Stages Of Thymocyte Development Research Articles

The Thymus in Chagas Disease: Molecular Interactions Involved in Abnormal T-Cell Migration and Differentiation.

Chagas disease, caused by the protozoan parasite T. cruzi, is a prevalent parasitic disease in Latin America. Presently, it is spreading around the world by human migration, thus representing a new global health issue. Chronically infected individuals reveal a dissimilar disease progression: while nearly 60% remain without apparent disease for life, 30% develop life-threatening pathologies, such as chronic chagasic cardiomyopathy (CCC) or megaviscerae. Inflammation driven by parasite persistence seems to be involved in the pathophysiology of the disease. However, there is also evidence of the occurrence of autoimmune events, mainly caused by molecular mimicry and bystander activation. In experimental models of disease, is well-established that T. cruzi infects the thymus and causes locally profound structural and functional alterations. The hallmark is a massive loss of CD4+CD8+ double positive (DP) thymocytes, mainly triggered by increased levels of glucocorticoids, although other mechanisms seem to act simultaneously. Thymic epithelial cells (TEC) exhibited an increase in extracellular matrix deposition, which are related to thymocyte migratory alterations. Moreover, medullary TEC showed a decreased expression of AIRE and altered expression of microRNAs, which might be linked to a disrupted negative selection of the T-cell repertoire. Also, almost all stages of thymocyte development are altered, including an abnormal output of CD4−CD8− double negative (DN) and DP immature and mature cells, many of them carrying prohibited TCR-Vβ segments. Evidence has shown that DN and DP cells with an activated phenotype can be tracked in the blood of humans with chronic Chagas disease and also in the secondary lymphoid organs and heart of infected mice, raising new questions about the relevance of these populations in the pathogenesis of Chagas disease and their possible link with thymic alterations and an immunoendocrine imbalance. Here, we discuss diverse molecular mechanisms underlying thymic abnormalities occurring during T. cruzi infection and their link with CCC, which may contribute to the design of innovative strategies to control Chagas disease pathology.

Sexual dimorphism in rat thymic involution: a correlation with thymic oxidative status and inflammation.

The study investigated mechanisms underlying sex differences in thymic involution in Dark Agouti rats. Adverse effects of aging on thymus were more pronounced in males than in females. Thymi from old males exhibited more prominent: (i) fibro-adipose degeneration which correlated with greater intensity of thymic oxidative stress and enhanced thymic TGF-β and IL-6 expression and (ii) decline in thymopoiesis, as suggested by the number of the most mature CD4+CD8-/CD4-CD8+ single positive (SP) TCRαβhigh thymocytes. The greater accumulation of adipose tissue in old male thymus was linked with greater age-related increase in thymic expression of PPARγ and STAT3, a transcription factor regulating the expression of PPARγ downstream genes, in male than in female rats. In aged thymi of both sexes the early CD4-CD8- double negative (DN) stage of thymocyte development was affected, so relative accumulation of the least mature CD45RC+CD2- cells followed by decreased frequency of their DN and CD4+CD8+ double positive (DP) TCRαβ- descendants was observed. Additionally, in old males, because of the increased thymic expression of Nur77, a nuclear receptor involved in negative selection, and decreased CD90 (a negative regulator of thymocyte selection threshold) MFI on DP TCRαβint thymocytes, less efficient positive/more efficient negative selection was found. Moreover, in male rats, thymocyte post-selection differentiation/maturation was skewed towards CD4-CD8+ SP TCRαβhigh cells compared with age-matched females, reflecting, at least partly, greater IL-15 expression in their thymi. The study indicated mechanisms underlying sex-based differences in age-related thymic changes and consequently necessity of sex-specific approaches in designing strategies to rejuvenate thymus.

Humanized Mice Reveal New Insights Into the Thymic Selection of Human Autoreactive CD8+ T Cells.

