Thymic Differentiation Research Articles

Towards Regenerative Therapy for Thymic Insufficiency after Hematopoietic Stem Cell Transplantation: Generation of MTS24 Positive Definitive Endoderm from Murine Embryonic Stem Cells.

Following hematopoietic stem cell transplantation (HSCT), longterm T-cell reconstitution should be established by thymus-dependent de-novo generation of naïve T-cells (thymopoiesis), which is especially important for generating a naïve T-cell pool with a broad T-cell receptor (TCR) repertoire. However, while erythroid and myeloid hematopoietic cell lineages recover rapidly following HSCT, T-cell development may severely lag behind due to thymic insufficiency. Recent studies in fetal mice have identified common thymic epithelial progenitor cells (TEPC) that were capable to re-establish a thymus in-vivo upon transplantation into a-thymic nude mice. These TEPC are characterized by expression of the transcription factor Foxn1 and by cell surface expression of MTS24. These TEPC arise exclusively from progenitors originating from the anterior foregut endoderm during embryogenesis. Therefore, we hypothesized that common TEPC may be generated in-vitro from embryonic stem (ES) cells that have differentiated towards definitive endoderm. Currently, the mechanisms underlying commitment of definitive endoderm towards a thymic fate are unknown. In order to differentiate murine ES cells towards definitive endoderm and TEPC and to identify the factors involved in the commitment of endoderm towards a thymic fate we investigated the expression of MTS24 and of genes associated with thymic differentiation in ES-cell derived endoderm using a Gcs–GFP/Sox17–huCD25 reporter ES cell line. Culture of these GscgfpSox17huCD25 ES cells in the presence of Activin A resulted in a rapid induction of mesendodermal differentiation. After 6 days of culture the majority of cells differentiated towards mesoderm (Gsc+Sox17−, 60%) and definitive endoderm (GSC+Sox17+, 35%). Apart from the addition of Activin A, the use of low passage number ES-cells and a seeding density between 200–300 cells/cm2 were the most important factors determining efficient differentiation towards definitive endoderm. Addition of insulin or WNT-3a had no significant effect on differentiation, while usage of a high passage number of ES-cells and/or a high seeding density mainly promoted development of visceral endoderm. Real-time quantitative PCR of the definitive endoderm fraction of these cultures not only showed expression of genes associated with definitive endoderm and gut tube formation (i.e. Sox17, Foxa2, Hnf4a and TCF2) but also of genes associated with anterior foregut endoderm (i.e. Hhex, Pax9) and a low, but significant, expression of Foxn1. Analysis of MTS24 expression within these cultures showed the presence of this antigen on all three cell types. The percentage of cells expressing MTS24 was highest in visceral endoderm (30–50%) and lowest in mesoderm (5–10%). The expression was approximately 12% in definitive endoderm. We conclude that murine ES cells cultured in the presence of Activin A can efficiently differentiate towards gut-tube like endoderm, including anterior forgut endoderm, and that a fraction of the generated endoderm also expresses the surface marker MTS24, suggesting the generation of epithelial progenitors with phenotypic characteristics of TEPC.

A New Subtype of T-Cell Acute Leukemia in Very Young Children Is Defined by a Translocation Targeting the C-MYB Oncogene, and a Specific Gene Expression Signature.

