Abstract
The roles of the reprogramming factors Oct4, Sox2, c-Myc and Klf4 in early T cell development are incompletely defined. Here, we show that Klf4 is the only reprogramming factor whose expression is downregulated when early thymic progenitors (ETPs) differentiate into T cells. Enforced expression of Klf4 in uncommitted progenitors severely impaired T cell development mainly at the DN2-to-DN3 transition when T cell lineage commitment occurs and affected the transcription of a variety of genes with crucial functions in early T cell development, including genes involved in microenvironmental signaling (IL-7Rα), Notch target genes (Deltex1), and essential T cell lineage regulatory or inhibitory genes (Bcl11a, SpiB, and Id1). The survival of thymocytes and the rearrangement at the Tcrb locus were impaired in the presence of enforced Klf4 expression. The defects in the DN1-to-DN2 and DN2-to-DN3 transitions in Klf4 transgenic mice could not be rescued by the introduction of a TCR transgene, but was partially rescued by restoring the expression of IL-7Rα. Thus, our data indicate that the downregulation of Klf4 is a prerequisite for T cell lineage commitment.
Similar content being viewed by others
Introduction
Mouse somatic cells can be reprogrammed into pluripotent stem cells by ectopic expression of the reprogramming factors Oct4, Sox2, c-Myc and Klf4, which are highly expressed in embryonic stem (ES) cells 1. These four genes are the most powerful factors involved in the regulation of the transcriptional network that is necessary for the maintenance of pluripotency and self-renewal of ES cells 2. Presumably, the expression of these genes, especially Oct4, Sox2 and Klf4 may need to be turned off or downregulated during the differentiation of pluripotent cells into a variety of differentiated derivatives.
Early T cell development is a multi-stage process in which progenitors gradually acquire T cell characteristics while losing their developmental plasticity or “stemness” 3. T cells are derived from lineage (Lin)−Sca1+Kit+Flt3− hematopoietic stem cells (HSCs) that reside in the bone marrow (BM). HSCs progress to multipotent progenitors (MPPs) and lymphoid-primed MPPs (LMPPs), 4 and then differentiate into Lin−Sca1lowKitlowFlt3hiIL-7Rαhi common lymphoid progenitors (CLPs). CCR7+ CCR9+ LMPPs and CLPs enter the thymus via the blood circulation and become ETPs with multi-lineage potential to give rise to T cells, dendritic cells (DCs), myeloid cells, natural killer (NK) cells, and B cells 3. As these progenitors receive signals, including Notch, c-Kit, and IL-7, from the thymic microenvironment 5, they initiate T cell differentiation and gradually lose their developmental potential for non-T cell lineages. CD4−CD8− double negative (DN) thymic progenitors consist of DN1 (CD44+CD25−), DN2 (CD44+CD25+), DN3 (CD44−CD25+), and DN4 (CD44−CD25−) populations 6, 7. DN1 and DN2 cells retain both T cell and non-T cell lineage developmental potential 8, whereas final T cell lineage commitment occurs after an irreversible transition from DN2 to DN3 when most T cell-specific genes are upregulated to a peak level 3, 5. Thus, the differentiation of uncommitted thymic progenitors into T cells is a process that can be used to explore the potential relationship between reprogramming factors and differentiation.
The four reprogramming factors have been shown to participate individually in developmental processes other than reprogramming. For example, c-Myc is required for the proliferation of thymocytes at the preTCR checkpoint 9, and Klf4 is a key regulator of monocyte development 10, B-cell numbers and activation-induced B-cell proliferation 11, and quiescence of CD8+ T cells 12, and is required for the production of IL-17 13. Gene expression profiling studies have also shown that Klf4 is expressed in mouse and human thymocytes 14, 15. However, with the exception c-Myc, the roles of the reprogramming factors in early T cell lineage commitment are not well-defined.
In this study, we examined the expression patterns of the reprogramming factors in BM progenitors and thymocyte populations. Klf4 was the only reprogramming factor that was downregulated during the differentiation of ETPs into T cells. Enforced Klf4 expression in thymocytes led to impaired transcription of many pivotal genes that specify T cell lineage and resulted in the inhibition of thymocyte survival and Tcrb rearrangement. Moreover, the impaired T cell lineage commitment in Klf4 transgenic mice could not be rescued by the introduction of a TCR transgene, but could be rescued to a certain extent by restoring IL-7Rα expression. Thus, downregulation of the transcription factor Klf4 is required for the lineage commitment of T cells.