Thymic selection constitutes the first checkpoint in T-cell development to purge autoreactive T cells. Most of our understanding of this process comes from animal models because of the challenges of studying thymopoiesis and how T cell receptor (TCR) specificity impacts thymocyte phenotype in humans. We developed a humanized mouse model involving the introduction of autoreactive TCRs and cognate autoantigens that enables the analysis of selection of human T cells in human thymic tissue in vivo. Here, we describe the thymic development of MART1-specific autoreactive CD8+ T cells that normally escape deletion and how their phenotype and survival are affected by introduction of the missing epitope in the hematopoietic lineage. Expression of the epitope in a fraction of hematopoietic cells, including all major types of antigen-presenting cells (APCs), led to profound yet incomplete deletion of these T cells. Upregulation of PD-1 upon antigen encounter occurred through the different stages of thymocyte development. PD-1 and CCR7 expression were mutually exclusive in both transgenic and non-transgenic thymocytes, challenging the view that CCR7 is necessary for negative selection in humans. In the presence of antigen, MART1-reactive T cells down-regulated TCR, CD3, CD8, and CD4 in the thymus and periphery. Moreover, expression of secondary TCRs influences MHC class I-restricted T cells to develop as CD4+, particularly regulatory T cells. This new model constitutes a valuable tool to better understand the development of autoreactive T cells identified in different human autoimmune diseases and the role of different APC subsets in their selection.

Deletion of the highly conserved functional region of the Cd4 enhancer NCE in RLM11 cells using CRISPR/Cas9 reduces CD4 expression

Abstract The glycoprotein cell surface marker CD4 is a key mediator in the adaptive immune response and T-cell development. Signaled CD4+ CD8+ double positive cells (DP) upregulate CD4 on the cell surface, allowing for a persistent TCR signal in the thymus and differentiating into the CD4+ single positive helper lineage. Previously, we have demonstrated that NCE (novel cis element), located in the first intron of Cd4, acts as an enhancer in RLM11 cells (CD4+, CD8−, Th-Pok− thymoma, arrested at the intermediate stage of development), but not in AKR-1G1 or VL3-3M2 cells (DP thymomas). We have identified a highly conserved region within NCE, labeled core-NCE, which may be vital to the enhancer’s function. To investigate the contribution of core-NCE to the level of CD4 expression, we utilized CRISPR/Cas9 technology to target the region for deletion. Here we describe the construction of plasmids containing guide sequences for targeted deletion of core-NCE by Cas9 cutting and NHEJ repair. We have successfully generated and sequence verified plasmids with three upstream and three downstream guides and introduced them in RLM11 cells. After single cell sorting, we obtained one clone that has the 300bp core-NCE deleted on one Cd4 allele, which resulted in 66% reduction in CD4 expression from that allele. As we removed the core-NCE in cells that have already established CD4 expression, we conclude that the core NCE has an enhancer function at the intermediate/transitional stage of thymocyte development that is separate from the previously described epigenetic function of the same intronic region, making NCE a good candidate for the regulatory element responsible for CD4 upregulation during differentiation into the CD4 lineage.

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3+ regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.

Loss of G\u03b1i proteins impairs thymocyte development, disrupts T-cell trafficking, and leads to an expanded population of splenic CD4+PD-1+CXCR5+/\u2212 T-cells

Thymocyte and T cell trafficking relies on signals initiated by G-protein coupled receptors. To address the importance of the G-proteins Gαi2 and Gαi3 in thymocyte and T cell function, we developed several mouse models. Gαi2 deficiency in hematopoietic progenitors led to a small thymus, a double negative (DN)1/DN2 thymocyte transition block, and an accumulation of mature single positive (SP) thymocytes. Loss at the double positive (DP) stage of thymocyte development caused an increase in mature cells within the thymus. In both models an abnormal distribution of memory and naïve CD4 T cells occurred, and peripheral CD4 and CD8 T cells had reduced chemoattractant responses. The loss of Gαi3 had no discernable impact, however the lack of both G-proteins commencing at the DP stage caused a severe T cell phenotype. These mice lacked a thymic medullary region, exhibited thymocyte retention, had a peripheral T cell deficiency, and lacked T cell chemoattractant responses. Yet a noteworthy population of CD4+PD-1+CXCR5+/− cells resided in the spleen of these mice likely due to a loss of regulatory T cell function. Our results delineate a role for Gαi2 in early thymocyte development and for Gαi2/3 in multiple aspects of T cell biology.

The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke

The protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.

