Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.     

Transforming growth factor-beta- and Activin-Smad signaling pathways are activated at distinct maturation stages of the thymopoeisis

Members of the transforming growth factor (TGF)-beta family play pivotal roles in the control of differentiation, proliferation and tolerance in peripheral T cells. Recently, they have been implicated in thymic selection, but their role is so far not well characterized. In the present study, we demonstrate that specific thymocyte populations are under the influence of either the TGF-beta and/or Activin pathway, and transduce signals into the nucleus via phosphorylated Smad2 (pSmad2). Thymocytes in the medulla and in the subcapsular zone expressed nuclear translocated pSmad2, a hallmark of active TGF-beta/Activin receptor signaling. When analyzed at the cellular level, the pSmad2(+) cells were confined to the double-negative (DN) and single-positive (SP) subpopulations. Moreover, the most immature DN thymocytes (CD44(+)CD25(-) and CD44(+)CD25(+)) expressed higher levels of pSmad2 compared to the more mature DN. In vitro stimulation demonstrated that pure CD44(+)CD25(-), CD44(+)CD25(+) and CD44(+)CD25(+) thymocytes respond to ActivinA, while the mature CD4(+) and CD8(+) SP thymocytes respond to TGF-beta stimulation measured as enhanced phosphorylation of Smad2. Double staining of pSmad2(+) cells with either the Activin type I receptor, ALK4, or the TGF-beta type I receptor, ALK5, demonstrated that pSmad2(+) DN cells exhibited high levels of immunoreactivity to ALK4 and moderate levels of immunoreactivity to the TGF-beta-responsive ALK5 receptor. In sharp contrast, the SP pSmad2(+) cells were predominately ALK5(+). Collectively, our results demonstrate that early and late thymocytes express pSmad2 in the nuclei in vivo. The functional experiments in vitro suggest that members of the TGF-beta family (TGF-beta or Activin) may play important non-redundant roles during different stages of thymopoiesis.     

The role of Notch and IL-7 signaling in early thymocyte proliferation and differentiation

We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCRbeta and surface TCRalpha/beta and TCRgamma/delta expressing T cells. Selection for cytoplasmic TCRbeta-positive cells is dependent on preTalpha expression. Both gamma/delta and alpha/beta TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7.     

Loss of Bim results in abnormal accumulation of mature CD4-CD8-CD44-CD25- thymocytes

The process of thymopoiesis is tightly regulated by a series of selection events which ensure that only functional T-lymphocytes directed against foreign antigens are exported into the periphery. The adaptive immune response largely depends on the regulation of thymocyte development, and thymocytes which fail selection in the thymus are removed by apoptosis. However, the roles of specific apoptotic proteins in early T-lymphocyte development are poorly understood. Here, we report a novel function for Bim in thymocyte development. There is an accumulation of thymocytes in Bim(-/-) mice that lack expression of CD4, CD8, CD44, and CD25 but express CD3 and TCRbeta. Further, the CD4(-)CD8(-)CD25(-)CD44(-)CD3(+)TCRbeta(+) thymocytes are smaller and do not proliferate. These data suggest that these thymocytes are mature DN thymocytes that may have down-regulated the expression of CD4 and CD8. The DN thymocyte phenotype in Bim(-/-) mice is unaffected by the additional loss of Bak or Bax and is similar to the thymic phenotype in mice lacking both Bak and Bax. These data demonstrate that Bim functions to ensure the proper homeostasis of mature thymocytes during selection and thymic export.     

MCL1 increases primitive thymocyte viability in female mice and promotes thymic expansion into adulthood

Increasing the pool of cells at early T-cell developmental stages enhances thymopoiesis and is especially beneficial when T-cell production is compromised by radiation or aging. Within the immature double-negative (DN; CD4(-)CD8(-)) thymocyte subpopulation, the DN1 subset contains the most primitive cells including the rare early T-cell progenitors (ETPs). In the present study, a human MCL1 transgene, under the control of its endogenous promoter, resulted in enlargement of an undistorted thymus in C57/BL6 mice. Enlargement occurred in females but not males, being seen at 1 month of age and maintained during progression into adulthood as the thymus underwent involution. The small DN1 subset was expanded disproportionally (ETPs increasing from ～0.016 to 0.03% of thymocytes), while more mature thymocytes were increased proportionally (1.5-fold) along with the stroma. DN1 cells from transgenic females exhibited increased viability with maintained proliferation, and their survival in primary culture was extended. Exposure of transgenic females to γ-irradiation also revealed an expanded pool of radioresistant DN1 cells exhibiting increased viability. While the viability of DN1 cells from transgenic males was equivalent to that of their non-transgenic counterparts directly after harvest, it was enhanced in culture-suggesting that the effect of the transgene was suppressed in the in vivo environment of the male. Viability was increased in ETPs from transgenic females, but unchanged in more mature thymocytes, indicating that primitive cells were affected selectively. The MCL1 transgene thus increases the viability and pool size of primitive ETP/DN1 cells, promoting thymopoiesis and radioresistance in peripubescent females and into adulthood.     