Results
Expression of reprogramming factors during the differentiation of HSCs into T cells
We examined the mRNA levels of the reprogramming factors (Oct4, Sox2, c-Myc and Klf4) in BM progenitor and thymocyte populations. All four genes were highly expressed in ES cells, as previously reported 1. Oct4 and Sox2 mRNA levels became undetectable in HSCs and at later stages of differentiation (Figure 1A and 1B), whereas c-Myc was expressed at higher levels at many differentiation stages, including in DN3 and DN4 cells, compared to ES cells (Figure 1C). This is in agreement with a previous report that c-Myc is required for pre-TCR-induced proliferation 9. Interestingly, Klf4 expression was decreased in HSCs, CLPs and DN1 thymocytes compared to ES cells, and Klf4 levels continued to decrease during the transition from DN1 to DN2. Klf4 levels finally became undetectable in DN3 cells and at later stages of differentiation (Figure 1D), which specifically correlates with T cell lineage specification.
Enforced expression of Klf4 in ETPs blocks T cell lineage commitment at the DN2-to-DN3 transition
To investigate whether the downregulation of Klf4 is required for T-cell lineage specification, we generated Klf4 transgenic mice (Klf4Tg), in which Klf4 expression was driven by the human CD2 promoter and enhancer, allowing continuous expression of Klf4 in thymocytes at the DN1 (including ETPs and CD117-DN1 cells, Figure 2A) and subsequent differentiation stages (Figure 2B) 16. Five transgenic founders with similar phenotypes were generated and one line with Klf4 protein levels comparable to the normal levels in DN1 was used in this study (Figure 2C and 2D).
To examine the effects of continuous expression of Klf4 on T cell lineage specification, we analyzed DN cells after gating out cells expressing lineage markers (Lin: CD4, CD8a, CD3e, B220, Mac-1, Gr-1 and Ter-119). In contrast to wild-type littermates (Litt), DN cells from Klf4Tg mice consisted of a very low percentage of DN3 and DN4 T-lineage-committed cells (Figure 2E). We also noticed that DN2 cells did not clearly separate from DN1 cells, and a DN1-DN2 transitional population characterized as Lin−CD44+CD25low accumulated, which suggests a partial arrest at the DN1-to-DN2 transition in Klf4Tg mice. In terms of absolute numbers, Klf4Tg mice did not have reduced levels of DN1 and DN2 thymocytes compared with Litt mice, but DN3 thymocytes were dramatically reduced by 28-fold (Figure 2F). Accordingly, the number of thymocytes at later stages of differentiation, including DN4, DP, CD4 SP and CD8 SP, and thus the total thymic cellularity was also significantly reduced (Figure 2F and 2G).
To further characterize DN2 thymocytes from Klf4Tg mice, we analyzed the surface expression of CD117 (c-KIT), which is essential in the earliest precursors (DN1 and DN2) but is gradually downregulated at the DN3 stage 17, 18. As shown in Figure 2H, CD117 protein levels were higher in Klf4Tg DN2 thymocytes compared to DN2 or DN3 thymocytes from Litt mice, indicating a blockage of the DN2-to-DN3 transition.
To exclude the possibility that enforced expression of Klf4 “disguises” DN3 cells as DN2 cells by upregulating CD44 expression, the Klf4 transgene was introduced into mice on a CD44−/− background 19. We found that the generation of T-lineage-committed cells (DN3) remained impaired in Klf4Tg CD44−/− mice (Supplementary information, Figure S1A and S1B). We also observed a certain percentage of DP thymocytes in adult Klf4Tg mice (Figure 2I) and we questioned if this percentage is age-dependent in Klf4Tg mice. To this end, we analyzed thymocytes from newborn Litt and Klf4Tg mice. In contrast to the predominance of CD4+CD8+ DP cells in Litt thymi, the thymi from newborn transgenic mice contained an overwhelming majority of DN cells (Figure 2I), which consisted only of uncommitted DN1 and DN2 thymic progenitors (Figure 2E). Moreover, DN2 thymocytes from newborn Klf4Tg mice also expressed higher levels of CD117 (Figure 2H). Thus, the impairment was mainly at the DN2-to-DN3 transition and not at the DN-to-DP transition. This was clearly observed in newborn Litt and Klf4Tg mice; a small number of DN3 and later stage (DN4, DP and SP) thymocytes accumulated with age, which led to a certain percentage of DP thymocytes in adult Klf4Tg mice. Taken together, these data suggest that enforced Klf4 expression in thymocytes severely impairs T cell development mainly at the DN2-to-DN3 transition during which T cell lineage commitment occurs.
