Thymocyte Subsets Research Articles

Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4(+) and CD8(+) T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide- induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4(+)CD8(+) double-positive (DP) thymocytes. Furthermore, the CD4(lo)CD8(lo), CD4(+)CD8(lo) and CD4(lo)CD8(+) thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4(+)CD8(+) DP thymocytes present during thymus regeneration were TCR(hi) and CD69(+) unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCR(hi/int+) and CD69(+/int) phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4(+)CD8(+) DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.

Deletion of Notch1 Converts Pro-T Cells to Dendritic Cells and Promotes Thymic B Cells by Cell-Extrinsic and Cell-Intrinsic Mechanisms

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

The Magnitude of Thymic Output Is Genetically Determined through Controlled Intrathymic Precursor T Cell Proliferation

The thymus plays a crucial role in providing the immune system with naive T cells showing a diverse TCR repertoire. Whereas the diversity of thymic production is mainly ensured by TCR rearrangement at both the TRA and TRB loci, the number of cells reaching the double-positive differentiation stage defines the extent of thymic output. A quantitative analysis of TCR excision circles (TREC; signal-joint TRECs and DJbetaTRECs) produced at different stages of thymopoiesis was performed in nine laboratory mouse strains. The results clearly demonstrate that the magnitude of thymic output is directly proportional to the extent of proliferation in the double-negative 4 thymocyte subset. Strikingly, intrathymic precursor T cell proliferation was found to be strain dependent, thus suggesting a genetic regulation of thymic output. The inherited character of thymic output was further confirmed by the transmission of the phenotype in a recessive fashion in F(1) progeny of the different parental strains. Our results provide the first demonstration of the genetic regulation of thymic output.

Age-Related Perturbation of Thymic Epithelial Cells (TEC) Homeostasis Is Caused by Increased TNFR/Fas-Mediated Apoptosis Coupled with Decreased Proliferation

Age-related thymic involution contributes to severe and clinically significant immune deficiency in the elderly. Proposed mechanisms of age-related thymic involution have focused on either intrinsic defects of lymphohematopoietic progenitors or primary defects of the thymic microenvironment with aging. Previously, we have demonstrated that keratinocyte growth factor (KGF) transiently reverses murine age-related thymic involution by regenerating the TEC compartment. We sought to understand the mechanisms of age-related loss of TECs and to evaluate a potential therapeutic strategy to durably regenerate the thymus. We found a significant increase in the frequency of apoptotic TECs (CD45- MHCII+) coupled with a decline in the frequency of S-phase TECs in young versus aged mice (11 vs 19% and 35% vs 17%, respectively). Telomere shortening was observed in both aged thymocytes and thymic stromal cells, indicating increased replicative senescence in both compartments. Previous studies have demonstrated lack of age-related thymic involution in Fas−/− mice. We found that the frequency of Fas+ TECs was 3-fold higher in aged mice compared to young mice. A distinct TEC subpopulation expressed Fas, but there was no increase in Fas+ CD45+ lymphoid cells. Although FasL was not expressed by thymocytes, expression of FasL by intrathymic mature T cells (ITMTs, CD4+CD8−CD44high or CD4−CD8+CD44high) increased 2-fold in aged mice. In contrast, there was no change in the frequency of FasL+ splenic memory T cells. To understand how Fas expression is induced in aged TEC, we examined the effects of two pro-inflammatory cytokines, TNF-α and IL-1β, which are known to induce Fas expression. Both TNF-α and IL-1β up-regulated Fas expression on K5+K8+ (possible precursor), K5−K8+ (cortical), or K5+K8− (medullary) TEC clones derived from young mice. RT-PCR and FACS analyses showed that the aged thymus had 4–5-fold higher levels of TNF-α, and TNF-α expression was increased in aged CD4+ and CD8+ ITMT, as well as all subsets of thymocytes. To elucidate the molecular mechanisms of apoptosis in the aged TEC, we analyzed levels of activated caspase-8, a key mediator of TNFR/Fas-induced apoptosis. Almost 50% of TECs in aged mice but only 25% in young mice had activated caspase-8. Furthermore, in vivo treatment (0.4mg/mouse, every 3rd day for 1 month) of a caspase-8-specific inhibitor (z-IETD-fmk) increased the number of TECs, decreased TEC apoptosis, and enhanced thymopoiesis in aged mice. The data suggest that thymic aging is mediated by decreased regenerative potential of TEC coupled with increased expression of inflammatory cytokines by both ITMT and thymocytes that make TECs more susceptible to apoptosis by either inducing Fas expression on TECs or directly triggering TNFR-mediated apoptosis. The inflammatory effects of ITMT on TEC death provides a feed-forward mechanism by which peripheral memory T-cell generation and immune senescence is linked to thymic involution.