Cytogenetics and expression studies have pointed out an increasing number of oncogenes in T-cell acute lymphoblastic leukemias (T-ALL), demonstrating the complexity of T-ALL oncogenesis and the requirement for a network of cooperative genomic events. Here, we identified two types of genomic alterations involving the C-MYB locus at 6q23 in human T-ALL, both associated with transcriptional deregulation. First, using a TCRB-based FISH screening, we found a new reciprocal translocation, t(6;7)(q23;q34), that juxtaposed the TCRB and C-MYB loci (n=6 cases). Breakpoints were fully characterized at the molecular level in 3 cases. Second, a genome wide copy-number analysis by array-CGH (customized 4K BAC/PAC arrays) identified short cryptic duplications which always include the C-MYB gene (MYBdup, n=11 out of 80 primary pediatric and adult T-ALL, 14%). Somatic origin of the genomic gain was demonstrated using paired leukemic and remission samples. The minimal region of gain was mapped to 230 Kb using 244K oligonucleotide a-CGH, and fiber-FISH demonstrated tandem duplication. Interestingly, these genomic events are reminiscent of the frequent myb retroviral insertions in murine leukemia. Expression analysis by RQ-PCR and microarrays data showed stronger C-MYB expression in the MYB-rearranged cases compared to other T-ALLs and normal controls (thymus, BM, and PBL). Moreover, allele-specific approaches showed a dramatically skewed allele expression in the TCRB-MYB cases, suggesting that the translocation-driven deregulated expression overcomes a cellular attempt to downregulate C-MYB. Considering that the C-MYB transcription factor has been involved at several key steps throughout normal thymic differentiation, these data strongly suggest that deregulation of C-MYB due to genomic events is oncogenic in T-ALL. Integrated analysis of clinical, genomic, and large-scale gene expression data showed that the MYB translocation defines a new T-ALL subtype. Strikingly, 5 out of 6 patients with the translocation had a very young age for T-cell leukemia (1.1, 1.3, 1.8, 2.5, and 2.9 year-old; median 2.2 versus 9.4 year-old for 355 pediatric T-ALL in the FRALLE 93/2000 pediatric trials, p<10−4). Gene expression profiling showed that these cases cluster in a new branch distinct from other subtypes and are characterized by a specific cell cycle/mitosis signature. By contrast, the MYBdup alteration was associated to previously defined T-ALL subtypes (TAL, TLX1/HOX11, TLX3/HOX11L2, MLL, CALM-AF10, and immature cases), suggesting an additional oncogenic event. NOTCH1 activating mutations and p16/CDKN2A/ARF deletions were found in both the TCRB-MYB and MYBdup cases, according to multi-step T-cell oncogenesis. This work is the first clear demonstration of the recurrent involvement of the C-MYB locus in a human neoplasia. Moreover the t(6;7) translocation defines a new T-ALL subtype associated with very young age for T-cell leukemia and a distinct biology.

FGFR2IIIb signaling regulates thymic epithelial differentiation

Heterogeneous epithelial populations comprising the thymic environment influence early and late stages of T-cell development. The processes that regulate the differentiation of thymic epithelium and that are responsible for this heterogeneity are not well understood, although mesenchymal/epithelial interactions are clearly involved. Here, we show that targeted expression by thymocytes of an fibroblast growth factor receptor-2IIIb (FGFR2IIIb) ligand, FGF10, profoundly alters the differentiation and function of thymic epithelium (TE). Reconstitution of irradiated lckFGF10 mice with normal bone marrow restores normal thymic organization and function, while wild-type mice reconstituted with lckFGF10 bone marrow recapitulates some of the thymic alterations seen in lckFGF10 mice. We also demonstrate that interference with FGFR2IIIb signaling in the thymus with a soluble FGFR2IIIb dominant-negative fusion protein leads to precocious reductions in thymic size and cellularity that resemble age-related thymic involution. These findings indicate that TE compartments are dynamically maintained and that FGF signals are involved in this process.

Unbiased analysis, enrichment and purification of thymic stromal cells

The microenvironment of the thymus consists of functionally discrete niches composed of distinct stromal cell subsets. Clinically relevant changes affecting T-cell differentiation occur within these niches with age and injury caused by irradiation and chemotherapy treatments. The study of thymic stromal cells has been hampered by the technical difficulty in isolating significant numbers of this important population. Here we present an improved protocol for enzymatic isolation of stromal cells that enables comparative flow cytometric analyses and their purification for downstream cellular or molecular analysis. Fractions analyzed throughout enzymatic digestion of the thymus revealed that various stromal subsets are isolated at characteristic intervals. This highlights the importance of pooling all cells isolated from the thymus for numerical and phenotypic analysis to avoid biased representation of subpopulations. We also describe refined magnetic bead separation techniques that yield almost pure preparations of CD45− stroma. Sorting of these suspensions using defined markers enabled purification of the major epithelial subsets, confirmed by keratin staining and PCR analysis. This three-step procedure represents a rapid, reproducible method for the unbiased purification of the stromal cells that direct thymic T-cell differentiation.