The transcription of a variety of genes pivotal in specifying the T cell lineage is altered in Klf4Tg mice
To understand the molecular mechanism behind the inhibition of the DN1-to-DN2 or DN2-to-DN3 transition by continuous expression of Klf4, we purified DN1 and DN2 thymocytes, and analyzed the differences in gene expression between Klf4Tg and Litt mice. We examined the expression of genes that are well-known for their roles in differentiation and regulation of early T-cell development 3, 20, including genes involved in microenvironmental signaling, Notch target genes, pre-TCR assembly and signaling genes, and essential T cell lineage regulatory genes (Figure 3).
We observed that the expression of some T cell-associated genes, including preTCRα, Rag2, Lat, Runx1, Bcl11b and HebCan, was higher in Klf4Tg DN1 and Klf4Tg DN2 cells than in Litt DN1 cells, but lower than in Litt DN2 cells. These characteristics are consistent with the Klf4Tg phenotype in which many DN1-DN2 transitional (Lin−CD44+CD25low) cells are inevitably included in the DN1 or DN2 population (Figure 2E). Remarkably, compared with Litt, there was a group of genes that exhibited similar changes in expression in both DN1 and DN2 cells from Klf4Tg mice; Deltex1, Ets2, SpiB and Id1 were upregulated, while IL-7Rα, Runx3, Ets1, Bcl11a and Id2 were downregulated. Collectively, these results suggest that enforced expression of Klf4 inhibits T cell lineage commitment by affecting the transcription of these key genes.
DN1 and DN2 cells do not deviate into other lineages in Klf4Tg mice
Given that Klf4 may function in regulating the cell cycle of lymphocytes 11, 12, we performed cell size and cell cycle analyses to determine whether enforced expression of Klf4 blocks the DN2-to-DN3 transition by inhibiting the proliferation of DN2 cells. As shown in Figure 4A, in Klf4Tg DN2 cells, the percentage of larger cells and cells in the S/G2/M phase of the cell cycle are not reduced. Thus, the proliferation of DN2 cells does not seem to be inhibited in Klf4Tg mice.
DN2 cells are able to differentiate into two different T cell lineages, namely, αβ T cells and γδ T cells 3, 21. To determine if enforced expression of Klf4 also impairs the generation of γδ T cells, we performed flow cytometry analysis of CD4−CD8− DN cells and observed a reduction of γδ T cells in Klf4Tg mice (Figure 4B and 4D). This suggests that enforced expression of Klf4 inhibits both αβ and γδ T cell lineage commitment. To further explore the fate of Klf4Tg DN1 and DN2 thymocytes, we analyzed thymocytes for markers of B cells, NK cells, NK-T cells, monocytes, granulocytes, mast cells and DCs by flow cytometry, but no significant augmentation of these non-T lineage cells was observed (Figure 4C and 4D). Taken together, these data indicate that Klf4Tg DN1 and DN2 cells do not differentiate into other lineages.
Thymocyte survival and Tcrb recombination are impaired in the presence of enforced Klf4 expression
We examined the survival of thymocytes and observed an increase in apoptosis in DN2 and DN3 cells from Klf4Tg mice (Figure 5A). Considering that the arrest at the DN2-to-DN3 transition might lead to an accumulation of DN2 cells, increased apoptosis could be the reason why Klf4Tg mice were able to retain similar DN2 cellularity as Litt mice, even though Klf4Tg DN2 thymocytes failed to differentiate further.