In Vivo Analysis of Protein Kinase B (PKB)/Akt Regulation in DNA-PKcs-null Mice Reveals a Role for PKB/Akt in DNA Damage Response and Tumorigenesis

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs(-/-) mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs(-/-) mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs(-/-) mice. Significantly, these tumors could be prevented by deletion of PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.

Altered ex vivo cytokine production in zinc-deficient, pair-fed and marginally zinc-deficient growing rats is independent of serum corticosterone concentrations

The objective of the present study was to examine the effects of dietary Zn deficiency on the ex vivo cytokine production (IL-2, interferon-gamma (IFN-gamma), IL-6 and IL-10) of isolated thymocytes and splenocytes after mitogenic stimulation with concavalin A and to explore the role of corticosterone in this regulation. Weanling rats were assigned to one of four dietary treatments for 3 weeks: Zn-deficient (< 1mg Zn/kg diet, ad libitum), pair-fed (30 mg Zn/kg diet, limited to amount of feed as consumed by the Zn-deficient group), marginally Zn-deficient (10 mg Zn/kg diet, ad libitum) and control (30 mg Zn/kg diet, ad libitum). Thymocytes and splenocytes were isolated for cytokine stimulation and determination of T-cell phenotypes. Serum corticosterone concentrations were determined by ELISA. The Zn-deficient and pair-fed groups had 14-fold higher serum corticosterone concentrations compared with the marginally Zn-deficient and control groups (P<0.0001). The proportions of thymocyte subsets were not altered in the Zn-deficient, pair-fed or marginally Zn-deficient groups; however, thymocyte IL-2 and IL-6 production in these groups was 33-54% lower compared with the control group (P<0.05). The Zn-deficient group had an 18-28% lower proportion of new T-cells (TCRalphabeta+CD90+), but no difference in the proportion of new T-cells that were cytotoxic or helper. The Zn-deficient group had a 49-62% lower production of Th1 cytokines (IL-2), but no difference in the production of Th2 cytokines (IL-6, IL-10) by stimulated splenocytes compared with the pair-fed, marginally Zn-deficient and control groups (P<0.01). These results indicate that Zn status is associated with altered cytokine production, while in vivo corticosterone concentrations are not associated with ex vivo cytokine production.

Increased Regulatory versus Effector T Cell Development Is Associated with Thymus Atrophy in Mouse Models of Multiple Myeloma

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play a central role in cancer tolerance. However, mechanisms leading to their accumulation in cancer remain unknown. Although the thymus is the main site of Treg development, thymic contribution to Treg expansion in cancer has not been directly examined. Herein, we used two murine models of multiple myeloma (MM), 5T2 MM and 5T33 MM, to examine Treg accumulation in peripheral lymphoid organs, including spleen, lymph nodes, bone marrow, and blood, and to explore thymic Treg development during malignancy. We found that peripheral ratios of suppressive-functional Tregs increased in both models of MM-inflicted mice. We found that thymic ratios of Treg development in MM increased, in strong association with thymus atrophy and altered developmental processes in the thymus. The CD4(+)CD8(+) double-positive population, normally the largest thymocyte subset, is significantly decreased, whereas the CD4(-)CD8(-) double-negative population is increased. Administration of thymocytes from MM-inflicted mice compared with control thymocytes resulted in increased progression of the disease, and this effect was shown to be mediated by Tregs in the thymus of MM-inflicted mice. Our data suggest that increased ratios of Treg development in the thymus may contribute to disease progression in MM-inflicted mice.

