Harnessing of the nucleosome-remodeling-deacetylase complex controls lymphocyte development and prevents leukemogenesis.

Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2ß nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage-determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell-specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.

Spatial mapping of thymic stromal microenvironments reveals unique features influencing T lymphoid differentiation.

Interaction of hematopoietic progenitors with the thymic microenvironment induces them to proliferate, adopt the T lineage fate, and asymmetrically diverge into multiple functional lineages. Progenitors at various developmental stages are stratified within the thymus, implying that the corresponding microenvironments provide distinct sets of signals to progenitors migrating between them. These differences remain largely undefined. Here we used physical and computational approaches to generate a comprehensive spatial map of stromal gene expression in the thymus. Although most stromal regions were characterized by a unique gene expression signature, the central cortex lacked distinctive features. Instead, a key function of this region appears to be the sequestration of unique microenvironments found at the cortical extremities, thus modulating the relative proximity of progenitors moving between them. Our findings compel reexamination of how cell migration, lineage specification, and proliferation are controlled by thymic architecture and provide an in-depth resource for global characterization of this control.

Chromosome choice for initiation of V-(D)-J recombination is not governed by genomic imprinting.

V-(D)-J recombination generates the antigen receptor diversity necessary for immune cell function, while allelic exclusion ensures that each cell expresses a single antigen receptor. V-(D)-J recombination of the Ig, Tcrb, Tcrg and Tcrd antigen receptor genes is ordered and sequential so that only one allele generates a productive rearrangement. The mechanism controlling sequential rearrangement of antigen receptor genes, in particular how only one allele is selected to initiate recombination while at least temporarily leaving the other intact, remains unresolved. Genomic imprinting, a widespread phenomenon wherein maternal or paternal allele inheritance determines allele activity, could represent a regulatory mechanism for controlling sequential V-(D)-J rearrangement. We used strain-specific single-nucleotide polymorphisms within antigen receptor genes to determine if maternal vs paternal inheritance could underlie chromosomal choice for the initiation of recombination. We found no parental chromosomal bias in the initiation of V-(D)-J recombination in T or B cells, eliminating genomic imprinting as a potential regulator for this tightly regulated process.

Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis.

Notch1 signaling is absolutely essential for steady-state thymic lymphopoiesis, but the role of other Notch receptors, and their potential overlap with the function of Notch1, remains unclear. Here we show that like Notch1, Notch3 is differentially expressed by progenitor thymocytes, peaking at the DN3 progenitor stage. Using mice carrying a gene-trapped allele, we show that thymic cellularity is slightly reduced in the absence of Notch3, although progression through the defined sequence of TCR-αß development is normal, as are NKT and TCRγδ cell production. The absence of a profound effect from Notch3 deletion is not explained by residual function of the gene-trapped allele because insertion mapping suggests that the targeted allele would not encode functional signaling domains. We also show that although Notch1 and Notch3 are coexpressed on some early intrathymic progenitors, the relatively mild phenotype seen after Notch3 deletion does not result from the compensatory function of Notch1, nor does Notch3 function explain the likewise mild phenotype seen after conditional (intrathymic) deletion of Notch1. Our studies indicate that Notch1 and Notch3 carry out nonoverlapping functions during thymocyte differentiation, and that while Notch1 is absolutely required early in the lymphopoietic process, neither receptor is essential at later stages.

Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes.