Pre-TCR signaling is necessary for DN3 cell survival 22, and rearrangement at the Tcrb locus is a hallmark of T cell lineage commitment 23. As shown in Figure 5B and 5C, an impairment of Dβ2-Jβ2 and V-DJβ rearrangements and a reduction of TCRβ intracellular protein levels in DN3 and DN4 cells from Klf4Tg mice were observed. Notably, the surface expression levels of TCRβ were lower in periphery CD4+ T or CD8+ T cells from Klf4Tg mice and the distribution of TCR Vβ was somewhat altered in Klf4Tg mice (Supplementary information, Figure S2). Therefore, we introduced a functional TCRαβ (OT-I) transgene into the Klf4Tg mice. As shown in Figure 5D and Supplementary information, Figure S3A-S3E, T cell development was not rescued. These results provided further validation that the main defect in Klf4Tg mice is at the DN1-to-DN2 and DN2-to-DN3 transition, which could not be rescued by the introduction of an OT-1 transgene that promotes β-selection.
It has been reported that Notch, CD117 and IL-7 signaling in the thymic microenvironment are required for the survival and differentiation of DN1 and DN2 thymocytes 5, 17, 24, 25. While Klf4Tg DN2 thymocytes expressed even higher levels of CD117 (Figure 2H) and Notch1 expression was slightly reduced in Klf4Tg DN2 thymocytes, IL-7Rα mRNA (Figure 3) and protein (Figure 6A) expression was severely impaired in Klf4Tg DN1 and DN2 thymocytes. Thus, Klf4Tg DN1 and DN2 thymocytes were infected with a retroviral construct encoding the Notch1 intracellular domain (NICD) or IL-7Rα and then transferred to fetal thymus organ culture (FTOC). After 2 days of culture, compared with vector-infected Litt DN1 and DN2 cells (35.2% of DN3, Figure 6B), neither NICD nor IL-7Rα could rescue the differentiation of Klf4Tg DN1 and DN2 cells into DN3 cells, although there were higher percentages of DN2 cells in cells infected with NICD (55.6%) or IL-7Rα (75.8%) compared to those infected with vector (45.4%). However, when the culture time was significantly increased (6 days, Figure 6B), IL-7Rα promoted the differentiation of Klf4Tg DN1 and DN2 cells into DN3 cells (33.3%) compared with vector-infected cells (9.12%) to some extent. To summarize, enforced expression of Klf4 inhibited the survival of T lineage cells, and the defect in the DN1-to-DN2 and DN2-to-DN3 transition in Klf4Tg thymocytes could be rescued to a certain extent by restoring the expression of IL-7Rα.
Discussion
The transcription factors Oct4, Sox2, c-Myc and Klf4 are four reprogramming factors that can recapture the pluripotency lost during differentiation by regulating the transcriptional network that controls the pluripotency and self-renewal of ES cells 26, 27. However, their roles in multipotent adult stem cells and specific lineage precursor cells are poorly understood. Our results show that c-Myc is highly expressed, and Sox2 and Oct4 expression are “turned off” in HSCs. Only Klf4 expression persists in the HSC stage and is finally “turned off” during T cell lineage specification. Enforced expression of Klf4 in thymocytes affects the transcription of many pivotal genes specifying T cell lineage, leading to increased apoptosis of T lineage cells, and inhibits T cell lineage commitment. Our studies here provide new insight into roles of reprogramming factors, especially Klf4, in early T cell development.
We have shown here that continuous expression of Klf4 in uncommitted progenitors severely impairs T cell lineage commitment. However, thymic progenitor DN1 cells express relatively high levels of Klf4 and downregulation of Klf4 is essential for normal T cell lineage commitment. Thus, our studies support the view that gene regulations during early T cell specification are mostly to downregulate prethymically inherited genes like Klf4, rather than to upregulate new T cell-specific factors 28, 29. Klf4 expression seems to be turned off at the DN1-to-DN2 transition. Enforced expression of Klf4 leads to an arrest at the DN1-to-DN2 transition, with the result that Klf4Tg DN2 cells are not bona fide DN2 cells, but closer to DN1-DN2 transitional (Lin−CD44+CD25low) cells, which is supported by the gene expression characteristic analysis of DN1 and DN2 cells from Litt and Klf4Tg mice. The incompetence of DN2 cells leads to a block at the DN2-to-DN3 transition in Klf4Tg mice. In humans, NOTCH signaling downregulates KLF4 expression in the gut 30, 31. Interestingly, Notch signaling is also a positive regulator of T cell lineage and Notch ligands are found on stromal cells throughout the thymus 5, 32. These findings raise the possibility that Notch signals from the thymic microenvironment may negatively regulate Klf4 expression, which could in turn influence DN1 and DN2 cells to specify T cell lineage fate.