Mesenchymal cells are required for functional development of thymic epithelial cells

Epithelial-mesenchymal interactions have essential roles in thymus organogenesis. Mesenchymal cells are known to be required for epithelial cell proliferation. However, the contribution of mesenchymal cells to thymic epithelial cell differentiation is still unclear. In the present study, we have investigated the roles of mesenchymal cells in functional development of epithelial cells in the thymus anlage in patch (ph) mutant mice, which have a primarily defect in mesenchymal cells caused by the absence of platelet-derived growth factor receptor alpha expression. In the ph/ph thymus anlage, T cell progenitors migrate normally among the epithelial cells, however, they are severely impaired to proliferate and differentiate to CD25-positive cells. Epithelial cells of the ph/ph thymus anlage show severely impaired proliferation and expression of functional molecules, such as SCF, Delta-like 4 and MHC class II, which have crucial roles in T cell development. Moreover, the cultured ph/ph thymus anlage fails to develop into a mature organ supporting full T cell development. Addition of intact thymic mesenchymal cells to organ culture induces development of the ph/ph thymus anlage. In the cultured lobes, added mesenchymal cells contribute to form not only the capsule but also the meshwork structure mingled with epithelial cells. Our present results strongly suggest the roles of mesenchymal cells in functional development of epithelial cells in thymus organogenesis. In addition, our data suggest that mesenchymal cells are required to create the thymic microenvironment and to maintain epithelial architecture and function.

The thymic theme of acetylcholinesterase splice variants in myasthenia gravis

AbstractCholinergic signaling and acetylcholinesterase (AChE) influence immune response and inflammation. Autoimmune myasthenia gravis (MG) is mediated by antibodies to the acetylcholine receptor and current therapy is based on anti-AChE drugs. MG is associated with thymic hyperplasia, showing signs of inflammation. The objectives of this study were to analyze the involvement of AChE variants in thymic hyperplasia. We found lower hydrolytic activities in the MG thymus compared with adult controls, accompanied by translocation of AChE-R from the cytoplasm to the membrane and increased expression of the signaling protein kinase PKC-βII. To explore possible causal association of AChE-R changes with thymic composition and function, we used an AChE-R transgenic model and showed smaller thymic medulla compared with strain-matched controls, indicating that AChE-R overexpression interferes with thymic differentiation mechanisms. Interestingly, AChE-R transgenic mice showed increased numbers of CD4+CD8+ cells that were considerably more resistant in vitro to apoptosis than normal thymocytes, suggesting possibly altered positive selection. We further analyzed microarray data of MG thymic hyperplasia compared with healthy controls and found continuous and discrete changes in AChE-annotated GO categories. Together, these findings show that modified AChE gene expression and properties are causally involved in thymic function and development.

Severe Defect in Thymic Development in an Insertional Mutant Mouse Model

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.

Aire-Dependent Alterations in Medullary Thymic Epithelium Indicate a Role for Aire in Thymic Epithelial Differentiation

The prevalent view of thymic epithelial differentiation and Aire activity holds that Aire functions in terminally differentiated medullary thymic epithelial cells (MTECs) to derepress the expression of structural tissue-restricted Ags, including pancreatic endocrine hormones. An alternative view of these processes has proposed that Aire functions to regulate the differentiation of immature thymic epithelial cells, thereby affecting tissue-restricted Ag expression and negative selection. In this study, we demonstrate that Aire impacts several aspects of murine MTECs and provide support for this second model. Expression of transcription factors associated with developmental plasticity of progenitor cells, Nanog, Oct4, and Sox2, by MTECs was Aire dependent. Similarly, the transcription factors that regulate pancreatic development and the expression of pancreatic hormones are also expressed by wild-type MTECs in an Aire-dependent manner. The altered transcriptional profiles in Aire-deficient MTECs were accompanied by changes in the organization and composition of the medullary epithelial compartment, including a reduction in the medullary compartment defined by keratin (K) 14 expression, altered patterns of K5 and K8 expression, and more prominent epithelial cysts. These findings implicate Aire in the regulation of MTEC differentiation and the organization of the medullary thymic compartment and are compatible with a role for Aire in thymic epithelium differentiation.

