Research Article

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

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Science  09 Mar 2012:
Vol. 335, Issue 6073, pp. 1195-1200
DOI: 10.1126/science.1216557

Making Immune Cells Young Again

Hematopoiesis, the development of the immune system, occurs in distinct waves. The immune system is first populated by cells that arise from fetal hematopoietic stem cells (HSCs) and then later by cells derived from adult HSCs. Furthermore, fetal HSCs give rise to lymphocytes with innate immunelike properties, whereas adult HSCs give rise to classical T and B cells. Yuan et al. (p. 1195, published online 16 February) now uncover the molecular mechanism behind these distinct waves of hematopoiesis. Expression of the RNA binding proteins Lin28 and Lin28b is enriched in fetal hematopoietic stem/progenitor cells (HSPCs) in mice and humans. Ectopic expression of Lin28 in mouse adult HSPCs was sufficient to induce the differentiation of both classical and innate-like lymphocyte lineages.

Abstract

The immune system develops in waves during ontogeny; it is initially populated by cells generated from fetal hematopoietic stem cells (HSCs) and later by cells derived from adult HSCs. Remarkably, the genetic programs that control these two distinct stem cell fates remain poorly understood. We report that Lin28b is specifically expressed in mouse and human fetal liver and thymus, but not in adult bone marrow or thymus. We demonstrate that ectopic expression of Lin28 reprograms hematopoietic stem/progenitor cells (HSPCs) from adult bone marrow, which endows them with the ability to mediate multilineage reconstitution that resembles fetal lymphopoiesis, including increased development of B-1a, marginal zone B, gamma/delta (γδ) T cells, and natural killer T (NKT) cells.

Hematopoietic stem cells (HSCs) give rise to all the erythroid, myeloid, and lymphoid lineages and are used clinically in transplants to treat patients with a wide variety of blood and immune system disorders that include immunodeficiencies and malignancies. In mice, the major site of hematopoiesis transitions from the fetal liver (FL) to the fetal spleen (FS) and bone marrow (BM) during late embryogenesis. FL HSCs differ phenotypically and functionally from adult BM HSCs, and a developmental switch is thought to occur within the first weeks of postnatal life (13). Adoptive transfer and fetal thymic organ culture studies established that FL HSPCs, unlike adult BM HSPCs, preferentially give rise to lymphocyte subsets that can be collectively referred to as innatelike lymphocytes. For example, CD5+ B cells, referred to as the B-1a subset, and certain γδ-T cell subsets develop almost exclusively in the fetus and/or neonate (47). More recently, a fetal source of HSPCs was found to preferentially give rise to the B-1a and marginal zone (MZ) B cell lineages (8). In contrast, adult BM HSPCs are more efficient at reconstituting conventional B and T lymphocyte pools as opposed to the B-1a and fetal γδ-T cell subsets. Although the development of these innatelike lymphocyte subsets requires recombination activating gene (RAG)–mediated V(D)J recombination at the T or B cell antigen receptor loci, their repertoire has been described as semi-invariant or oligoclonal (9). Similarly to cells of the innate immune system, they are often strategically localized in the body to respond rapidly to a limited set of conserved antigens. Despite their roles as potent mediators of host defense, the molecular basis for their preference to develop in the fetus or neonate largely remains a mystery. The ability to generate B-1a B cells and Vγ3+ γδ-T cells [see (10) for nomenclature used] is intrinsically programmed in the FL HSCs (5, 10, 11). Sox17 was identified as a transcription factor that is specifically expressed in and required for the maintenance of fetal but not adult HSCs (12). Thus, transcriptional control can distinguish the fetal from adult HSC fate. In this study, we show that a layer of posttranscriptional control involving Lin28b and let-7 microRNAs (miRNAs) contributes to the distinct differentiation potential of these two stem cell types.

