Abstract
Thymic T cell development is initiated from bone-marrow-derived multi potent thymus-seeding progenitors. During the early stages of thymocyte differentiation, progenitors become T cell restricted. However, the cellular environments supporting these critical initial stages of T cell development within the thymic cortex are not known. Here we use the dependence of early, c-Kit-expressing thymic progenitors on Kit ligand (KitL) to show that CD4−CD8−c-Kit+CD25− DN1-stage progenitors associate with, and depend on, the membrane-bound form of KitL (mKitL) provided by a cortex-specific KitL-expressing vascular endothelial cell (VEC) population. In contrast, the subsequent CD4−CD8−c-Kit+CD25+ DN2-stage progenitors associate selectively with cortical thymic epithelial cells (cTECs) and depend on cTEC-presented mKitL. These results show that the dynamic process of early thymic progenitor differentiation is paralleled by migration-dependent change to the supporting niche, and identify VECs as a thymic niche cell, with mKitL as a critical ligand.
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Acknowledgements
We thank C. Blackburn (Institute for Stem Cell Research, UK), J. Nathans (Johns Hopkins Medical School, Maryland, USA) and M. Fruttiger (National Institute of Medical Research, UK) for providing Foxn1–Cre, Rosa26–CreERT2 and Pdgfb–CreERT2 mice, respectively, A. Samraj for screening of targeted embryonic stem cells, and the Wolfson Imaging Centre for image analysis. This work was supported by an MRC Strategic Award (G0701761) and MRC Program Grant (G0900892) to C.N., by an MRC Program Grant to S.E.W.J., by Cancer Research UK and by Leukemia and Lymphoma Research. A.J.M. is the recipient of an MRC Senior Clinical fellowship.
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M.B. carried out mouse genetic experiments and imaging, and associated data analysis; R.F. and S.M. generated Sl/Sld andΔEx7 mice and measured KitL levels and activity; S.T. analysed RNASeq data; T.C.L. provided protocols for single-cell profiling; A.G. generated RNAseq libraries; D.W. performed image analysis; P.B. provided ICKitL-specific antibody; A.J.M. provided protocols for single-cell profiling; S.E.W.J. and C.N. conceived the project and wrote the manuscript with M.B.
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Integrated supplementary information
Supplementary Figure 5 Heat maps of stromal cell gene expression.
(a) Heat map of gene expression data from Fig. 3a–g. RPKM values were normalized for each individual gene across all cell types. (b) mRNA expression in single KT+ VECs, KT− VECs (h) and cTECs and mTECs (i) using microfluidics-based qPCR. Expression values are normalized to B2m for each gene, and subsequently to the average value for each individual gene across all cell types. Each column represents a single cell.
Supplementary Figure 6 Lack of detectable sKitL expression from Steel-Dickie allele.
(a) Serum KItL levels in 3-week-old wild-type (+/+; n = 18), Sl/+ (n = 7), Sld/+ (n = 7) and Sl/Sld (n = 4) mice measured by ELISA. Error bars show standard errors. Student’s t-test P values are shown. For source data see Supplementary Table 5. (b) Schematic of the exon 7 floxed Kitl allele. (c) Concentration of sKItL in supernatants of HEK293 cells transfected with the empty MSCV-IRES-GFP vector (N = 6) or MSCV-IRES-GFP vectors encoding Sld KitL (N = 3) and ΔEx7 KitL (N = 3), respectively. Error bars show standard errors. For source data see Supplementary Table 5. (d) Bioassay measuring proliferation of the KitL-dependent MC/9 cell line exposed to the supernatants from (c). Values are relative to MC/9 cells grown under optimal proliferation conditions. Error bars show standard errors. N = 3 for each condition. For source data see Supplementary Table 5. (e) Serum KItL levels in 3 week old control (+/+; n = 6) and KitlΔEx7/ΔEx7 (n = 4) mice measured by ELISA. Error bars show standard errors. Student’s t-test P value is shown. For source data see Supplementary Table 5. (f) Total thymic cellularity of 4–5 weeks old +/+ mice (n = 3) and their Sl/+ littermates (n = 3). Error bars show standard errors. The difference between +/+ and Sl/+ genotypes is not statistically significant (P = 0.71). For source data see Supplementary Table 5. (g) Absolute number of phenotypically defined Flt3 + DN1, Flt3–DN1 and DN2 cells in thymi of mice from (f). Error bars show standard errors. No statistical significant differences were observed (P = 0.21, P = 0.48 and P = 0.35, respectively). For source data see Supplementary Table 5. (h) Total bone marrow cells in mice from (f). Error bars show standard errors. The difference between +/+ and Sl/+ is not statistically significant (P = 0.206). For source data see Supplementary Table 5. (i) Number of cells in the indicated phenotypically defined lymphoid progenitor populations in mice from (f). Values are group averages; error bars show standard errors. P-values relative to the control group are shown. For source data see Supplementary Table 5.