In the thymus, Notch signaling is essential for T lymphopoiesis, with Delta-like (Dll)4 uniquely involved in this process. However, using cocultures, either Dll4 or Dll1 were shown to support T lymphopoiesis. To address which Dll is more effective at inducing hematopoietic progenitor cells to give rise to T lineage cells in vitro, we generated OP9 cells expressing a series of incrementally discrete and equivalent levels of Dll1 or Dll4. In keeping with previous findings, OP9 cells expressing high levels of either Dll1 or Dll4 gave rise to T lineage cells with similar efficacy, and prevented the differentiation of B and myeloid-lineage cells. However, at limiting levels, Dll4 maintained its ability to inhibit B lineage choice and induce T lineage commitment and differentiation at lower levels than Dll1. This manifest property of Dll4 is evident despite lower levels of steady-state surface expression than Dll1 on OP9 cells. The heightened effectiveness of Dll4 over Dll1 also corresponded to the induction of Notch target genes, and inhibition of B and myeloid-specific transcription factors. Furthermore, we show that OP9 cells expressing levels of Dll4 equivalent to those present in thymic epithelial cells, as expected, gave rise to T lineage cells, but were also permissive for the differentiation of myeloid cells; whereas, still inhibiting B lymphopoiesis. Our findings show that Dll4 expressed at physiological levels on OP9 cells is functionally distinct from similarly expressed levels of Dll1, illustrating the unique properties of Dll4 in supporting the combined T lineage and specific myeloid-lineage outcomes that underpin its function within the thymus.

Many roads, one destination for T cell progenitors.

The thymus manufactures new T cells throughout life but contains no self-renewing potential. Instead, replenishment depends on recruitment of bone marrow-derived progenitors that circulate in the blood. Attempts to identify thymic-homing progenitors, and to assess the degree to which they are precommitted to the T cell lineage, have led to complex and sometimes conflicting results. As described here, this probably reflects the existence of multiple distinct types of T cell lineage progenitors as well as differences in individual experimental approaches.

Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice.

Upon thymus entry, thymic-homing progenitors undergo distinct phases of differentiation as they migrate through the cortex to the capsule, suggesting that the signals that induce these differentiation steps may be stratified in corresponding cortical regions. To better define these regions, we transplanted purified stem cells into nonirradiated congenic recipients and followed their differentiation with respect to both tissue location and time. The earliest progenitors (DN1) remained confined to a very narrow region of the cortex for about the first 10 d of intrathymic residence; this region virtually overlaps the sites of thymic entry, suggesting that DN1 cells move very little during this lengthy period of proliferation and lineage commitment. Movement out of this region into the deeper cortex is asynchronous, and corresponds to the appearance of DN2 cells. Differentiation to the DN3 stage correlates with movement across the midpoint of the cortex, indicating that stromal signals that induce functions such as TCR gene rearrangement reside mainly in the outer half of the cortex. The minimum time to reach the capsule, and thus transit to the DP stage, is approximately 13 d, with the average time a few days longer. These findings reveal for the first time the kinetics of steady-state progenitor differentiation in the thymus, as well as defining the boundaries of cortical regions that support different phases of the differentiation process. We also show that the first lineage-positive progeny of transplanted stem cells to appear in the thymus are dendritic cells in the medulla, suggesting that each new wave of new T cell production is preceded by a wave of regulatory cells that home to the medulla and ensure efficient tolerance and selection.

Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions.

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

Thymic T cell development and progenitor localization depend on CCR7.

T cell differentiation in the adult thymus depends on sequential interactions between lymphoid progenitors and stromal cells found in distinct regions of the cortex and medulla. Therefore, migration of T cell progenitors through distinct stromal environments seems to be a crucial process regulating differentiation and homeostasis inside the thymus. Here we show that CCR7-deficient mice are distinguished by a disturbed thymic architecture, impaired T cell development, and decreased numbers of the thymocytes. Analysis of developing double negative (CD4-CD8-) pool of wild-type thymus reveals that CCR7 expression is restricted to a CD25intCD44+ subpopulation. Correspondingly, CCR7 deficiency results in an accumulation of this population in mutant thymus. Furthermore, immunohistology shows that in CCR7-deficient mice CD25+CD44+ cells accumulate at the cortico-medullary junction, suggesting that CCR7 signaling regulates the migration of early progenitors toward the outer thymic cortex, thereby continuing differentiation. Results obtained from mixed bone marrow chimeras support this view, since the development of CCR7-deficient thymocytes is also disturbed in a morphologically intact thymus. Thus, our findings establish an essential role for CCR7 in intrathymic migration and proper T cell development.

Dynamic changes in epithelial cell morphology control thymic organ size during atrophy and regeneration.