Lin28b and the let-7 family of miRNAs are differentially expressed in lymphoid progenitors originating from fetal and adult HSCs. To identify the molecular differences between fetal and adult lymphoid progenitors, we performed global miRNA-expression profiling of FL and adult BM progenitor B (pro-B) cells [B220+CD19+CD24+CD43+ lacking immunoglobulin M (IgM)]. This population was chosen because we could readily sort sufficient numbers of these progenitors to high purity, and this choice led to the observation that expression of the let-7 family of miRNAs was largely absent in FL pro-B cells. In the mouse genome, there are 12 let-7 paralogs that result in the expression of nine distinguishable mature miRNA isoforms (isomiRs) (fig. S1). The let-7 miRNAs are broadly and highly expressed in the hematopoietic system (13). To our surprise, the eight let-7 isomiRs that were detectable in adult BM pro-B cells were either significantly reduced in expression or undetectable in FL pro-B cells (Fig. 1A). From the same fluorescence-activated cell sorting (FACS), we had isolated the B220+CD19CD24CD43+IgMprecursors to the pro-B cells (pre-pro-B cells). We determined that mature let-7g and let-7c isomiRs were abundantly expressed in pre-pro-B cells from adult BM but not from FL using quantitative reverse transcription polymerase chain reaction (QRT-PCR) (Fig. 1B). Next, we examined whether the observed global repression of let-7 miRNAs in fetal B cell progenitors was mediated by endogenous Lin28 or Lin28b, encoded by two evolutionarily conserved paralogs, known to specifically block the biogenesis of let-7 miRNAs posttranscriptionally by binding to the terminal loop region of the let-7 primary or precursor miRNAs (pri- or pre-miRNAs) (1417). We determined in FACS-sorted populations that Lin28b mRNA was abundant in all stages of FL B cell development (Fig. 1C) and also in FL lineage (Lin)Sca-1+c-Kit+ (LSK) HSCs and LinSca-1intc-Kitint interleukin-7 receptor α chain–positive (IL7Rα+) common lymphoid progenitors (CLPs) (Fig. 1D) but absent in the corresponding populations in adult BM. In contrast, Lin28 mRNA was not detectable in fetal HSPCs (fig. S2A). These findings indicate that Lin28b expression is limited to HSPCs of fetal origin and could account for the observed reduction in mature let-7 expression.

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), γδ-T (γδ-TCR+CD3+), DP (CD4+CD8+CD3lo), and CD4SP (CD4+CD8CD3+). All thymocyte subsets except for the γδ-T cells were also gated through a γδ-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.

Classic fetal thymus (FT) graft studies suggest that fetal HSPCs seed the thymus between 10 and 13 days of gestation, whereas cells derived from adult HSPCs begin to substantially dilute the first-generation thymocytes at day 7 after birth and have largely replaced them by 2 weeks of age (18). To test whether Lin28b mRNA expression in the thymus correlates with the presence of fetal HSC-derived cells, we performed QRT-PCR on various FACS-sorted thymocyte populations ranging from the double-negative (DN)2 and DN3 (CD4CD8CD25+CD44int) cells to the CD4+CD8 single-positive (CD4SP) stage from 1-day-old, 1-week-old, and 4-week-old mice. We observed abundant Lin28b transcripts in all the tested thymocyte subsets from 1-day-old thymi, with the greatest amount observed in the γδ-T cell receptor–positive (γδ-TCR+) thymocytes (Fig. 1E). The abundance of endogenous Lin28b mRNA expression was dramatically decreased in thymocytes by 7 days after birth and was undetectable by 4 weeks of age. Declining Lin28b expression coincided with rising levels of mature let-7g (fig. S2B). These data demonstrate that endogenous Lin28b mRNA expression declines in concordance with the decrease in the proportion of thymocyte progenitors of fetal origin in the postnatal mouse. It is noteworthy that Lin28b expression was not maintained in the thymus or peripheral lymphoid populations in sublethally irradiated adult Rag1–/– mice reconstituted with FL HSPCs (fig. S2C). These findings suggest that Lin28b expression is turned on during fetal hematopoiesis but not sustained during ontogeny.

To determine whether these observed fetal signatures in mouse HSPCs are conserved in humans, we compared Lin28b and let-7g expression in human FL, FT, FS, and CD34+ umbilical cord blood (CB) cells with that in adult BM, lymph node, and thymus. As in mice, Lin28b mRNA expression was exclusively detected in fetal human hematopoietic tissues (Fig. 1F), whereas mature let-7g was more abundant in the adult (fig. S2D). Taken together, these expression analyses reveal a previously unknown molecular difference between HSPCs of fetal and adult origin that is conserved between human and mice and prompted us to identify a heterochronic role of the Lin28b–let-7 axis in regulating hematopoiesis during mammalian ontogeny.