Peroxisome Proliferator-Activated Receptor Delta (PPARδ) activity is required for normal T cell development

Abstract PPARδ is a nuclear receptor that functions as a transcriptional regulator of various processes including metabolic homeostasis, cell proliferation and differentiation; however, whether PPARδ regulates T cell development is not known. We observed that PPARδ-deficient (PPARδ−/−) mice have smaller spleens and thymi compared to wild-type (WT) mice. This reduction in thymus cellularity was apparent across all stages of thymocyte development. To further narrow in on the cellular basis of this effect, radiation bone marrow chimeras were generated where WT or PPARδ−/− mice were reconstituted with WT or PPARδ−/− bone marrow. These experiments clearly revealed that the defects in thymus and spleen mapped to the radio-resistant compartment. This was furthered confirmed by the finding that mice with T cell-restricted deficiency of PPARδ did not exhibit defective thymocyte homeostasis. Given that thymic epithelial cells (TECs) are the major radio-resistant cell population in the thymus, we next generated mice with a TEC-restricted deficiency of PPARδ (PPARδflox/floxFoxN1-Cre+). We observed that PPARδflox/floxFoxN1-Cre+ mice exhibit a trend for reduced thymus and spleen cellularity compared to PPARδflox/flox mice; however, this phenotype was not as striking as seen in PPARδ−/− mice. These results suggest that, in addition to acting within TECs, PPARδ may regulate the production of a circulating factor that in turn regulates thymocyte development. Large-scale serum profiling studies are underway to identify this factor. Altogether, this work highlights a role for PPARδ in T cell development.

Insights into the Transcriptional Regulation of the Unrearranged and Rearranged Tcra and Tcrd Genes

The combined T-cell receptor α and δ locus, Tcra/Tcrd, encodes the TCRα and TCRδ chains of the αβ or γδ T-cell receptors (TCRαβ and TCRγδ), respectively, which define the two distinct T-cell lineages, αβ and γδ T lymphocytes. Like other antigen receptor loci, this locus must recombine its variable (V), diversity (D), and joining (J) gene segments to generate a diverse range of TCR that allow vertebrates to respond to an unlimited number of antigens. The Tcra/Tcrd germline transcription and subsequent V(D)J gene segment rearrangements are strictly regulated by two distant transcriptional enhancers, Eα and Eδ, respectively, during thymocyte development. Once the Tcra locus is productively rearranged, it is assumed Eα remains active for the transcription of the rearranged locus and the expression of the functional TCRα chain in αβ T lymphocytes. However, our recent experiments have shown Eα is significantly inhibited during the final stage of thymocyte development, concomitantly with the expression of the rearranged Tcra locus, and remains inhibited in αβ T lymphocytes. These results imply the existence of an Eα-independent mechanism to activate transcription of the rearranged Tcra locus in αβ T lymphocytes. Interestingly, Eα is essential for the normal expression of the rearranged Tcrd locus in γδ T lymphocytes. In this review, the current knowledge about the regulation of Tcra/Tcrd germline transcription and gene segment rearrangement during thymocyte development and the possible mechanisms for transcription of the rearranged Tcra locus in mature αβ T lymphocytes are discussed. The knowledge of the detailed mechanisms involved in the regulation of transcription at the Tcra/Tcrd locus by distant enhancers is important to understand the cases in which deregulation this process resulta in disease.

Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

Differential Development Of Donor Bone Marrow-Derived Thymocytes After Allogeneic In Utero Hematopoietic Cell Transplantation In The Murine Model

Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development during postnatal ontogeny. Methods Bone marrow (BM) cells (10x106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. Results Our findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of immature TCRβ-CD4+CD8- and TCRβ-CD4-CD8+ single positive (SP) cells, a slightly higher proportion of CD4-CD8- (DN) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor DN cells and TCR gamma/delta cells which do not require positive selection based on MHC recognition are increased compared to the host. Donor T-regulatory cells were present at a higher frequency in the thymus and secondary lymphoid organs of chimeric animals compared to T-regulatory cells in host and normal strain controls. Conclusion Our data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development. In contrast, generation of donor T-regulatory cells was not impaired and appeared to be increased in chimeric animals. The alloreactivity, regulatory suppression, and MHC restriction of the donor derived T cells, and the potential importance of these observations for clinical IUHCT remain to be determined. Disclosures: No relevant conflicts of interest to declare.

Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4‐T‐cells

After thymic emigration CD4-T-cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4-T-cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T-cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4-T-cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T-cells involve infection of in vitro activated cells with Cre expressing lenti-, retro-, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4-T-cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER(T2) ) under the control of the CD4 gene promoter. We show here that in CD4CreER(T2) mice Cre is inducibly and selectively activated in CD4-T-cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4-T-cells into effector or suppressor sub-lineages.

Role of Hedgehog signalling at the transition from double‐positive to single‐positive thymocyte

In the thymus, developing T cells receive signals that determine lineage choice, specificity, MHC restriction and tolerance to self-antigen. One way in which thymocytes receive instruction is by secretion of Sonic hedgehog (Shh) from thymic epithelial cells. We have previously shown that Hedgehog (Hh) signalling in the thymus decreases the CD4:CD8 single-positive (SP) thymocyte ratio. Here, we present data indicating that double-positive (DP) thymocytes are Hh-responsive and that thymocyte-intrinsic Hh signalling plays a role in modulating the production of CD4+ (SP4), CD8+ (SP8) and unconventional T-cell subsets. Repression of physiological Hh signalling in thymocytes altered the proportions of DP and SP4 cells. Thymocyte-intrinsic Hh-dependent transcription also attenuated both the production of mature SP4 and SP8 cells, and the establishment of peripheral T-cell compartments in TCR-transgenic mice. Additionally, stimulation or withdrawal of Hh signals in the WT foetal thymus impaired or enhanced upregulation of the CD4 lineage-specific transcription factor Gata3 respectively. These data together suggest that Hh signalling may play a role in influencing the later stages of thymocyte development.

The pre-TCR signal induces transcriptional silencing of the TCR locus by reducing the recruitment of STAT5 and Runx to transcriptional enhancers

The mouse TCRγ locus is positively regulated by the transcription factors STAT5 and Runx. While the locus undergoes frequent rearrangements in T lymphocytes, TCRγ transcription is repressed in αβ T cells. This phenomenon, known as TCRγ silencing, depends on pre-TCR-induced thymocyte proliferation. The molecular basis for TCRγ silencing, however, is largely unknown. Here, we show that pre-TCR signaling reduces transcription and histone acetylation of the TCRγ locus irrespective of V-J rearrangements. We also demonstrate that Runx is recruited to Eγ and HsA enhancer elements of the TCRγ locus, primarily at the CD4(-)CD8(-) double-negative stage and that Runx binding to these elements decreases at later stages of thymocyte development. Importantly, anti-CD3 antibody treatment decreased IL-7R expression levels, STAT5 phosphorylation and recruitment of STAT5 and Runx to Eγ and HsA elements in RAG2-deficient thymocytes, suggesting that pre-TCR signaling triggers reduced binding of STAT5 and Runx to the enhancer elements. Furthermore, we observed that misexpression of STAT5 or Runx in the CD4(+)CD8(+) double-positive cell line DPK induces TCRγ gene transcription. Finally, we showed that TCRγ transcription is induced in αβ T cells from Runx3 transgenic mice, suggesting that Runx3 counteracts TCRγ silencing in αβ T cells in vivo. Our results suggest that pre-TCR signaling indirectly inactivates TCRγ enhancers by reducing recruitment of STAT5 and Runx and imply that this effect is an important step for TCRγ silencing in αβ T cells.

Over‐expression of Runx1 transcription factor impairs the development of thymocytes from the double‐negative to double‐positive stages

Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.

Costimulatory Role of CXCR4 with Pre-TCR and Its Crosstalk with PI3K in β-Selection of Thymocytes

Chemokines were initially thought of only as regulators of the migration of developing thymocytes between different compartments of the thymus and were predicted also to be involved in early stages of thymocyte development. Recent data have suggested additional functions for the chemokine receptor CXCR4-a G protein-coupled receptor-in thymocyte development, particularly during the beta-selection developmental checkpoint. Phosphotidylinositol 3-kinase interacts with the pre-T cell receptor and CXCR4 during migration, proliferation, and differentiation of developing thymocytes. CXCR4 and Notch signaling, along with inhibition of glycogen synthase kinase-3beta (GSK-3beta), are essential for the proliferation and differentiation of thymocytes during beta-selection. Thus, a critical role of chemokines and its downstream signaling in the beta-selection of thymocytes has been demonstrated.