T lymphocytes must be produced throughout life, yet the thymus, where T lymphocytes are made, exhibits accelerated atrophy with age. Even in advanced atrophy, however, the thymus remains plastic, and can be regenerated by appropriate stimuli. Logically, thymic atrophy is thought to reflect senescent cell death, while regeneration requires proliferation of stem or progenitor cells, although evidence is scarce. Here we use conditional reporters to show that accelerated thymic atrophy reflects contraction of complex cell projections unique to cortical epithelial cells, while regeneration requires their regrowth. Both atrophy and regeneration are independent of changes in epithelial cell number, suggesting that the size of the thymus is regulated primarily by rate-limiting morphological changes in cortical stroma, rather than by their cell death or proliferation. Our data also suggest that cortical epithelial morphology is under the control of medullary stromal signals, revealing a previously unrecognized endocrine-paracrine signaling axis in the thymus.

Electronically subtracting expression patterns from a mixed cell population.

MOTIVATION: Biological samples frequently contain multiple cell-types that each can play a crucial role in the development and/or regulation of adjacent cells or tissues. The search for biomarkers, or expression patterns of, one cell-type in those samples can be a complex and time-consuming process. Ordinarily, extensive laboratory bench work must be performed to separate the mixed cell population into its subcomponents, such that each can be accurately characterized. RESULTS: We have developed a methodology to electronically subtract gene expression in one or more components of a mixed cell population from a mixture, to reveal the expression patterns of other minor or difficult to isolate components. Examination of simulated data indicates that this procedure can reliably determine the expression patterns in cell-types that contribute as little as 5% of the total expression in a mixed cell population. We re-analyzed microarray expression data from the viral infection of macrophages and from the T-cells of wild type and Foxp3 deletion mice. Using our subtraction methodology, we were able to substantially improve the identification of genes involved in processes of subcomponent portions of these samples.

Foxn1[Cre] Expression in the Male Germline.

Foxn1 (forkhead box N1), also known as the nude gene or winged-helix nude (Whn), is a forkhead transcription factor thought to be restricted to keratinocytes in the skin and thymus. Consistent with this tissue distribution, spontaneous or targeted mutation of Foxn1 results in the absence of both hair and a thymus. Genetic manipulation of the Foxn1 locus thus represents a powerful tool for tissue specific gene control in the skin and thymus, and tools such as Cre recombinase under control of the Foxn1 locus are widely used for this purpose. Unexpectedly, we show that Foxn1[Cre] exhibits unexpected activity in male germ cells, resulting in ubiquitous targeting of loxP-flanked alleles in all tissues in offspring from Foxn1[Cre] expressing male mice. Inheritance of recombined loxP alleles occurs independently of Cre inheritance (i.e., offspring lacking Cre nonetheless exhibit recombined alleles), suggesting that Foxn1[Cre] induced recombination in male germ cells must occur prior to meiosis in diploid germ cells. Together with previously published data, our results show that Foxn1, and alleles under its control, are expressed in the pre-meiotic male germline, revealing a new tool for germline targeting of genes, and raising important concerns for gender selection when using Foxn1 regulatory elements.

Metabolic Damage and Premature Thymus Aging Caused by Stromal Catalase Deficiency.

T lymphocytes are essential mediators of immunity that are produced by the thymus in proportion to its size. The thymus atrophies rapidly with age, resulting in progressive diminution of new T cell production. This decreased output is compensated by duplication of existing T cells, but it results in gradual dominance by memory T cells and decreased ability to respond to new pathogens or vaccines. Here, we show that accelerated and irreversible thymic atrophy results from stromal deficiency in the reducing enzyme catalase, leading to increased damage by hydrogen peroxide generated by aerobic metabolism. Genetic complementation of catalase in stromal cells diminished atrophy, as did chemical antioxidants, thus providing a mechanistic link between antioxidants, metabolism, and normal immune function. We propose that irreversible thymic atrophy represents a conventional aging process that is accelerated by stromal catalase deficiency in the context of an intensely anabolic (lymphoid) environment.

A simple method for detecting up to five immunofluorescent parameters together with DNA staining for cell cycle or viability on a benchtop flow cytometer.