Ectopic Lin28 expression in adult BM HSPCs represses the let-7 family of miRNAs and allows multilineage reconstitution. To test whether ectopic expression of Lin28 can confer characteristics of fetal lymphopoiesis in adult mice, we used a retrogenic BM chimera system. Lin28 was chosen because an excellent antibody was available to confirm its expression. HSPCs from lineage-depleted adult BM of wild-type C57BL/6 mice were transduced with a Lin28-encoding retrovirus and transplanted into sublethally irradiated Rag1–/– recipients. Full-length mouse Lin28 cDNA was cloned into a mouse stem cell virus–based vector upstream of an internal ribosomal entry site followed by green fluorescent protein (GFP) (Lin28-RV) (19). Lineage-depleted C57BL/6 bone marrow cells from adult mice were transduced with Lin28-RV or control GFP-RV ex vivo and injected intravenously into sublethally irradiated Rag1–/– recipients (fig. S3A). The initial transduction efficiency varied between 20 and 60%, as determined by the frequency of GFP+ cells 24 hours postinfection (Fig. 2A). HSPCs transduced with Lin28-RV mediated multilineage reconstitution with grossly normal proportions of T, B, and myeloid cells in the peripheral lymphoid organs 6 to 8 weeks after transplantation in recipient mice hereafter referred to as Lin28-RV BM chimeras (Fig. 2B and fig. S3). Ectopic Lin28 protein expression was confirmed by Western blot in thymocytes of Lin28-RV BM chimeras (Fig. 2C). To assess the effect of ectopic Lin28 protein on miRNA expression, we performed global miRNA profiling of sorted GFP+ and GFP preselection double-positive (DP) thymocytes (CD3loCD4+CD8+) (fig. S4A). Our results demonstrate that the impact of Lin28-RV on miRNA expression is dramatic and highly specific to the let-7 family members (Fig. 2D). Diminished expression of mature let-7a and let-7g was further validated by QRT-PCR (Fig. 2E). To assess the impact on the transcriptome, we performed deep sequencing of cDNA generated from poly(A)+ RNA (RNA-Seq), comparing sorted GFP+ and GFP preselection DP thymocytes in Lin28-RV BM chimeras (fig. S4). Statistical analysis using the Sylamer software demonstrated enrichment only of the complementary let-7 family heptameric seed sequence TACCTCA in 3′ untranslated regions (3′UTRs) of genes up-regulated in GFP+ DP thymocytes (fig. S5) and not the 969 other heptameric seed sequences that were also tested. Out of the 175 up-regulated genes containing this seed motif and passing Bonferroni-corrected E-value threshold of 0.01 identified using Sylamer, we observed a higher-than-expected overlap of 47 and 67 genes with let-7 target genes phylogenetically predicted by PicTar and TargetScan, respectively (tables S1 and S2) (20, 21). Thus, Lin28-RV mediated a dramatic decrease in steady-state levels of mature let-7 miRNAs in our retrogenic system with minimal effects on other miRNAs and resulted in the specific global derepression of putative let-7 target mRNAs. These data reveal that, despite abundant let-7 expression throughout the adult hematopoietic system, we can readily knock down their expression by Lin28-RV transduction without grossly inhibiting multilineage reconstitution.

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.

Lin28 reprograms adult BM HSPCs to undergo fetal-like B lymphopoiesis. It has been reported that FL HSPCs preferentially differentiate into B-1a cells compared with adult HSPCs (6, 7). To test for the effect of Lin28-RV transduction on the development of B-1a cells in adult mice, the peritoneal cavities of Lin28 retrogenic BM chimeras were analyzed 6 to 8 weeks posttransplant for donor-derived B cells. Remarkably, Lin28-RV–transduced adult HSPCs efficiently generated the B-1a subset, whereas untransduced and GFP-RV–transduced adult HSPC controls were biased toward the generation of conventional B-2 cells (Fig. 3, A and B, and fig. S6). In addition to the B-1a subset, growing evidence suggests that a substantial component of the MZ B cell compartment is also of fetal origin (8, 22, 23). Consistent with this notion, Lin28-RV–transduced HSPCs reproducibly gave rise to an increased percentage of B220+CD1d+CD23 splenic MZ B cells compared with their untransduced (GFP) or GFP-RV–transduced controls (Fig. 3, C and D). Similar results were obtained from human Lin28b-RV BM chimeras (fig. S7, A and B). These data indicate that the Lin28b–let-7 axis may account for the long-known differences between fetal and adult fate bias in B cell development and that ectopic Lin28 or Lin28b expression in HSPCs is sufficient to confer fetal-like B lymphopoiesis.

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.

