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. 2012 Feb 19;13(4):412-9.
doi: 10.1038/ni.2255.

The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential

Sidinh Luc  1 , Tiago C Luis, Hanane Boukarabila, Iain C Macaulay, Natalija Buza-Vidas, Tiphaine Bouriez-Jones, Michael Lutteropp, Petter S Woll, Stephen J Loughran, Adam J Mead, Anne Hultquist, John Brown, Takuo Mizukami, Sahoko Matsuoka, Helen Ferry, Kristina Anderson, Sara Duarte, Deborah Atkinson, Shamit Soneji, Aniela Domanski, Alison Farley, Alejandra Sanjuan-Pla, Cintia Carella, Roger Patient, Marella de Bruijn, Tariq Enver, Claus Nerlov, Clare Blackburn, Isabelle Godin, Sten Eirik W Jacobsen
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The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential

Sidinh Luc et al. Nat Immunol. .

"V体育平台登录" Abstract

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus. VSports手机版.

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Figures

Figure 1
Figure 1
ETPs are multipotent lympho-myeloid restricted progenitors. (a) Flow cytometry profiles and gating strategies for the detection of Lin−CD4−CD8α−CD25−c-KithiFlt3hi ETPs from young adult mice (4–6 weeks). Numbers in plots indicate percent ETPs among total thymocytes. DAPI, DNA-intercalating dye; FSC, forward scatter; SSC, side scatter; -W, width; -H, height. (b) Frequency of B cell potential of cultures seeded with a single Lin−Sca-1+c-Kit+Flt3hi bone marrow cell (LMPP; n = 320); a single Flt3hi ETP (n = 73 cells) or ten Flt3hi ETPs (n = 960 cells); ten Flt3− ETPs (n = 960 cells); or other DN thymocyte progenitor populations (DN1–DN4; n = 2,400 cells (seeded with 100 cells per well)), all from adult mice. (c) Flow cytometry profiles and gating strategies as in a, for cells from newborn mice (1 d). (d) Frequency of B cell potential as in b, for cultures of cells from newborn mice, seeded as single Flt3hi ETPs (n = 348 cells) or single Flt3− ETPs (n = 210 cells), and other DN thymocyte progenitor populations seeded at 100 cells per culture (n = 4,200–6,000 cells). (e) Expression of enhanced yellow fluorescent protein (eYFP) in ETPs from neonatal mice (n = 4) expressing Cre from the Cd79a promoter. (f) Frequency of cells with B cell potential (B; n = 348 cells), T cell potential (T; n = 204 cells), natural killer cell potential (NK; n = 48 cells), GM potential (grown in liquid (GM(L); n = 600 cells) or on stroma (GM(S); n = 64 cells)), megakaryocyte potential (grown in liquid (Mk(L); n = 1,080 cells) or on semisolid support (Mk(SS); n = 6; 200 cells per replicate)) or erythroid potential (E; n = 8; 500–1,000 cells per replicate) among Flt3+ ETPs from neonatal mice (positive controls, Supplementary Fig. 5). (g) Expression of myeloid markers Mac-1, Gr-1 and lysozyme M (reported as eGFP expression; left and middle), and morphological analysis (right) of sorted granulocytes (top) and monocytes (bottom) from cultured Flt3+ ETPs from neonatal mice. Scale bars, 5 μM. (h) Quantitative analysis of the expression of genes associated with lymphoid cells, myeloid cells and megakaryocytes–erythroid cells by purified Flt3+ ETPs from newborn mice (n = 6; 25 cells per replicate); results are presented relative to the expression of Hprt (encoding hypoxanthine guanine phosphoribosyl transferase). *, ≤0.001 (below detection limit). Data are representative of four experiments (a); fourteen experiments (c); seven (b) or sixteen (d) experiments (Flt3hi ETPs); sixteen experiments (bone marrow; b); four experiments (Flt3− ETPs (b) and other DN populations (b,d)); ten experiments (Flt3− ETPs; d); one experiment (e); two to sixteen experiments (f); one experiment (g); or two experiments (h; mean and s.e.m. in b,d,f; average and s.d. of six replicates in h).
Figure 2
Figure 2
ETPs possess combined T, B and GM lineage potentials. (a) Single cell gene expression analysis of lymphoid, myeloid and MkE genes in purified newborn Flt3hi ETPs. Mean (± s.d.) frequency of cells expressing specified genes among cells positive for c-Kit gene expression (96-98% of total cells) (left; n=176 from 2 exp). The frequency (± s.d.) of ETPs with combined lymphoid-GM lineage transcriptional priming (right) based on co-expression of one or more genes for the lymphoid program (Il7r, sterile IgH), myeloid/GM program (Csf1r, Mpo) but not the MkE program (Gata1, Epor). (b) A representative clone from a single newborn WT Flt3+ETP cell with combined T, B and myeloid lineage potential determined by FACS and morphology analysis (myeloid, black arrowhead, lymphocyte, white arrowhead). (c,d) Cloning frequencies (left), of ETPs generating CD45+ cells (white bars) and CD45+ cells with a definitive lineage readout (grey bars). Lineage distribution of clones (right) from (c) single WT (n=132 from 3 exp) and (d) single vavP-mcl-1 ETPs (n=167 from 2 exp).
Figure 3
Figure 3
Absence of pluripotent HSCs in newborn thymus. (a) Flow cytometry profiles of CD150, CD201 and Mpl stem cell marker expression in Lin−CD4−CD8α−CD25−c-Kithi ETPs in newborn mice (n=2 exp). (b) Flow cytometry profiles of CCR9 and Rag1 GFP co-expression in ETPs, LMPPs and HSCs (n=2 exp). (c-h) Peripheral blood analyses showing mean (±s.d.) thymocyte contribution to T, B, myeloid and platelet (Vwf eGFP+) lineages at 3-4 weeks (c; n=9, d; n=7), 16 weeks (e; n=8, f; n=7) and 12 weeks (g; n=8,h; n=8) after intravenous or intrafemoral competitive transplantation of total or CD4+ and CD8+ depleted neonatal thymocytes from Vwf eGFP or WT mice. The frequency of reconstituted animals is indicated above each lineage.
Figure 4
Figure 4
ETPs cluster closer to candidate TSPs in the BM than other thymic progenitors. (a,b) Two- and three-dimensional principal component analyses of normalized global gene expression profiles of (a) purified HSC, LMPP, ETP, DN2 and DN3 from neonatal mice (n=3) and (b) purified neonatal HSC (n=3), LMPP (n=3), CLP (n=4), ETP (n=3), DN3 (n=3), ProB (n=3) and adult ETP (n=2). Each symbol represents a separate biological sample (sorted from different pools of mice). Euclidean distances between average x and y values for each population measured in the first two principal components are shown in panel (a).
Figure 5
Figure 5
ETPs, LMPPs and CLPs have closely related T and myeloid lineage transcriptional profiles. (a,b) Heatmap representation of T (a) and GM (b) lineage-affiliated gene expression represented as normalized median expression values from purified neonatal HSC (n=6), LMPP (n=6), CLP (n=4), ETP (n=6), DN2 (n=3), DN3 (n=6) and adult ETP (n=2) populations. Gene lists were established as described in the Online Methods section and Supplementary Methods).
Figure 6
Figure 6
Quantitative expression analysis of lymphoid and myeloid genes in neonatal ETPs. Quantitative gene expression analysis showing lymphoid and myeloid genes in purified neonatal HSC (n=6), LMPP (n=4), ETP (n=6), DN2 (n=6) and DN3 (n=6) populations (25 cells/replicate). Average expression levels (± s.d.). (*) ≤0.001, below detection.
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