T cell specification takes place under a tightly controlled transcriptional hierarchy. The E protein (also named helix-loop-helix protein) and Notch signaling cooperate to promote early T cell development 33. HEBAlt, a long form of the E protein HEB gene, is specifically expressed in lymphoid precursor cells 34. In thymic precursors, HEBAlt collaborates with Notch signals to promote early T cell development by suppressing B cell or myeloid potential 35, 36. HEB−/− T cells possess compromised Notch1 function and lose T cell potential 37. The activity of E proteins is crucial for the DN2-to-DN3 transition and TCR gene rearrangement; loss of E2A leads to a reverse differentiation from DN3 cells to DN2 cells 38. E proteins lose their function under high levels of Id1 protein, which can inhibit the DNA-binding activity of E proteins. It has been reported that Id1 transgenic mice expressed high levels of Detex1, which inhibits the DN1-to-DN2 transition and leads to an accumulation of CD4−CD8−CD44+CD25low cells 39. This is similar to what is observed in Klf4Tg mice. Moreover, SpiB overexpression in DN2 thymocytes has been shown to lead to a blockage at the DN2-to-DN3 transition 40. Thus, the defects in Klf4Tg mice may partly be caused by elevated levels of Id1 and SpiB or decreased levels of HEBalt.
T cell lineage specification is driven by signals unique to the thymic microenvironment 5. IL-7 signaling is necessary to regulate the accessibility of the locus of TCRγ 41 and this might explain the reduction of γδ T cells in Klf4Tg mice, in which IL-7Rα expression is severely repressed. In addition, the IL-7/IL-7R pathway also plays a nonredundant role in early T cell development by facilitating survival, proliferation and TCR expression.5, 42, 43, 44, 45 Our results show that restoring the expression of IL-7Rα could rescue, to some extent, the defects in T cell lineage commitment in Klf4Tg mice, raising the possibility that enforced expression of Klf4 inhibits T lineage cell survival by repressing the transcription of IL-7Rα.
Materials and Methods
Generation of Klf4 transgenic mice
The Klf4 transgenic construct consisted of Klf4 cDNA driven by the human CD2 promoter and locus control region in the transgenic expression vector p29Δ2 (a gift from Paul Love). The transgenic mice were generated on a DBA/2 × C57BL/6 background and then backcrossed to C57BL/6 for at least ten generations. Transgene integrations of tail DNA were identified by PCR with the following primers. P1: 5′-TTGGCAAAGGAGCACATCAGA-3′; and P2: 5′-GTCTGGCAGGAAAGGAGGGTA-3′.
Mice
CD44−/− mice were obtained from the Model Animal Research Center, Nanjing University, China. C57BL/6 and OT-I TCR transgenic mice were obtained from Jackson Laboratory. Mice were analyzed between 4 and 8 weeks of age, except where otherwise indicated. All animal experiments were approved by the institutional animal use committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Flow cytometric analysis, cell purification and antibodies
Single-cell thymocyte or BM cell suspensions were prepared and stained as described 46. Lineage negative cells were enriched by magnetic bead depletion using a lineage cocktail containing biotin-conjugated antibodies, anti-biotin beads and a MACS separator (Miltenyi Biotec). Individual subsets of Lin− thymocytes or BM cells were obtained by cell sorting using a FACSAria II cell sorter (BD Biosciences) for Lin−Sca1+Kit+Flt3− (HSC), Lin−Sca1lowKitlowFlt3hiIL-7Rαhi (CLP), DN1 (Lin−CD44+CD25-), DN2 (Lin−CD44+CD25+), DN3 (Lin−CD44−CD25+) and DN4 (Lin−CD44−CD25−) subsets. DP thymocytes were isolated by cell sorting CD4+CD8+. Aliquots of sorted cells were reanalyzed to ensure purity, which was greater than 90%.