Signaling through IL7Rα has been reported to be required for adult B cell development but dispensable for fetal B lymphopoiesis (1). To further examine the notion that ectopic Lin28 expression reprograms adult BM HSPCs to acquire fetal-like B cell development, we investigated whether Lin28-RV could restore B cell potential in adult Il7rα–/– BM HSPCs. Recipients reconstituted with Il7rα–/– HSPCs transduced with Lin28-RV were analyzed 7 weeks posttransplant for the presence of B cells in the peritoneal cavity and the spleen. In accordance with previous reports, control GFP IL7Rα-deficient donor cells failed to give rise to any detectable population of mature CD19+B220+ B cells. In contrast, sizable populations of GFP+ Lin28-induced B cells were detected in peritoneal cavities and spleens of Lin28-RV BM chimeras (Fig. 3E). Reminiscent of fetal lymphopoiesis, we observed an overrepresentation of the B-1a and MZ B cell lineages. These results demonstrate that Lin28 acts as a potent reprogramming factor in adult HSPCs to confer fetal-like aspects of B cell development that include independence from IL7Rα-mediated signaling.

Ectopic Lin28 expression favors the development of innatelike T cells. Adult thymocytes mainly differentiate into αβ-T cells but also Vγ2-, Vγ1.1-, and Vγ5-expressing γδ-T cell subsets, whereas fetal thymocytes preferentially rearrange the Vγ3 and Vγ4 TCR gene segments (24). In addition, one subpopulation of Vγ1.1+ T cells is the CD4+Vγ1.1+Vδ6.3+ innatelike T cell subset and has been described to develop perinatally, originating primarily from late embryonic precursors (25). We observed a >15-fold increase in the percentage of total γδ-TCR+ CD3+ cells among GFP+ thymocytes in Lin28-RV BM chimeras compared with either the GFP thymocytes in the same mouse or GFP+ thymocytes in the GFP-RV BM chimeras (Fig. 4, A and B). The Lin28-induced γδ-T cells are primarily CD4SP (Fig. 4C), which contributes to an expansion of the overall CD4SP thymocyte compartment (fig. S8A), and do not express the adult-specific Vγ2-TCR chain or the fetal-specific Vγ3-TCR chain. The latter is expected, as the development of Vγ3+ T cells is known to require both fetal HSCs and a fetal thymus microenvironment (5). Remarkably, we identified Lin28-induced γδ-T cells as almost exclusively carrying the Vγ1.1+Vδ6.3+ γδ-TCR that normally develops perinatally (Fig. 4D). Both the innatelike CD4+Vγ1.1+Vδ6.3+ and Vα14+Jα18+ invariant NKT (iNKT) cells are known to express the promyelocytic leukemia zinc finger protein (PLZF) transcription factor, and the development of PLZF+ CD4 T cells is a physiological process in humans during fetal and perinatal stages of ontogeny (26). Indeed, we were able to demonstrate an increased representation of PLZF+ γδ-TCR+CD3+ thymocytes in Lin28-RV BM chimeras (fig. S8B). The ability of Lin28 to induce γδ-T cell development correlates with the finding that γδ-TCR+ thymocytes naturally express the highest levels of Lin28b in neonatal mice (Fig. 1E). In line with our findings regarding B cell development, these data support the ability of Lin28 to confer fetal-like lymphopoiesis in the γδ-T cell lineage.

Fig. 4

Ectopic Lin28 expression in adult BM HSPCs promotes the development of Vγ1.1+Vδ6.3+ T cells. (A) Flow cytometric analysis to enumerate γδ-TCR+ CD3+ cells in thymi of GFP-RV and Lin28-RV BM chimeras. (B) Percentages of γδ-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+ γδ-TCR+ CD3+ thymocytes in the Lin28-RV (red line) and GFP-RV BM (blue line) chimeras. (D) Flow cytometric clonotype analysis of γδ-TCR+ CD3+ thymocytes in the GFP+ and GFP fractions of the Lin28-RV BM chimeras.