Neuropilin 1 and CD25 co-regulation during early murine thymic differentiation

Neuropilin 1 (NP1) is a receptor for both semaphorin and vascular endothelial growth factor expressed by subpopulations of neuronal and endothelial cells. In the immune system, NP1 is present on dendritic and regulatory T cells. Here, we show that NP1 is expressed in the murine thymus, starting on day 12.5 of gestation. In the adult, NP1 is mainly expressed by CD4 −CD8 − double negative cells, CD4 +CD8 + double positive cells, and CD4 +CD25 + regulatory T cells but barely detected in single CD4 + and CD8 + positive thymocytes. Within the CD4 −CD8 −CD3 − (triple-negative, TN) immature cells, NP1 expression starts in TN3 (CD44 −CD25 +) and increases in TN4 (CD44 −CD25 −) cells. In order to study the role of NP1 in thymocyte differentiation, we generated mice in which the np1 gene is selectively disrupted in the T-cell lineage. The mutant mice display normal thymocyte, peripheral, conventional and CD4 +CD25 +Foxp3 + regulatory T-cell populations. However, we observe a down-regulation of the CD25 expression between the TN3 and TN4 stages that is (i) correlated to increased expression of NP1 in control mice and (ii) altered in mutant mice, suggesting that NP1 is co-regulated with CD25 during early immature thymocyte differentiation.

An MHC-linked locus modulates thymic differentiation of CD4+CD25+Foxp3+ regulatory T lymphocytes.

CD4+CD25+Foxp3+ regulatory T lymphocytes are crucial for maintenance of immunological tolerance to self and innocuous non-self, are known to modulate immunity to tumors and infectious agents and can induce transplantation tolerance. Surprisingly, only a single genetic polymorphism is known to modulate regulatory T cell (Treg) development in the thymus, leading to a lethal autoimmune disorder. Here, we show that considerably different levels of Tregs are found in the thymi of distinct common laboratory mouse strains. We demonstrate that distinct levels of phenotypically and functionally identical Tregs develop with similar kinetics in the studied mice, that the responsible locus acts in a thymocyte-intrinsic manner and that levels of thymic Foxp3+ Tregs correlate to those found in the periphery. Using several congenic mouse strains, we mapped one of the at least two genetic loci capable of quantitatively modulating thymic Treg development to a <or=2.2 Mb region telomeric to the MHC. Our data indicate that polymorphic genes closely linked to the MHC locus substantially modulate differentiation of Tregs. Identification of responsible genes should help in understanding the mechanisms involved in commitment to the Treg lineage as well as selection of these cells in the thymus.

Ontogeny of American paddlefish lymphoid tissues

The temporal and spatial distribution of American paddlefish Polyodon spathula immune cell populations was determined using enzyme cytochemistry and immunohistochemistry. Monocytes and macrophages were present in the renal haematopoietic tissue, spleen, meningeal myeloid tissue, cardiac myeloid tissue and lamina propria of the spiral valve at 7 days post‐hatch (dph). Immature lymphocytes were present in the renal haematopoietic tissue, spleen, meningeal myeloid tissue, cardiac myeloid tissue, thymus and lamina propria of the spiral valve at 7 dph. Type A lymphocytes (T‐cell like) were demonstrated in the thymus by 21 dph. Type B immunoglobulin positive lymphocytes (B‐cell like) were present in the renal haematopoietic tissue, cardiac myeloid tissue and lamina propria of the spiral valve by 7 dph, the thymus at 21 dph, the spleen by 56 dph, and were not observed in the meningeal myeloid tissue of paddlefish aged 7–28 dph. Granulocytes were present in the renal haematopoietic tissue, thymus, spleen, meningeal myeloid tissue, cardiac myeloid tissue and lamina propria of the spiral valve by 7 dph. The spleens in 7–28 dph fish were predominately red pulp. Differentiation of leukocytic and erythrocytic compartments (white and red pulp, respectively) was not apparent in the spleen until 56 dph. Remarkable thymic cortical and medullary differentiation was not yet present at 28 dph, and the thymus was not sampled at 56, 84 or 112 dph. The cardiac myeloid tissue was not developed until 56 dph. Peyer’s patches were present in the lamina propria by 56 dph. Paddlefish lympho‐myeloid structures are therefore slow to develop, and vaccination procedures should be performed at 2–4 months post‐hatch.