Antibodies (Abs) in lineage cocktails included Abs against CD11b, B220, CD4, CD8a, CD3e, Gr-1 and TER-119. Additional Abs used included Abs against CD44, CD25, TCRβ, TCRγδ and CD117. The Abs mentioned above were purchased from BD Pharmingen. Abs against IL7Ra, Sca1, Flt3 and Annexin V-FITC were from BioLegend. The anti-Klf4 (GKLF, H-180) Ab was from Santa Cruz Biotechnology.
Real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen). Contaminating genomic DNA was removed from RNA samples with the DNA-Free kit (Ambion). Reverse transcription was performed using the SuperScript III First-Strand kit (Invitrogen). Real-time PCR was performed on a Rotor-Gene 6000 (Corbett Life Sciences) with the SYBR green real-time PCR master mix (Toyobo). Relative expression levels were normalized to Gapdh or β-actin transcript levels and calculated using the 2−ΔΔCT method. The sequences of the primers used have been previously published 1, 20, 34, 47, 48, 49, 50 and are listed under Supplementary information, Table S1.
Retroviral transduction and fetal thymic organ culture
Murine cDNAs for the Notch 1 intracellular domain and Il-7Rα were amplified and verified by sequencing, and then cloned into the pMCs-IRES-GFP retroviral vector. Retroviral transduction and FTOC were performed as described previously 40. Briefly, sorted cells were infected, transduced with viral supernatant and seeded onto deoxyguanosine-depleted day 15 C57BL/6 host lobes in Terasaki microwell plate hanging drop cultures for 1 day, and the thymus lobes were then transferred to a freshly prepared filter/sponge for regular thymus organ culture conditions.
Statistical analysis
Student's t-test was used for the comparison of two independent groups, and probability values of P < 0.05 were considered significant. Statistical analyses of all numerical values in main figures are presented in Supplementary information, Figure S4.
References
Takahashi K, Yamanaka S . Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663–676.
Kim J, Chu J, Shen X, Wang J, Orkin SH . An extended transcriptional network for pluripotency of embryonic stem cells. Cell 2008; 132:1049–1061.
Rothenberg EV, Moore JE, Yui MA . Launching the T-cell-lineage developmental programme. Nat Rev Immunol 2008; 8:9–21.
Adolfsson J, Månsson R, Buza-Vidas N, et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 2005; 121:295–306.
Petrie HT, Zúñiga-Pflücker JC . Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu Rev Immunol 2007; 25:649–679.
Sitnicka E, Buza-Vidas N, Ahlenius H, et al. Critical role of FLT3 ligand in IL-7 receptor-independent T lymphopoiesis and regulation of lymphoid-primed multipotent progenitors. Blood 2007; 110:2955–2964.
Porritt HE, Rumfelt LL, Tabrizifard S, et al. Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages. Immunity 2004; 20:735–745.
Taghon T, Yui MA, Rothenberg EV . Mast cell lineage diversion of T lineage precursors by the essential T cell transcription factor GATA-3. Nat Immunol 2007; 8:845–855.
Dose M, Khan I, Guo Z, et al. c-Myc mediates pre-TCR-induced proliferation but not developmental progression. Blood 2006; 108:2669–2677.
Feinberg MW, Wara AK, Cao Z, et al. The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation. EMBO J 2007; 26:4138–4148.
Klaewsongkram J, Yang Y, Golech S, Katz J, Kaestner KH, Weng NP . Kruppel-like factor 4 regulates B cell number and activation-induced B cell proliferation. J Immunol 2007; 179:4679–4684.
Yamada T, Park CS, Mamonkin M, Lacorazza HD . Transcription factor ELF4 controls the proliferation and homing of CD8(+) T cells via the Kruppel-like factors KLF4 and KLF2. Nat Immunol 2009; 10:618–626.
Lebson L, Gocke A, Rosenzweig J, et al. Cutting edge: the transcription factor Kruppel-like factor 4 regulates the differentiation of Th17 cells independently of RORγt. J Immunol 2010; 185:7161–7164.