Supplementary Figure 7 Specificity of Cre lines used.
(a) Schematic of the Tie2-Cre lineage tracing using the Rosa26-EYFP Cre reporter. (b) Flow cytometric analysis of thymic stromal cell subsets from Tie2-Cre; Rosa26-EYFP mice (green lines) and Rosa26-EYFP control mice (blue lines). Percentage of YFP + cells observed in the VEC (right plot) and combined MCs and TECs populations (left plot) is shown. (c) Schematic of the Pdgfb − CreERT2-IRES-iGFP reporter. (d) Flow cytometric analysis of thymic stromal cell subsets from Pdgfb − CreERT2-IRES-iGFP mice (green lines) and non-transgenic control mice (blue lines). Percentage of GFP + cells detected in the VECs (right plot) and the combined MC and TEC populations (left plot) is shown. (e) Schematic of Foxn1-Cre lineage tracing using the Rosa26-EYFP Cre reporter. (f) Flow cytometry analysis of thymic stromal cell subsets in the Foxn1-Cre/Rosa26-EYFP mouse model. Percentage of EYFP + cells in the TEC (right plot) and MC plus VEC lineages (left plot) is displayed. (g) Total thymic cells from 6 weeks old controls (n = 3 biological replicates pooled over 2 independent experiments) and Pdgfb-CreERT2 transgenic (n = 3) littermates, treated with tamoxifen and analysed one week after the last injection. The Cre driver is shown below the x-axis. Values are group averages. Error bars show standard errors. No statistical significant differences were observed. For source data see Supplementary Table 5. (h) Absolute number of phenotypic Flt3 + DN1, Flt3- DN1 and DN2 thymocytes from the mice in (g). Error bars show standard errors. No statistical significant differences were observed. For source data see Supplementary Table 5. (i) Number of total bone marrow cells from the mice analysed in (g). Error bars show standard errors. No statistical significant differences were observed. For source data see Supplementary Table 5. (j) Absolute number of phenotypic bone marrow lymphoid progenitor populations from the mice in (g). Error bars show standard errors. No statistically significant differences were observed. For source data see Supplementary Table 5. (k) Absolute number of phenotypic DAPI−lineage−B220−CD4−CD8− (DN), DAPI−CD4 + CD8 + (DP), DAPI−CD4+CD8− (SP4), DAPI−CD4−CD8+ (SP8) from 4–5 weeks old uninjected (n = 6) and tamoxifen injected (n = 6) control mice pooled from 3 independent experiments. Error bars show standard errors. No statistically significant differences were observed.
Supplementary Figure 8 Loss of mKitL does not affect thymic architecture or morphology.
Immunofluorescence analysis of thymic sections by using antibodies against CD31 (green), Ly51 (red), UEA-1 (blue) to assess the overall thymus architecture in 4–5 weeks old control (a–d) (KitlLEx7/LEx7) and FoxN1ΔEx7 mice (e–h). Scale bar: 300 μm. Immunofluorescence analysis of thymic sections by using antibodies against CD31 (green), Ly51 (red), UEA-1 (blue) in 4–5 weeks old, tamoxifen injected control (i–l) (KitlLEx7/LEx7), Tie2/PdgfbΔEx7 mice (m–p) and Tie2/Pdgfb/FoxN1ΔEx7 mice. Scale bar: 300 μm.
Supplementary Figure 9 Impact of mKitL depletion on thymic stromal cell and thymic progenitor populations.