In this manuscript, we describe modifications to a commercial three-laser benchtop flow cytometer, as well as relevant biological methods, that allow analysis of up to five immunofluorescent parameters together with an ultraviolet (UV)-excitable DNA stain. This method allows expanded capacity for multiparameter immunophenotyping of complex mixed cell populations, together with accurate measurements of DNA content (cell cycle) or cell viability, on a stable, end-user operated platform.

Activation kinetics and off-target effects of thymus-initiated cre transgenes.

The bacteriophage enzyme Cre is a site-specific recombinase widely used to delete loxP-flanked DNA sequences in lineage-specific fashion. Several mouse lines that direct Cre expression to lymphoid progenitors in the thymus have been established, but a side-by-side comparison of when they first become active, and/or their relative efficiency at various developmental stages, has been lacking. In this study, we evaluated these in four common Cre transgenic strains with thymus-initiated promoters (Lck, Cd2, or Cd4). We found that while all of them eventually labeled nearly all thymocytes, their kinetics were dramatically different, and other than Cd4[Cre], did not faithfully recapitulate the expression pattern of the corresponding endogenous gene. Perhaps even more importantly, while thymuses from some strains compared favorably to thymuses from control (Cre-negative) mice, we found that Cre expression could also result in off-target effects, including moderate to severe decreases in thymic cellularity. These effects occurred in the absence of loxP-flanked DNA target genes, and were dose and copy number dependent. Loss of cellularity was attributable to a specific decrease in CD4(+)8(+) immature cells, and corresponds to an increased rate of programmed cell death. In addition to a comprehensive analysis of activation kinetics in thymus-initiated Cre transgenes, our data show that Cre is toxic to CD4(+)8(+) cells in a dose-dependent fashion, and emphasize that the choice of thymus-initiated Cre strain is critically important for minimizing off-target effects of Cre.

Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth.

The thymus is the most rapidly aging tissue in the body, with progressive atrophy beginning as early as birth and not later than adolescence. Latent regenerative potential exists in the atrophic thymus, because certain stimuli can induce quantitative regrowth, but qualitative function of T lymphocytes produced by the regenerated organ has not been fully assessed. Using a genome-wide computational approach, we show that accelerated thymic aging is primarily a function of stromal cells, and that while overall cellularity of the thymus can be restored, many other aspects of thymic function cannot. Medullary islet complexity and tissue-restricted antigen expression decrease with age, representing potential mechanisms for age-related increases in autoimmune disease, but neither of these is restored by induced regrowth, suggesting that new T cells produced by the regrown thymus will probably include more autoreactive cells. Global analysis of stromal gene expression profiles implicates widespread changes in Wnt signaling as the most significant hallmark of degeneration, changes that once again persist even at peak regrowth. Consistent with the permanent nature of age-related molecular changes in stromal cells, induced thymic regrowth is not durable, with the regrown organ returning to an atrophic state within 2 weeks of reaching peak size. Our findings indicate that while quantitative regrowth of the thymus is achievable, the changes associated with aging persist, including potential negative implications for autoimmunity.

Early commitment: T cell progenitors in the blood.

Thymic lymphopoiesis depends on blood-borne, marrow-derived progenitors, but characterization of these cells, and their lineage potentials, has been difficult. In this issue, Krueger and von Boehmer show some blood cells that are already T lineage restricted, suggesting that T lineage commitment can occur pre-thymically.

Zoned out: functional mapping of stromal signaling microenvironments in the thymus.

All hematopoietic cells, including T lymphocytes, originate from stem cells that reside in the bone marrow. Most hematopoietic lineages also mature in the bone marrow, but in this respect, T lymphocytes differ. Under normal circumstances, most T lymphocytes are produced in the thymus from marrow-derived progenitors that circulate in the blood. Cells that home to the thymus from the marrow possess the potential to generate multiple T and non-T lineages. However, there is little evidence to suggest that, once inside the thymus, they give rise to anything other than T cells. Thus, signals unique to the thymic microenvironment compel multipotent progenitors to commit to the T lineage, at the expense of other potential lineages. Summarizing what is known about the signals the thymus delivers to uncommitted progenitors, or to immature T-committed progenitors, to produce functional T cells is the focus of this review.