Tabrzifard S, Olaru A, Plotkin J, Fallahi-Sichani M, Livak F, Petrie HT . Analysis of transcription factor expression during discrete stages of postnatal thymocyte differentiation. J Immunol 2004; 173:1094–1102.
Dik WA, Pike-Overzet K, Weerkamp F, et al. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J Exp Med 2005; 201:1715–1723.
Boer Jd, Williams A, Skavdis G, et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol 2003; 33:314–325.
Massa S, Balciunaite G, Ceredig R, Rolink AG . Critical role for c-kit (CD117) in T cell lineage commitment and early thymocyte development in vitro. Eur J Immunol 2006; 36:526–532.
Ceredig R, Rolink T . A positive look at double-negative thymocytes. Nat Rev Immunol 2002; 2:888–897.
Graham VA, Marzo AL, Tough DF . A role for CD44 in T cell development and function during direct competition between CD44+ and CD44- cells. Eur J Immunol 2007; 37:925–934.
Franco CB, Scripture-Adams DD, Proekt I, et al. Notch/Delta signaling constrains reengineering of pro-T cells by PU.1. Proc Natl Aca Sci USA 2006; 103:11993–11998.
Rothenberg EV, Taghon T . Molecular genetics of T cell development. Annu Rev Immunol 2005; 23:601–649.
Michie AM, Zuniga-Pflucker JC . Regulation of thymocyte differentiation: pre-TCR signals and beta-selection. Semin Immunol 2002; 14:311–323.
Dik WA, Pike-Overzet K, Weerkamp F, et al. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J Exp Med 2005:1715–1723.
Kim K, Lee CK, Sayers TJ, Muegge K, Durum SK . The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways. J Immunol 1998; 160:5735–5741.
Ciofani M, Zuniga-Pflucker JC . A survival guide to early T cell development. Immunol Res 2006; 34:117–132.
Pei D . Regulation of pluripotency and reprogramming by transcription factors. J Biol Chem 2009; 284:3365–3369.
Yamanaka S . A fresh look at iPS cells. Cell 2009; 137:13–17.
David-Fung E-S, Butler R, Buzi G, et al. Transcription factor expression dynamics of early T-lymphocyte specification and commitment. Dev Biol 2009; 325:444–467.
Kawazu M, Yamamoto G, Yoshimi M, et al. Expression profiling of immature thymocytes revealed a novel homeobox gene that regulates double-negative thymocyte development. J Immunol 2007; 179:5335–5345.
Ghaleb AM, Aggarwal G, Bialkowska AB, Nandan MO, Yang VW . Notch inhibits expression of the Kruppel-like factor 4 tumor suppressor in the intestinal epithelium. Mol Cancer Res 2008; 6:1920–1927.
Real PJ, Tosello V, Palomero T, et al. Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia. Nat Med 2009; 15:50–58.
Rothenberg EV, Scripture-Adams DD . Competition and collaboration: GATA-3, PU.1, and Notch signaling in early T-cell fate determination. Semin Immunol 2008; 20:236–246.
Ikawa T, Kawamoto H, Goldrath AW, Murre C . E proteins and Notch signaling cooperate to promote T cell lineage specification and commitment. J Exp Med 2006; 203:1329–1342.
Dionne CJ, Tse KY, Weiss AH, et al. Subversion of T lineage commitment by PU.1 in a clonal cell line system. Dev Biol 2005; 280:448–466.
Wang D, Claus CL, Rajkumar P, et al. Context-dependent regulation of hematopoietic lineage choice by HEBAlt. J Immunol 2010; 185:4109–4117.
Braunstein M, Rajkumar P, Claus CL, et al. HEBAlt enhances the T-cell potential of fetal myeloid-biased precursors. Int Immunol 2010; 22:963–972.
Braunstein M, Anderson MK . HEB-deficient T-cell precursors lose T-cell potential and adopt an alternative pathway of differentiation. Mol Cell Biol 2011; 31:971–982.
Wojciechowski J, Lai A, Kondo M, Zhuang Y . E2A and HEB are required to block thymocyte proliferation prior to pre-TCR expression. J Immunol 2007; 178:5717–5726.