Although the iNKT cell lineage has not been directly demonstrated to preferentially arise from fetal precursors, several groups have described a transient burst of rapidly proliferating iNKT cell precursors around day 12 after birth (27, 28), consistent with a biased ontogenic window of development. Our discovery of an increased PLZF+ thymocyte population in thymi of Lin28-RV BM chimeras prompted us to further evaluate the effect of Lin28 on iNKT cell development. We observed a 100-fold increase in the percentage of iNKT (CD1dPBS57+CD3+) cells among GFP+ thymocytes in the Lin28-RV BM chimeras compared with the GFP thymocytes within the same mouse or a 30-fold increase compared with the GFP+ thymocytes in the GFP-RV BM chimeras (Fig. 5, A and B). This dramatic increase in thymic iNKT cells contributes to a Lin28-induced expansion of the overall CD4SP and DN thymocyte compartments (fig. S8A), as well as the PLZF+ thymocyte pool (fig. S8B). A dramatic increase in the frequency of GFP+ iNKT cells was also observed in the spleen, lymph node, and liver of Lin28-RV BM chimeras (Fig. 5B), translating to higher-than-expected iNKT cell absolute numbers in the spleen (fig. S8C). Positive selection of iNKT cells relies on thymocyte-thymocyte interactions mediated by CD1d (29). To determine whether CD1d is required for the development of Lin28-induced iNKT cells, we reconstituted Rag1–/– recipients with Cd1d–/– HSPCs transduced with Lin28-RV. No iNKT cells were detectable in these recipients, which was consistent with the notion that their positive selection requires CD1d (fig. S9).

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.

Because it is not clear whether iNKT cells develop early during ontogeny, we performed flow cytometric analysis to resolve the proposed developmental stages of the iNKT cell lineage subsequent to the DP thymocyte stage: stage 1 (CD44NK1.1), stage 2 (CD44+NK1.1), and stage 3 (CD44+NK1.1+) (27). The composition of the Lin28-induced thymic CD3+CD1dPBS57+ population resembled that of a 2-week-old thymus, characterized by a marked increase in the percentage of immature stage 1 iNKT cells (Fig. 5, C and D). Because DP thymocytes are precursors of stage 1 iNKT cells, we quantified the efficiency with which DP thymocytes in wild-type C57BL/6 mice of varying ages give rise to stage 1 iNKT by calculating the ratio of stage 1 iNKT cells to DP thymocytes. Consistent with previous reports, our results indicate a peak in iNKT cell potential between 2 and 4 weeks of age, followed by a sharp decline (Fig. 5, D and E). Thus, our data point toward a higher iNKT cell potential in DP thymocytes of embryonic or neonatal HSPC origin.

Increased numbers of thymic PLZF+ innatelike T cells can produce large amounts of IL-4 and thereby promote thymic development of Eomesodermin (Eomes)+ memory-like CD8+ T cells, characterized by high surface expression of CD44 and low surface expression of CD24 (3032). This phenomenon is not normally detectable in adult wild-type C57BL/6 mice because of the infrequency of PLZF+ thymocytes in this strain (31, 33). However, we observed a significant increase in memory-like CD8+ T cells among both GFP+ and GFP thymocytes in Lin28-RV BM chimeras but not in the GFP-RV control chimeras (fig. S10). This result demonstrates the ability of Lin28-induced PLZF+ innatelike T cells to support the differentiation of memory-like CD8+ T cells within the thymus and is consistent with the well-characterized IL4-mediated bystander effect that is not cell intrinsic (34).

Ectopic Lin28 confers fetal-like signatures to adult LSK HSPCs. It has been reported that the potential to develop into innatelike lymphocytes is intrinsically programmed in HSCs (5, 11). Therefore, we performed cell cycle and gene expression analyses to look for Lin28-induced fetal characteristics in the BM HSCs of Lin28-RV chimeras. Cell cycle analysis of Lin28-RV–transduced BM HSPCs in culture compared with controls did not reveal any significant differences (fig. S11B). However, this is an artificial system that is highly mitogenic for HSPCs. Furthermore, a number of surface markers, including CD34, CD93 (AA4.1), and CD11b, are preferentially expressed on FL HSCs, likely because of their heightened proliferative state (3540). We did not detect any Lin28-induced changes in the expression of these markers, nor that of endothelial cell-selective adhesion molecule (ESAM), CD48, and Tie-2 within the LSK HSC compartment in Lin28-RV chimeras (fig. S12A). Taken together, our results suggest that ectopic Lin28 does not grossly affect the cell intrinsic proliferative capacity or the overall composition of the adult LSK HSC compartment and that increased proliferation alone cannot account for the fetal-like developmental potential of HSPCs.

To characterize the effect of ectopic Lin28 on the long-term competence of HSCs, we performed FACS and QRT-PCR analysis of known long-term reconstituting (LT) HSC markers on GFP+ and GFP LSK HSCs from aged Lin28-RV and GFP-RV BM chimeras 10 months after transplantation. CD150 surface expression marks LT HSCs (41), and Mecom (Evi1) and Tal1 encode for transcription factors critical in regulating HSC quiescence and long-term competence (42, 43). The presence of GFP+ Lin28-RV–transduced LSK HSCs 10 months after adoptive transfer, along with comparable expression of surface CD150 protein and Evi1 and Tal1 mRNA between Lin28-RV and control LSK HSCs, suggests that ectopic Lin28 does not interfere with LT HSCs (fig. S12).