Alterations in the thymocyte phenotype of EphB‐deficient mice largely affect the double negative cell compartment

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T-cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4- CD8-) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44- CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25-) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15-day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T-cell subsets defined by CD4/CD8 marker expression, in all cases.

LEF-1 Negatively Controls Interleukin-4 Expression through a Proximal Promoter Regulatory Element

Lymphoid enhancer-binding factor 1 (LEF-1) and T cell factor (TCF-1) are downstream effectors of the Wnt signaling pathway and are involved in the regulation of T cell development in the thymus. LEF-1 and TCF-1 are also expressed in mature peripheral primary T cells, but their expression is down-regulated following T cell activation. Although the decisive roles of LEF-1 and TCF-1 in the early stages of T cell development are well documented, the functions of these factors in mature peripheral T cells are largely unknown. Recently, LEF-1 was shown to suppress Th2 cytokines interleukin-4 (IL-4), -5, and -13 expression from the developing Th2 cells that overexpress LEF-1 through retrovirus gene transduction. In this study, we further investigated the expression and functions of LEF-1 and TCF-1 in peripheral CD4+ T cells and revealed that LEF-1 is dominantly expressed in Th1 but not in Th2 cells. We identified a high affinity LEF-1-binding site in the negative regulatory element of the IL-4 promoter. Knockdown LEF-1 expression by LEF-1-specific small interfering RNA resulted in an increase in the IL-4 mRNA expression. This study further confirms a negative regulatory role of LEF-1 in mature peripheral T cells. Furthermore, we found that IL-4 stimulation possesses a negative effect on the expressions of LEF-1 and TCF-1 in primary T cells, suggesting a positive feedback effect of IL-4 on IL4 gene expression.

Thiorphan-Induced Survival and Proliferation of Rat Thymocytes by Activation of Akt/Survivin Pathway and Inhibition of Caspase-3 Activity

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK(1)R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5(+) thymocytes, namely on the CD4(+)CD8(+) (double positive; DP) and CD4(+) subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK(1)R mRNA expression as well as SP and NK(1)R protein levels in an NK(1)R-dependent manner. Thiorphan increased CD10(+)CD4(+) and CD10(+)DP thymocyte numbers, and an NK(1)R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor alpha chain expression, and concanavalin A-induced proliferation of CD5(+) thymocytes, and it inhibited spontaneous and NK(1)R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

The Signaling Protein Wnt4 Enhances Thymopoiesis and Expands Multipotent Hematopoietic Progenitors through β-Catenin-Independent Signaling

Despite studies based on deletion or activation of intracellular components of the canonical Wingless related (Wnt) pathway, the role of Wnts in hematolymphopoiesis remains controversial. Using gain-of-function and loss-of-function models, we found that Wnt4 differentially affected diverse subsets of hematopoietic stem and progenitor cells. Bone-marrow and thymic Lin(-)Sca1(+)Kit(hi) cells (LSKs) were the key targets of Wnt4. In adult mice, Wnt4-induced expansion of Flt3(+) bone-marrow LSKs (lymphoid-primed multipotent progenitors) led to a sizeable accumulation of the most immature thymocyte subsets (upstream of beta-selection) and a major increase in thymopoiesis. Conversely, Wnt4(-/-) neonates showed low frequencies of bone-marrow LSKs and thymic hypocellularity. We provide compelling evidence that Wnt4 activates noncanonical (beta-catenin-independent) signaling and that its effects on hematopoietic cells are mainly non-cell-autonomous. Our work shows that Wnt4 overexpression has a unique ability to expand Flt3(+) LSKs in adults and demonstrates that noncanonical Wnt signaling regulates thymopoiesis.