Wang HC, Perry SS, Sun XH . Id1 attenuates Notch signaling and impairs T cell commitment by elevating Deltex1 expression. Mol Cell Biol 2009; 29:4640–4652.
Lefebvre JM, Haks MC, Carleton MO, et al. Enforced expression of Spi-B reverses T lineage commitment and blocks beta-selection. J Immunol 2005; 174:6184–6194.
Durum SK, Candeias S, Nakajima H, et al. Interleukin 7 receptor control of T cell receptor gamma gene rearrangement: role of receptor-associated chains and locus accessibility. J Exp Med 1998; 21:2233–2241.
Yu Q, Erman B, Park JH, Feigenbaum L, Singer A . IL-7 receptor signals inhibit expression of transcription factors TCF-1, LEF-1, and RORgammat: impact on thymocyte development. J Exp Med 2004; 200:797–803.
Peschon JJ, Morrissey PJ, Grabstein KH, et al. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 1994; 180:1955–1960.
Gonzalez-Garcia S, Garcia-Peydro M, Martin-Gayo E, et al. CSL-MAML-dependent Notch1 signaling controls T lineage-specific IL-7R{alpha} gene expression in early human thymopoiesis and leukemia. J Exp Med 2009; 206:779–791.
Saba I, Kosan C, Vassen L, Möröy T . IL-7R-dependent survival and differentiation of early T-lineage progenitors is regulated by the BTB/POZ domain transcription factor Miz-1. Blood 2011; 117:3370–3381.
Wang X, Xiao G, Zhang Y, et al. Regulation of Tcrb recombination ordering by c-Fos-dependent RAG deposition. Nat Immunol 2008; 9:794–801.
Agata Y, Tamaki N, Sakamoto S, et al. Regulation of T cell receptor beta gene rearrangements and allelic exclusion by the helix-loop-helix protein, E47. Immunity 2007; 27:871–884.
Taghon T, Yui MA, Pant R, Diamond RA, Rothenberg EV . Developmental and molecular characterization of emerging beta- and gammadelta-selected pre-T cells in the adult mouse thymus. Immunity 2006; 24:53–64.
Tydell CC, David-Fung ES, Moore JE, Rowen L, Taghon T, Rothenberg EV . Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway. J Immunol 2007; 179:421–438.
Yui MA, Rothenberg EV . Deranged early T cell development in immunodeficient strains of nonobese diabetic mice. J Immunol 2004; 173:5381–5391.
Acknowledgements
The authors thank their colleagues Jinxiu Rui for helpful discussions, and Man Zhang and Jinsong Li for kindly providing ES cells. This work was supported in part by the Ministry of Science and Technology (2007CB815802 and 2012CB518700), the National Natural Science Foundation of China (30925031and 30872290), and the Shanghai Municipal Government (09QH1402500).
Author information
Authors and Affiliations
Corresponding author
Additional information
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Enforced expression of Klf4 blocks the DN2-to-DN3 transition, which is independent of CD44 expression. (PDF 37 kb)
Supplementary information, Figure S2
T cell repertoire in periphery. (PDF 117 kb)
Supplementary information, Figure S3
Klf4/OT-I Tg DN1 and DN2 cells were still unable to normally give rise to T cell lineage committed DN3 thymocytes. (PDF 79 kb)
Supplementary information, Figure S4
Statistic analyses of all numerical values in main figures. (PDF 73 kb)
Supplementary information, Table S1
Primers for Real-time PCR (PDF 11 kb)
Rights and permissions
About this article
Cite this article
Wen, X., Liu, H., Xiao, G. et al. Downregulation of the transcription factor KLF4 is required for the lineage commitment of T cells. Cell Res 21, 1701–1710 (2011). https://doi.org/10.1038/cr.2011.183
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/cr.2011.183
Keywords
This article is cited by
-
RBPJ-dependent Notch signaling initiates the T cell program in a subset of thymus-seeding progenitors
Nature Immunology (2019)
-
Genome-wide analyses identify KLF4 as an important negative regulator in T-cell acute lymphoblastic leukemia through directly inhibiting T-cell associated genes
Molecular Cancer (2015)
-
Haploinsufficiency of Hedgehog interacting protein causes increased emphysema induced by cigarette smoke through network rewiring
Genome Medicine (2015)