Hmga2 and Igf2 have been reported to be targets of the Lin28–let-7 axis (4446). Consistent with the finding that Lin28b is critical in conferring fetal HSPC identity, both Hmga2 and Igf2 are highly expressed in fetal but not adult HSCs (47). To assess whether ectopic Lin28 confers a fetal-like gene expression profile in adult BM HSCs, we performed QRT-PCR of FACS-sorted LSK HSCs and detected marked enhancement in the expression of both genes in the GFP+ fraction from Lin28-RV BM chimeras (fig. S12B). Collectively, these results demonstrate the ability of ectopic Lin28 expression to confer fetal-like gene expression signatures in LSK HSCs.

In summary, these results identify Lin28b expression as a key molecular feature distinguishing fetal from adult BM HSPCs in mice and humans, which sheds new understanding on a long-standing question in the field of lymphocyte development (fig. S13). We demonstrate that ectopic expression of Lin28 in adult murine BM HSPCs dramatically skews the lymphocyte repertoire toward the production of innatelike lymphocyte subsets. Thus, we propose that Lin28b is a master regulator gene for fetal HSPC identity that is capable of reprogramming adult BM HSPCs to acquire fetal-like characteristics in a cell autonomous manner. As a result, Lin28b is a switch that turns on the development of major subsets of innatelike lymphocytes. Together, these findings highlight how posttranscriptional regulation of gene expression can be pivotal in determining cell-fate decisions.

Our findings have potential clinical implications for improving the reconstitution of innatelike lymphocyte populations on adult BM transplantation. Most HSC transplants utilize adult BM, and it is not known whether B-1 and MZ B cells develop after hematopoietic reconstitution. Because of the scarcity and ethical limitations of human fetal HSCs, they have not been seriously considered for clinical use. Umbilical cord blood is used clinically, but typically contains too few HSCs to reconstitute an adult (48). In the mouse, innatelike B-1 cell subsets play a role in T-independent host defense against pathogens such as Streptococcus pneumoniae (49); protective innate response activator B cells that produce granulocyte-macrophage colony-stimulating factor differentiate from B-1a cells during sepsis (50); Vδ6.3+ γδ-T cells provide critical protection against Listeria monocytogenes (51). Furthermore, the ability of iNKT cells to produce large amounts of cytokines, including interferon-γ, IL-4, and tumor necrosis factor, allows them to modulate a broad spectrum of diseases, including cancer, graft rejection, autoimmunity, and infectious diseases (52). Thus, given the growing awareness of the ability of innatelike lymphocytes to influence immunity, it is of clinical interest to determine whether fetal-like HSCs can more effectively achieve reconstitution of the full continuum of the innate and adaptive immune system.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1216557/DC1

Materials and Methods

Figs. S1 to S13

Tables S1 and S2

References (5359)

References and Notes

Acknowledgments: We thank W. E. Paul for his generous support and advice throughout; T. Bender, J. Daniel, B. J. Fowlkes, R. Germain, M. Lenardo, W. E. Paul, and M. Schlissel for critical reading of this manuscript and constructive suggestions; K. Laky and B. J. Fowlkes for valuable advice and antibodies; NIH Tetramer Core Facility for reagents; M. Holt for NKT cell discussions; P. Burr for sequencing; J. Edwards and C. Eigsti for cell sorting; M. Foster for animal care; R. Zahr for technical support; and N. Bartonicek (EMBL-EBI, Hinxton, UK) for providing the RefSeq 3′UTR library for Sylamer analysis. We gratefully acknowledge the high-performance computational capabilities of the Biowulf Linux cluster at the NIH (http://biowulf.nih.gov) and NIAID Office of Cyber Infrastructure and Computational Biology High-Performance Computing cluster required for massively parallel sequencing analyses. The data reported in this paper are tabulated in the main text and in the Supporting Online Material. The RNA-Seq and NanoString data are available in the Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo) under the accession nos. GSE34854, GSE35081, and GSE35107. The materials and reagents used in the paper are available under a Materials Transfer Agreement. We apologize that we could not cite all the relevant references because of space limitations. The Integrative Immunobiology Unit is supported by the Intramural Research Program of the NIAID, NIH. The authors declare no competing financial interests.
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