Bmp4 and Noggin expression during early thymus and parathyroid organogenesis

The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have previously shown that thymus and parathyroid-specific organ domains are established within the primordium prior to formation of the organs proper: Gcm2 expression defines the prospective parathyroid cells in the dorsal pouch from E9.5, while Foxn1 is expressed in the thymus domain from E11.25. Bmp (bone morphogenetic protein) signaling has been implicated in thymic epithelial cell differentiation and thymus organogenesis. In the present study, we report expression patterns of Bmp4 and Noggin, a Bmp4 antagonist, in the third pharyngeal pouch using two lacZ transgenic mouse strains. Results from this gene expression study revealed localization of Bmp4 expression to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5, in those cells that will express Foxn1 and form the thymus. Conversely, the expression of Noggin was confined to the dorsal region of the pouch and primordium at these stages, and thus appeared to be co-expressed with Gcm2 in the parathyroid domain. This represents the first detailed study of Bmp4 and Noggin expression during the early stages of thymus and parathyroid organogenesis.

Organic Chemicals Adsorbed onto Diesel Exhaust Particles Directly Alter the Differentiation of Fetal Thymocytes Through Arylhydrocarbon Receptor but Not Oxidative Stress Responses

Diesel exhaust particles (DEP) were reported to have adverse effects on the immune system of laboratory animals and to induce thymic involution, particularly when exposure occurred during the fetal or lactational period. DEP consist of a carbon core to which many organic compounds are adsorbed, including polyaromatic hydrocarbons (PAHs) and their derivatives (e.g., dioxins and quinones). Although it has been suggested that these organic compounds were responsible for mediating the effects of DEP through their regulation of gene expression, the molecular mechanism of action of DEP has not been fully elucidated. In this study, we examined the direct effect of DEP extracts and their constituents on gene expression and phenotype in the fetal thymus. Fetal thymuses from C57BL/6 mice were exposed to DEP extracts for 24 hrs, after which their gene expression was analyzed using an Affymetrix GeneChip system. DEP extracts up-regulated several genes known as arylhydrocarbon receptor (AhR)-target genes, including cytochrome P450 1a1 (Cyp1a1), 1b1 (Cyp1b1), TCDD-inducible poly(ADP-ribose) polymerase (Tiparp), and scinderin (Scin). Similarly, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (B[a]P), which are AhR ligands, induced remarkably similar changes in gene expression compared to DEP extracts. In addition, our data showed little contribution of quinones to DEP extracts-induced changes in gene expression in fetal thymus through oxidative stress responses. These changes in gene expression were also confirmed by semi-quantitative RT-PCR. Furthermore, DEP extracts skewed thymic T-cell differentiation in favor of the production of CD8 T-cells, which was also observed when exposed to AhR ligands. Our results suggest that organic compounds adsorbed onto DEP alter thymic gene expression and directly affect thymocyte development by activating the AhR.

Regulated Costimulation in the Thymus Is Critical for T Cell Development: Dysregulated CD28 Costimulation Can Bypass the Pre-TCR Checkpoint

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.

Expansion and long-range differentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes

Unlike conventional major histocompatibility complex–restricted T cells, Vα14-Jα18 NKT cell lineage precursors engage in cognate interactions with CD1d-expressing bone marrow–derived cells that are both necessary and sufficient for their thymic selection and differentiation, but the nature and sequence of these interactions remain partially understood. After positive selection mediated by CD1d-expressing cortical thymocytes, the mature NKT cell lineage undergoes a series of changes suggesting antigen priming by a professional antigen-presenting cell, including extensive cell division, acquisition of a memory phenotype, the ability to produce interleukin-4 and interferon-γ, and the expression of a panoply of NK receptors. By using a combined transgenic and chimeric approach to restrict CD1d expression to cortical thymocytes and to prevent expression on other hematopoietic cell types such as dendritic cells, macrophages, or B cells, we found that, to a large extent, expansion and differentiation events could be imparted by a single-cognate interaction with CD1d-expressing cortical thymocytes. These surprising findings suggest that, unlike thymic epithelial cells, cortical thymocytes can provide unexpected, cell type–specific signals leading to lineage expansion and NKT cell differentiation.