Role of CRTAM during mouse early T lymphocytes development

CRTAM was reported as a novel receptor expressed in activated NKT and CD8 T lymphocytes. However, we have recently shown that it is also expressed in several non-immune tissues. In opposition to what has been stated for lymphoid cells, CRTAM expression is constitutive in epithelia, suggesting a role in cell–cell interactions. Given the importance of cell interactions during T lymphocyte development, we evaluated CRTAM during T lymphocyte ontogeny. Here we show that CRTAM has an unexpected constitutive expression in adult thymocytes and, remarkably, it is sustained during all stages of thymocyte development. CRTAM expression is restricted to CD8 and all DN subpopulations, with a consistent pattern from E13.5 stage to adult mice. Blocking CRTAM interaction with CADM1 impairs thymus growth, uncovering a novel role in thymus development, with a consequent impact in thymocyte maturation. Thus, CRTAM interaction with CADM1 is involved in structural maintenance of the thymic lobes.

Role of TLX1 in T‐cell acute lymphoblastic leukaemia pathogenesis

The diverged homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11 or TCL3) is activated in ∼5-10% of childhood and up to 30% of adult T-cell acute lymphoblastic leukemia (T-ALL) cases, most frequently by t(10;14)(q24;q11) and t(7;10)(q35;q24) chromosomal translocations which juxtapose the intact TLX1 coding region downstream of T cell receptor (TCR) δ (TRD@) or TCRβ (TCRB) gene regulatory sequences. TLX1+ T-ALL samples are virtually all arrested at the early cortical (CD1+) CD4+CD8+ “double-positive” (DP) stage of thymocyte development (Ferrando et al, 2002; Asnafi et al, 2004).

Challenges and Opportunities for Effective NOTCH1 Targeting in T-ALL.

The NOTCH pathway is an evolutionarily conserved mechanism responsible for the direct transduction of developmental signals at the cell surface into changes in gene expression in the nucleus. In the hematopoietic system, the NOTCH1 signaling pathway is required for the commitment of multipotent hematopoietic progenitors to the T cell lineage and, later on, to support cell growth, proliferation, and survival at multiple stages of thymocyte development. These physiological functions are disrupted by activating mutations in the NOTCH1 gene in over 50% of T-cell acute lymphoblastic leukemias (T-ALL). NOTCH1 mutations result in ligand-independent activation or increased protein stability, leading to deregulated cell proliferation and survival in T cell progenitors and T cell transformation. Importantly, small molecule inhibitors of the γ-secretase complex (GSIs) effectively abrogate NOTCH1 signaling, making NOTCH1 a promising therapeutic target for the treatment of T-ALL. However, the clinical development of GSIs has been hampered by our incomplete understanding of the effector pathways controlled by NOTCH1, the lack of clinical responses to GSI therapy, and the development of gastrointestinal toxicity secondary to inhibition of NOTCH signaling in the gut. Recent progress in the identification of the transcriptional regulatory networks that control T cell transformation downstream of NOTCH1 has shown a close relationship between oncogenic NOTCH1 signaling and the transcriptional control of cell growth and metabolism. Thus, NOTCH1 directly controls multiple genes involved in anabolic pathways and further promotes cell growth via direct transcriptional upregulation of MYC and indirect upregulation of the PI3K-AKT signaling pathway via HES1-mediated downregulation of PTEN. Moreover, oncogenic NOTCH1 signaling promotes activation of the NFKB pathway and interacts with the function of p53. Although the specific mechanisms mediating some of these interactions have not been elucidated yet, these results suggest that combinations of GSIs with chemotherapy or drugs targeting the PI3K-AKT and NFKB pathways may have a synergistic effect in the treatment of T-ALL. Moreover, inhibitory antibodies capable of selectively blocking the activation of NOTCH1 and inhibitors of the nuclear transcriptional complex mediating the activation of NOTCH1 target genes offer alternative approaches for the inhibition of oncogenic NOTCH1 signaling. Each of these strategies offers the promise to induce potent antileukemic effects and/or to ameliorate the gastrointestinal toxicity associated with systemic inhibition of NOTCH signaling and may ultimately lead to the development of rationally-designed and highly effective anti-NOTCH1 therapies for T-ALL.