is representative of a pool of 3 independent experiments. (d) qRT-PCR gene expression analysis of HSAhi signature- genes Ebf1, Pax5, Hes1 (top panel) and of HSAlo signature-genes Glis3, Pira11, Cd7 (bottom panel) in HSAlo, HSAint and HSAhi iCLPs from E14 RBPJfx/+ and RBPJfx/fx. Histograms display relative quantity of expression normalized to Hprt. Data is a pool of 3 independent experiments. (e) Plots showing CD4 and CD19 expression in the progeny of α4β7- CLP IL7cre:RBPJfx/+ and RBPJfx/fx in culture for 10 days with OP9dll4, IL7, KitL and Flt3L. (b, c, d) mean±SEM *p<0.05 N.S. non significant (Student’s t test).

Supplementary Figure 3 Single-cell multiplex qPCR analysis of T cell–, B cell– and ILC-biased transcripts in FL CLPs.

Hierarchical unsupervised clustering of the multiplex single cell transcriptional analysis of 17 single HSAlo, HSAint and HSAhi iCLP and α4β7+ CLP for 28 transcripts of the HSAlo, HSAhi and α4β7+ CLP signatures and of Eltd1.

Supplementary Figure 4 PIRA/B-expressing FL CLPs and CRLPs co-express all T cell–bias genes similar to DN1a fetal thymocytes.

(a) E13 fetal blood and (b) thymocytes were isolated and stained for Lin (GR1, TER119, CD3ε, CD4, CD8, CD19, CD11c and NK1.1) CD44 CD25, c-kit, HSA and PIRA/B. DN1 were defined as Lin- CD44+ CD25- c-Kit+ and divided into DN1a (HSA-) and DN1b (HSA+), DN2 were defined as Lin- CD44+ CD25+ c-Kit+, and PIRA/B expression was analyzed in DN1a, DN1b and DN2. Single cell multiplex qRT-PCR on single sorted E13 (c) α4β7- HSAint PIRA/B+ CLPs (far left pannel), α4β7- HSAlo PIRA/B+ CLPs (middle left panel), α4β7- HSAlo PIRA/B+ CRLPs (middle right panel) and DN1a PIRA/B+ thymocytes (far right panel). The same set of 30 genes as in Fig 6 was analyzed by qRT-PCR using the Biomark Fluidigm system after 20 cycles of specific pre-amplification. Expression of Hprt, Pira11, Cd7, Ccr9, Glis3 and Ebf1 is displayed. Each dot represent detectable expression of a given gene and each horizontal line represent gene expression pattern in a given single cell. Frequency and absolute number of single cells expressing a given gene are displayed at the top of each column. (d) Plot showing the Ct values of Gata3 expression in HSAhi, HSAint and HSAlo PIR+ and PIR- CLP and in CRLP. Data is representative of 2 independent experiments. (d) mean±SEM **p<0.01 N.S. non significant (Student’s t test).

Supplementary Figure 5 PIRA/B+ T cell–biased LMPPs differentiate into PIRA/B+ T cell–biased iCLPs.

(a) Histogram displays frequency of single Flt3hi LMPP expressing Il7ra transcripts in WT and Il7-/- FL13. Data is representative of 82 to 89 single cells analyzed in 2 independent experiments. (b) Ct values of Il7ra transcripts in WT LMPP that express or not Ebf1 compared to iCLP. (c) Post sorting purity controls of FL E13 Il7-/- PIRA/B- (top panel) and PIRA/B+ (bottom panel) Flt3hi LMPPs. (d) Dot plots display the phenotype of the IL7Rα- compartment of cultured FL13 Il7-/- PIRA/B- and PIRA/B+ Flt3hi LMPPs after 24 hours of culture with saturating amounts of Flt3L and c-KitL, in the absence of stromal cells. Data is representative of 3 independent experiments. (a,b) mean±SEM. ****p<0.0001 (Student’s t test).

Supplementary Figure 6 IL-7 deficiency does not affect the proliferation, survival or B cell potential of E13 FL CLPs.

(a) Absolute numbers of CLPs per E13 FL in WT and Il7-/-embryos. Data is representative of 9 embryos analyzed in 3 independent experiments, each dot represents a single embryo. (b) qRT-PCR gene expression analysis of the HSAhi signature-genes Ebf1, Pax5, Hes1, Foxo1 and Rag2, of Notch1, and HSAlo signature-genes Glis3 and Pira11 in HSAlo, HSAint and HSAhi iCLPs from WT and Il7-/-embryos. Histograms display relative quantity of expression normalized to Hprt. Data is representative of 3 independent experiments. (c) Histogram displays frequency of B-lineage positive wells after culture of HSAint and HSAhi iCLPs from WT and Il7-/-embryos. Data is representative of a pool of 3 independent experiments. (d) Single cell multiplex qRT-PCR on single sorted E13 WT and Il7-/-HSAhi iCLPs, expression of Hprt, Ebf1, Pax5, Pira11, Cd7 and Glis3 is displayed. Data is representative of 3 independent experiments. (e,f) KI67 and DAPI staining and frequency of sorted HSAint and HSAlo iCLPs in S/G2/M (KI67+ DAPI+). Data is representative of 2 independent experiments. (g) Histogram display Annexin V staining on sorted HSAlo, HSAint and HSAhi WT (red line) and Il7-/-(blue line) iCLPs compared to adult thymocytes (gray tinted line). Data is representative of 2 independent experiments. (h) qRT-PCR gene expression analysis of Bcl2 transcripts in HSAlo, HSAint and HSAhi WT and Il7-/-iCLPs. Histograms display relative quantity of expression normalized to Hprt. Data is representative of 4 independent experiments. (a,b,c,f,h) mean±SEM *p<0.01 N.S. non significant (Student’s t test).

Supplementary Figure 7 Flt3 deficiency does not affect the commitment or priming of FL CLPs.

(a) WT and Flt3l-/- E13 FL were stained as described in Fig 1 and Flt3+ α4β7- CLP compartments were analyzed. Data is representative of 4 independent experiments. (b) Dot plots display PIRA/B expression in WT (left panel) and Flt3l-/-right panel) LSK compartment. Data is representative of 3 independent experiments. (c) Histograms display absolute numbers of total CLPs and HSAlo, HSAint and HSAhi iCLP fractions per E13 FL in WT and Flt3l-/-embryos. (d) Single cell multiplex qRT-PCR on single sorted E13 Flt3l-/- HSAint iCLPs, experiment was performed as described in Fig 3. Expression of Hprt, Ebf1, Pax5, Pira11, Cd7 and Glis3 is displayed. Data is representative of a pool of 2 independent experiments. (e) Display frequencies of single cell expressing T only, B only or T and B genes in Flt3l-/- HSAint iCLPs. “None” includes no B and no T gene expression. (f) Il7-/- and Il7-/-/Tslp-/- double KO E13 FL were stained as described in Fig 1 and Flt3+ α4β7- CLP compartments were analyzed. (g) Histograms display absolute numbers of HSAlo, HSAint and HSAhi iCLP fractions per E13 FL in Il7-/- and Il7-/-/Tslp-/-double KO embryos. Data is representative of 4 embryos per group analyzed independently. (c,g) mean±SEM **p<0.01***p<0.001 N.S. non significant (Student’s t test).

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Berthault, C., Ramond, C., Burlen-Defranoux, O. et al. Asynchronous lineage priming determines commitment to T cell and B cell lineages in fetal liver. Nat Immunol 18, 1139–1149 (2017). https://doi.org/10.1038/ni.3820

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