HOXA genes are included in genetic and biologic networks defining human acute T-cell leukemia (T-ALL)

Using a combination of molecular cytogenetic and large-scale expression analysis in human T-cell acute lymphoblastic leukemias (T-ALLs), we identified and characterized a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA and the TCRB locus. Real-time quantitative polymerase chain reaction (RQ-PCR) analysis showed that the expression of the whole HOXA gene cluster was dramatically dysregulated in the HOXA-rearranged cases, and also in MLL and CALM-AF10-related T-ALL cases, strongly suggesting that HOXA genes are oncogenic in these leukemias. Inclusion of HOXA-translocated cases in a general molecular portrait of 92 T-ALLs based on large-scale expression analysis shows that this rearrangement defines a new homogeneous subgroup, which shares common biologic networks with the TLX1- and TLX3-related cases. Because T-ALLs derive from T-cell progenitors, expression profiles of the distinct T-ALL subgroups were analyzed with respect to those of normal human thymic subpopulations. Inappropriate use or perturbation of specific molecular networks involved in thymic differentiation was detected. Moreover, we found a significant association between T-ALL oncogenic subgroups and ectopic expression of a limited set of genes, including several developmental genes, namely HOXA, TLX1, TLX3, NKX3-1, SIX6, and TFAP2C. These data strongly support the view that the abnormal expression of developmental genes, including the prototypical homeobox genes HOXA, is critical in T-ALL oncogenesis.

A novel role for CD28 in thymic selection: elimination of CD28/B7 interactions increases positive selection

While the importance of the CD28/B7 costimulation pathway is well established for mature T cells, the role of CD28 in thymocyte selection is less well defined. The role of CD28 in both negative and positive selection was assessed using H-Y-specific TCR-transgenic (Tg) RAG-2-deficient (H-Yrag) mice. Negative selection in male H-Yrag mice was not affected by deficiency in CD28 or B7. Surprisingly, absence of CD28 or B7 in H-Yrag females resulted in increased numbers of CD8 single-positive (SP) thymocytes. The CD8 SP thymocytes found in these females were mature and functionally competent. Furthermore, double-positive (DP) thymocytes from CD28-knockout (CD28KO) or B7.1/B7.2 double-KO (B7DKO) females had higher levels of both CD5 and TCR than those from WT females, consistent with a stronger selecting signal. CD28KO H-Yrag fetal thymic organ cultures also had elevated numbers of thymic CD8 SP cells, reflecting increased thymic differentiation and not recirculation of peripheral T cells. Finally, increased selection of mature CD4 and CD8 SP T cells was observed in non-TCR-Tg CD28KO and B7DKO mice, indicating that this function of CD28-B7 interaction is not unique to a TCR-Tg model. Together these findings demonstrate a novel negative regulatory role for CD28 in inhibiting differentiation of SP thymocytes, probably through inhibition of thymic selection.

P53 prevents maturation of T cell development to the immature CD4 −CD8 + stage in Bcl11b−/− mice

Signaling pathways such as the pre-TCR and Wnt pathways regulate α/β T cell differentiation in thymus. Mice lacking an essential component of the pre-TCR exhibit arrest at the (CD4 −CD8 −) (CD44 −CD25 +) stage (DN3) of thymocyte development, and introduction of p53 deficiency into those mice abrogates this arrest, resulting in transition to the (CD4 +CD8 +) double-positive (DP) stage. This paper examines the effect of inactivation of p53 on thymocyte development in Bcl11b −/− mice that exhibit arrest at the DN3 or (CD4 −CD8 +) immature single-positive (ISP) stage. No DP thymocytes were detected in thymocytes of adoptive transfer experiments in scid mice that were derived from p53 −/− Bcl11b −/− precursors but ISP thymocytes increased in the proportion and in the cell number approximately three times higher than those from Bcl11b −/− precursors. Consistently, the level of apoptosis decreased to the level of wild-type precursors. These results suggest that inactivation of p53 is sufficient for DN3 thymocytes to differentiate into the ISP, but not to DP, stage of thymocyte development in Bcl11b −/− mice. This provides evidence for a novel p53-mediated checkpoint that regulates the transition from the DN3 to ISP stage of thymocyte development.

Inclusion of HOXA Genes in Genetic and Biological Networks Defining Human Acute T-Cell Leukemia.