Disruption of thymopoiesis in ST6Gal I-deficient mice

Thymocyte development is accompanied by sequential changes in cell surface glycosylation. For example, medullary thymocytes have increased levels of alpha2,3-linked sialic acid and a loss of asialo core 1 O-glycans as compared to cortical thymocytes. Some of these changes have been linked to fine tuning of the T cell receptor avidity. We analyzed ST6Gal I transcript abundance and levels of alpha2,6-linked sialic acid across thymocyte subsets. We found that ST6Gal I transcript levels increased following T cell receptor beta-selection suggesting that this sialyltransferase may influence the development of early thymocyte populations. Indeed, low levels of alpha2,6-linked sialic acid were found in the earliest T lineage cells, and then increased in T cell receptor beta-selected cells. To determine whether ST6Gal I influences T cell development, we analyzed ST6Gal I-deficient mice for disruptions in thymocyte populations. We found reduced thymic cellularity in the ST6Gal I-deficient mice starting in the early thymocyte compartments.

Identification of Thymocyte Subset by Multicolor Flow Cytometry ED LSR II FACSDriva - FlowJo Software Analysis

In their development, thymocytes express different cell surface molecules that important for identification of thymocyte subset. Itrs not easy to detect this cell surface molecules to determine the thymocyte subpopulation for research. Here we used multicolor flow cytometry ED LSR II FACSDriva - FlowJo software to identify of thymocyte subset from thymocyte sample solution using several antibodies such as mouse anti rat CD2-FITC, mouse anti rat CD45RC-PE, mouse anti rat CD4-APC, mouse anti rat CD8a-PerCP, mouse anti rat CD3-Biotin + PE-Cy7 or APC-Cy7. We determined double negative and single positive thymocyt subset (CD4 or CD8), found that the double negative thymocyte subset express CD2 and CD45RC. It was useful to determine the thymocyte subset using multicolor flow sitometry ED LSR II FACSDriva - FlowJo software.

Structural Features of Galectin-9 and Galectin-1 That Determine Distinct T Cell Death Pathways

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.

Structural Features of Galectin‐9 and Galectin‐1 that Determine Distinct T cell Death Pathways

Galectin‐9 and galectin‐1 both kill thymocytes, peripheral T cells and T cell lines. However, differences in target cell specificity, glycoprotein receptor requirements, and intracellular death pathways for these two galectins are not well understood. We found that galectin‐9 and galectin‐1 required different glycan ligands and glycoprotein receptors to trigger T cell death. They also triggered different intracellular death pathways, as only galectin‐9 T cell death was blocked by intracellular bcl‐2, while only galectin‐1 T cell death was blocked by intracellular galectin‐3. Galectin‐9 and galectin‐1 were both expressed in murine thymus, but killed different thymocyte subsets, with mature thymocytes more susceptible to galectin‐9. To define the structural features responsible for distinct activities of galectin‐1 and galectin‐9, we created a series of bivalent neo‐galectins with galectin‐1 and galectin‐9 carbohydrate recognition domains on different peptide linkers. The N‐terminal carbohydrate recognition domain and linker peptide contributed to potency of the galectins. Strikingly, the C‐terminal carbohydrate recognition domain determined receptor recognition, death pathway signaling and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency all contribute to specific effects of different galectins in regulating T cell death.

Characterization of the In Vivo Dynamics of Medullary CD4+CD8− Thymocyte Development

Our previous studies have defined a differentiation program followed by the newly generated single-positive (SP) thymocytes before their emigration to the periphery. In the present study, we further characterize the development of CD4SP cells in the thymic medulla using mainly intrathymic adoptive transfer assays. By analyzing the differentiation kinetics of the donor cells, which were shown to home correctly to the medullary region following adoptive transfer, we established the precursor-progeny relationship among the four subsets of CD4SP thymocytes (SP1-SP4) and demonstrated that the progression from SP1 to SP4 was unidirectional and largely synchronized. Notably, while the phenotypic maturation from SP1 to SP4 was achieved in 2-3 days, a small fraction of donor cells could be retained in the thymus for a longer period, during which they further matured in function. BrdU incorporation indicated that cell expansion occurred at multiple stages except SP1. Nevertheless, CFSE labeling revealed that only a limited number of cells actually divided during their stay in the medulla. As to the thymic emigration, there was a clear bias toward cells with increasing maturity, but no distinction was found between dividing and nondividing thymocytes. Collectively, these data not only provide solid evidence for a highly ordered differentiation program for CD4SP thymocytes, but they also illustrate several important features associated with the developmental process.