(a) Relative percentage of phenotypic TECs, VECs and MCs from 4–5 weeks old control, Foxn1ΔEx7 and Tie2/PdgfbΔEx7 mice analyzed as in Fig. 5a. Number of biological replicates displayed. Error bars show standard errors. No statistically significant differences were observed. For source data see Supplementary Table 5. (b) Absolute number of DN, DP, SP4 and SP8 from 4–5 week old control (n = 7) and Foxn1ΔEx7 mice (n = 3). Error bars show standard errors. P-values relative to the control group are shown. For source data see Supplementary Table 5. (c) Absolute number of phenotypic DN, DP, SP4 and SP8 from 4–5 weeks old control (n = 5), Tie2/PdgfbΔEx7 (n = 4) and Tie2/Pdgfb/FoxN1ΔEx7 (n = 5), analyzed as in Fig. 5a. Error bars show standard errors. P-values relative to the control group are shown. For source data see Supplementary Table 5.
Supplementary Figure 10 Effect of stromal mKitL deletion on bone marrow lymphopoiesis.
(a) Number of total bone marrow cells from KitlLEx7/LEx7 (n = 4) and Tie2/PdgfbΔEx7 (n = 4) mice from 2 separate experiments, treated with tamoxifen and analysed as in Fig. 5a. Error bars show standard errors. No statistically significant differences were observed. (b) Number of phenotypic bone marrow lymphoid progenitor populations in mice from (a). Error bars show standard errors. No statistically significant differences were observed. (c) Number of total bone marrow cells from KitlLEx7/LEx7 (n = 7) and Foxn1ΔEx7 (n = 7) mice from 3 separate experiments. Error bars show standard errors. P-values relative to the control group are shown (Student’s t-test). (d) Number of phenotypic bone marrow lymphoid progenitor populations in mice from (c). Error bars show standard errors. No statistically significant differences were observed. (e) Number of total bone marrow cells from KitlLEx7/LEx7 (n = 6) and Tie2/Pdgfb/Foxn1ΔEx7 (n = 5) mice analyzed as in (a) from 4 separate experiments. Error bars show standard errors. No statistically significant differences were observed. (f) Number of phenotypic bone marrow lymphoid progenitor populations in mice from (e). Error bars show standard errors. No statistically significant differences were observed.
Supplementary Figure 11 Thymic stromal cells FACS analysis.
Gating strategy used to define thymic stromal cell populations within the total thymic live (DAPI −) singlet cell population: VECs: Lin−EpCAM−CD31+; MCs Lin−EpCAM−CD31−,; cTECs: Lin−EpCAM+Ly51+UEA-1−; mTECs: Lin−CD31−EpCAM+Ly51−UEA-1+. Lin− is CD45−Ter119−.
Supplementary Figure 12 Thymic T cell progenitors and bone marrow lymphoid progenitors FACS analysis.
(a) Gating strategy used to define Flt3 + DN1 (Lin − CD4 − CD8α − CD25 − c-KithiFlt3 +), Flt3–DN1 (Lin − CD4 − CD8α − CD25 − c-KithiFlt3 −) and DN2 (Lin − CD4 − CD8α − CD25 + c-KithiFlt3 −) thymocytes in young adult mice (4–6 weeks). (b) Gating strategy used to define lymphoid progenitor populations within the bone marrow live (DAPI −) singlet cell population. The following populations were analysed: NK/T progenitors (LIn–B220 − CD49b − NK1.1 − CD122 +), Lymphoid-biased multi-potent progenitors (LMPPs: Lin − B220 − CD49b − NK1.1 − CD122 − Flt3 + CD127 − c-KithiSca1hi), All Lymphoid Progenitors (ALPs: Lin − B220 − CD49b − NK1.1 − CD122 − Flt3 + CD127 + Ly6D − c-Kitlo), B-Lymphoid Progenitors (BLPs: Lin − B220 − CD49b − NK1.1 − CD122 − Flt3 + CD127 + Ly6D + c-Kit −), Common Lymphoid Progenitors (CLPs: Lin − B220 − CD49b − NK1.1 − CD122 − Flt3 + CD127 + Ly6D − c-KitloSca1lo).
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Buono, M., Facchini, R., Matsuoka, S. et al. A dynamic niche provides Kit ligand in a stage-specific manner to the earliest thymocyte progenitors. Nat Cell Biol 18, 157–167 (2016). https://doi.org/10.1038/ncb3299
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DOI: https://doi.org/10.1038/ncb3299
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