We have identified a new recurrent chromosomal translocation, targeting the major homeobox gene cluster HOXA in human T-cell acute lymphoblastic leukemia (T-ALL). Four cases were characterized using a combination of FISH, Southern blot, breakpoint region sequencing, and a large scale expression analysis of a series of T-ALL. Specific RQ-PCR analysis of the HOXA1 to HOXA13 transcripts showed that the whole HOXA gene cluster expression was dramatically deregulated in the HOXA-rearranged cases, and also in the MLL and CALM-AF10-related T-ALL, strongly suggesting that HOXA genes are oncogenic in these types of leukemia. The HOXA-rearranged cases were included in a general portrait of T-ALL based on large scale expression analysis, showing that a new homogeneous T-ALL subgroup is defined by this chromosomal rearrangement. Moreover, patterns of gene expression associated to the distinct T-ALL oncogenic subgroups were compared with gene expression in normal human thymic sub-populations (11 purified sub-populations). Inappropriate use or perturbation of some specific molecular networks involved in thymic differentiation could be detected in the T-ALL cells. Also, we found that abnormal, frequently ectopic, expression of at least one developmental gene, including HOXA, TLX1/HOX11, TLX3/HOX11L2 and a few more, could be identified in most of the T-ALL cases. Our data strongly support the view that the abnormal expression of developmental genes, including the prototypical major homeobox genes HOXA in some cases, is critical in T-ALL oncogenesis.

Cbfb-MYH11 Induces Expansion of a Lin-Kit+Sca1- Abnormal Progenitor Compartment that Predisposes Acute Myeloid Leukemia in Mice.

Acute myeloid leukemia (AML) samples with chromosome 16 inversion express the CBFb-MYH11 fusion gene. One of three RUNX genes (RUNX1, RUNX2, and RUNX3) encode the α-subunit and CBFb encodes the β subunit of the heterodimeric transcription factor CBF. This transcription factor is a key regulator of multiple steps on hematopoietic differentiation. Studies in the mouse have determined that Cbfb-MYH11 expression impairs hematopoiesis, and that it induces AML in collaboration with other mutations.To further study the effects of Cbfb-MYH11 expression in hematopoiesis and leukemogenesis, we created a Cbfb-MYH11 conditional knock-in model (Cbfb56M), using the Cre-loxP recombination system. In this model, the floxed Cbfb allele expresses wildtype Cbfb, and the downstream Cbfb-MYH11 is induced upon Cre-mediated deletion. Floxed heterozygous and homozygous Cbfbfb56M mice are disease-free, indicating that Cbfb expressed from the floxed allele is functional. In addition, Cbfb-MYH11 was efficiently induced in over 80% of bone marrow cells from Cbfb56M/+/Mx1-Cre mice after pIpC injection.The preleukemic effects of Cbfb-MYH11 in hematopoiesis were analyzed using induced mice and non-competitive repopulation assays. First, circulating B-cells were reduced soon after Cbfb-MYH11 induction, and a significant differentiation block at the pre-pro B-cell stage was detected in the bone marrow. Second, thymic T-cell differentiation of induced mice showed impairment of DN2 to DN3 stage and reduction of thymic size in 3/10 induced mice analyzed. Interestingly, the number of circulating T cells was unaffected in repopulation assays. Third, platelets were reduced 50% in peripheral blood and megakaryocyte number was reduced in bone marrow. Fourth, we found an expanded abnormal progenitor compartment (Lin-kit+Sca1-) that accumulated in the bone marrow and spleen. In vitro differentiation assays showed a 2-to-3 fold increase of Cbfb-MYH11 colonies when compared to controls. The colony size was smaller, and showing partial differentiation deficiency. Interestingly, the colony numbers declined upon serial plating below controls. Taken together these results indicate that Cbfb-MYH11 induce accumulation of late progenitors (primarily myeloid progenitors) with limited self-renewal potential.Acute myeloid leukemia arised spontaneously 4 to 6 months after Cbfb-MYH11 induction, with expansion of blast- and monoblastic-like leukemic cells defined as Lin-kit+Sca1-. Leukemic mice showed infiltration in several tissues, including spleen, liver, brain, and lungs. To test whether a second “hit” is necessary in this model, we used bone marrow transduction assays to co-express Cbfb-MYH11 and the candidate cooperating gene Runx2. Recipient mice developed AML with similar phenotype 6 to 12 weeks post transplantation, while control mice remained healthy for 6 months.This study demonstrates that Cbfb-MYH11 expression (i) defines a preleukemic stage with hematopoietic differentiation block at stages associated with Runx function, (ii) the accumulation of an abnormal progenitor cell population (Lin-/kit+/Sca1-), (iii) induces additional mutations to efficiently develop AML, and (iv) synergizes with Runx2 in AML development.