Developmental pathway of CD4+CD8- medullary thymocytes during mouse ontogeny and its defect in Aire-/- mice.

The newly generated single-positive (SP) thymocytes undergo further maturation in the thymic medulla before their emigration to the periphery. The present study was undertaken to validate a developmental program we proposed for CD4SP medullary thymocytes and to explore the mechanisms regulating this process. During mouse ontogeny, the emergence of different subsets of CD4SP thymocytes followed a strict temporal order from SP1 to SP4. Parallel to the transition in surface phenotype, a steady increase in function was observed. As further evidence, purified SP1 cells were able to sequentially give rise to SP2, SP3, and SP4 cells in intrathymic adoptive transfer and in culture. Notably, the development of CD4SP cells in the medulla seemed to be critically dependent on a functionally intact medullary epithelial cell compartment because Relb and Aire deficiency were found to cause severe blockage at the transition from SP3 to SP4. Taken together, this work establishes an ontogenetically and functionally relevant maturation program for CD4SP thymocytes. Precise dissection of this program should facilitate further inquiry into the molecular mechanisms governing normal thymocyte development and its disturbance in pathological conditions.

Antagonistic Interactions between Ikaros and the Chromatin Remodeler Mi-2β Determine Silencer Activity and Cd4 Gene Expression

Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.

Deletion of PKBα/Akt1 Affects Thymic Development

BackgroundThe thymus constitutes the primary lymphoid organ for the majority of T cells. The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development. Defects in single components of this pathway prevent thymocytes from progressing beyond early T cell developmental stages. Protein kinase B (PKB) is the main effector of the PI3K pathway.Methodology/Principal FindingsTo determine whether PKB mediates PI3K signaling in the thymus, we characterized PKB knockout thymi. Our results reveal a significant thymic hypocellularity in PKBα −/− neonates and an accumulation of early thymocyte subsets in PKBα −/− adult mice. Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBα within the lymphoid component of the thymus. Microarray analyses show that the absence of PKBα in early thymocyte subsets modifies the expression of genes known to be involved in pre-TCR signaling, in T cell activation, and in the transduction of interferon-mediated signals.Conclusions/SignificanceThis report highlights the specific requirements of PKBα for thymic development and opens up new prospects as to the mechanism downstream of PKBα in early thymocytes.

Expression Profiling of Immature Thymocytes Revealed a Novel Homeobox Gene That Regulates Double-Negative Thymocyte Development

Intrathymic development of CD4/CD8 double-negative (DN) thymocytes can be tracked by well-defined chronological subsets of thymocytes, and is an ideal target of gene expression profiling analysis to clarify the genetic basis of mature T cell production, by which differentiation of immature thymocytes is investigated in terms of gene expression profiles. In this study, we show that development of murine DN thymocytes is predominantly regulated by largely repressive rather than inductive activities of transcriptions, where lineage-promiscuous gene expression in immature thymocytes is down-regulated during their differentiation. Functional mapping of genes showing common temporal expression profiles implicates previously uncharacterized gene regulations that may be relevant to early thymocytes development. A small minority of genes is transiently expressed in the CD44(low)CD25(+) subset of DN thymocytes, from which we identified a novel homeobox gene, Duxl, whose expression is up-regulated by Runx1. Duxl promotes the transition from CD44(high)CD25(+) to CD44(low)CD25(+) in DN thymocytes, while constitutive expression of Duxl inhibits expression of TCR beta-chains and leads to impaired beta selection and greatly reduced production of CD4/CD8 double-positive thymocytes, indicating its critical roles in DN thymocyte development.