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Science 335 (6073): 1195-1200

Copyright © 2012 by the American Association for the Advancement of Science

Lin28b Reprograms Adult Bone Marrow Hematopoietic Progenitors to Mediate Fetal-Like Lymphopoiesis

Joan Yuan, Cuong K. Nguyen, Xiuhuai Liu, Chrysi Kanellopoulou, and Stefan A. Muljo*

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD 20892, USA.


Figure 1
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Fig. 1. Differential let-7 and Lin28b expression in fetal and adult lymphocyte progenitors. Mouse expression data represent RNA of FACS-sorted populations pooled from at least three adult or neonatal mice or 12 FLs. (A) Relative expression of mature let-7 isomiRs in FL and adult BM pro-B cells (B220+CD19+CD24+CD43+IgM) determined by NanoString global miRNA-expression profiling. (B) QRT-PCR analysis of mature let-7g and let-7c expression in pre-pro-B cells (B220+CD19CD24CD43+IgM) from FL and adult BM. (C) QRT-PCR analysis of Lin28b mRNA expression in FACS-sorted B cell precursor subsets from the indicated organs. Pre-pro-B (B220+CD19CD24CD43+IgM), pro-B (B220+CD19+CD24+CD43+IgM), pre-B (B220+CD19+CD24+CD43IgM), and immature B (B220+CD19+CD24+CD43IgM+). E14.5, embryonic day 14.5. (D) QRT-PCR analysis of Lin28b mRNA in sorted FL and BM HSPC populations: LSK (LinSca-1+c-Kit+) and CLP (LinSca-1intc-KitintCD127+). (E) QRT-PCR analysis of Lin28b mRNA expression in FACS-sorted thymocyte subsets from mice of the indicated age. DN2 and DN3 (CD4CD8CD25+CD44int), DN4 (CD4CD8CD25CD44), {gamma}{delta}-T ({gamma}{delta}-TCR+CD3+), DP (CD4+CD8+CD3lo), and CD4SP (CD4+CD8CD3+). All thymocyte subsets except for the {gamma}{delta}-T cells were also gated through a {gamma}{delta}-TCR CD1dPBS57– gate. (F) QRT-PCR analysis of human Lin28b mRNA expression in the indicated fetal and adult organs: FL, FT, FS, CD34+ CB, BM, thymus (Thy), lymph nodes (LN), and spleen (SPL). Human samples contain commercially obtained RNA pooled from at least three donors each. For all panels, error bars represent standard error of triplicate experimental replicates. n.d. indicates not detectable or below background signal level.

 

Figure 2
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Fig. 2. Lin28-mediated depletion of let-7 and multilineage reconstitution in Lin28-RV BM chimeras. (A) FACS plot shows representative frequency of GFP+ cells among lineage-depleted adult BM cells enriched in HSPCs 24 hours posttransduction with Lin28-RV. (B) Absolute numbers of GFP+ (green) and GFP (white) CD19+ B- and CD4+ and CD8+ T-lymphocyte subsets in lymph nodes of three Lin28-RV BM chimeras 6 to 8 weeks after adoptive transfer are plotted. Data are representative of >5 independent reconstitution experiments. (C) (Top) Lin28 Western blot of total thymocyte lysate from GFP-RV and Lin28-RV BM chimeras. (Bottom) Tubulin Western blot as a loading control. (D) NanoString global miRNA-expression profiling analysis of FACS-sorted GFP+ and GFP DP thymocytes (CD4+CD8+CD3lo). Normalized counts of individual mature miRNAs in each population are plotted on x and y axis, respectively (log scale). RNA was pooled from FACS-sorted populations of three individual Lin28-RV BM chimeras. (E) QRT-PCR validation of mature let-7a and let-7g expression levels in GFP and GFP+ DP thymocytes from Lin28-RV BM chimera. Error bars indicate standard error of triplicate experimental replicates.

 

Figure 3
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Fig. 3. Ectopic Lin28 expression in adult BM HSPCs confers fetal-like B cell development. Plots depict flow cytometric analyses of (A) CD19+ peritoneal cavity (PerC) and (C) B220+ splenic (SPL) B cell subsets in recipients of adult BM HSPCs transduced with the indicated retroviral particles. (B) Percentages of B-2 (CD19+B220hiCD5), B-1a (CD19+B220loCD5+), and B-1b (CD19+B220loCD5) B lymphocytes among the GFP+ and GFP fractions of peritoneal cavity CD19+ B cells in eight independent BM chimeras. (D) Percentages of marginal zone (MZ) (B220+CD1d+CD23) and follicular B-2 (FoB) cells (B220+CD1dCD23+) among GFP+ and GFP splenic CD19+ B cells in six independent BM chimeras. (E) FACS analyses show the presence of B cells in the peritoneal cavity and spleen of BM chimeras reconstituted with Lin28-RV–transduced Il7rα–/– BM HSPCs. Data are representative of two independent BM chimeras. ***P < 0.0001 represents statistical significance calculated by t test; n.s., not statistically significant.

 

Figure 4
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Fig. 4. Ectopic Lin28 expression in adult BM HSPCs promotes the development of V{gamma}1.1+V{delta}6.3+ T cells. (A) Flow cytometric analysis to enumerate {gamma}{delta}-TCR+ CD3+ cells in thymi of GFP-RV and Lin28-RV BM chimeras. (B) Percentages of {gamma}{delta}-TCR+ CD3+ thymocytes among GFP+ and GFP cells in the thymi of five independent Lin28-RV BM chimeras. *P < 0.05 represents statistical significance calculated by t test. (C) Flow cytometric analysis shows distribution of surface CD4 expression among GFP+ {gamma}{delta}-TCR+ CD3+ thymocytes in the Lin28-RV (red line) and GFP-RV BM (blue line) chimeras. (D) Flow cytometric clonotype analysis of {gamma}{delta}-TCR+ CD3+ thymocytes in the GFP+ and GFP fractions of the Lin28-RV BM chimeras.

 

Figure 5
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Fig. 5. Ectopic Lin28 expression in adult BM HSPCs promotes the development of iNKT cells. (A) Plots depict flow cytometric analysis of thymic iNKT cells defined by double staining of CD1d tetramer loaded with PBS57 (CD1dPBS57+) and antibody against CD3 in GFP-RV and Lin28-RV BM chimeras. (B) Percentages of iNKT cells among the GFP+ and GFP cells in the indicated organs from Lin28-RV BM chimeras. *P < 0.05; **P < 0.01; ***P < 0.005. Indicated P values represent statistical significance calculated by t test. (C) Plots depict flow cytometric analysis of developmental stages 1 to 3 within the iNKT cell compartment among GFP+CD1dPBS57+CD3+ thymocytes in GFP-RV (left) and Lin28-RV BM chimeras (right). Stage 1 (CD44NK1.1), 2 (CD44+NK1.1), and 3 (CD44+NK1.1+). (D) Plots depict flow cytometric analysis of developmental stages 1 to 3 iNKT cells in the thymi of intact wild-type C57BL/6 mice of the indicated age. (E) Precursor frequency analysis shows iNKT cell potential as a function of age. Left y axis (blue line) indicates the ratio of stage 1 to DP thymocytes in intact C57BL/6 mice of the indicated age (equation shown above graph). Right y axis (gray line) indicates the percentage of DP thymocytes.

 


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