Beta-selection: abundance of TCRbeta-/gammadelta- CD44- CD25- (DN4) cells in the foetal thymus

Expression of TCRbeta and pre-TCR signalling are essential for differentiation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double-positive (DP) stage. Thymocyte development in adult Rag1, Rag2 or TCRbetadelta-deficient mice is arrested at the DN3 stage leading to the assumption that pre-TCR signalling and beta-selection occur at, and are obligatory for, the transition from DN3 to DN4. We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta. These foetal icTCRbeta-/gammadelta- DN4 cells were T lineage as determined by expression of Thy1 and icCD3 and TCRbeta DJ rearrangement. In addition, in the foetal Rag1-/- thymus, a normal percentage of DN4 cells were present. In wild-type mice after hydrocortisone-induced synchronisation of differentiation, the majority of DN4 cells that first emerged did not express icTCRbeta/gammadelta, showing that adult thymocytes can also differentiate to the DN4 stage independently of pre-TCR signalling. Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis. Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.     

A role for E2-2 at the DN3 stage of early thymopoiesis

Roles for the E-proteins E2A and HEB during T lymphocyte development have been well established. Based on our previous observations of counter selection against T cells lacking E2-2, it seemed reasonable to assume that there would be a function also for E2-2 in thymocyte development. Aiming at assigning such a role for E2-2, we analyzed the expression of E2-2, E2A, HEB as well as Id mRNA during T cell development. Interestingly, whereas all three E-proteins were expressed during early thymocyte development, significant expression beyond the DP stage was detected only for E2A. Among the Id proteins, Id2 displayed a prominent expression exclusively in DN1, whereas Id3 showed some expression in DN1, followed by a down regulation and then a prominent induction, peaking in the DP stage. E2-2 was expressed during the DN stages, as well as in the DP stage, suggesting that E2-2 operates in concert with the other E-proteins during early thymocyte development. We found that E2-2 null thymocytes displayed a partial block at the DN3 stage of development, as well as a reduced expression of pre-T alpha, known to be regulated also by E2A and HEB. The fact that E2-2 deficient thymocytes develop without gross abnormalities is likely to stem from redundancy due to the co-expression of E2A and HEB.     

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation

ABSTRACT: BACKGROUND: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. RESULTS: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRalphabeta+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vbeta repertoire. CONCLUSIONS: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.     

Inhibition of thymopoiesis of CD34+ cell maturation by HIV-1 in an in vitro CD34+ cell and thymic epithelial organ culture model

The mechanisms by which HIV-1 affects thymopoiesis were determined by preincubating CD34+ cells or cultured thymic epithelial (CTE) cells with lymphotropic (T-) and monotropic (M-) strains of HIV-1 in an in vitro CTE organ and CD34+ cell coculture model that allows for analysis of development of thymocytes and mature T cells. When purified CD34+ cells were precultured with either T- or M-tropic strains of HIV-1, thymopoiesis was impaired in a two-week coculture manifested by decreased cell number of thymocytes generated. However, the percentages of thymocyte subpopulations were comparable to control uninfected cocultures. Furthermore, HIV infection of thymocytes was predominantly observed in the CD44+CD3- population. However, in a four-week coculture experiment, HIV infection and depletion of more mature thymocytes were also observed. When CTE cells were preincubated with T- and M-tropic strains of HIV before addition of CD34+ cells, the number of thymocytes and subpopulations of thymocytes at early and later stages of maturation were markedly decreased. Furthermore, CD34+ and CD44+CD3- cells become HIV-infected. In summary, HIV-1 infection inhibited thymocyte maturation at early stages of thymocyte maturation CD44+CD25-CD3-. In addition, HIV also depleted later stages of CD4+ thymocyte subpopulations.     

Metalloproteinase-dependent control of thymocyte differentiation and proliferation

The development of T cells in the thymus is dependent on interactions between thymocytes and thymic stromal cells, on stimulation by growth factors, and on the binding to and migration along extracellular matrix (ECM) components. As metalloproteinases (MP) are involved in processes such as growth factor release and ECM modelling, we assessed the effect of MP inhibitors on T-cell development using fetal thymic organ culture systems. MP inhibitors significantly reduced the numbers of CD4/CD8 double-positive (DP) and mature single-positive thymocytes generated, correlated with a reduced number of cell cycles between the double-negative (DN)3 and DP stages. The progression of early thymocyte progenitors through the DN1-4 stages of development was also severely affected, including incomplete upregulation of CD25, decreased DN3 cell numbers, reduced rearrangement of the T-cell receptor (TCR)-beta locus and expression of intracellular TCR-beta by fewer DN3 cells. When purified DN1 cells were utilized as donor cells in reaggregate thymic organ cultures, essentially no DP thymocytes were produced in the presence of MP inhibitors. The results suggest that MP inhibitors affect the differentiation of developing thymocytes before, and reduce proliferation after, pre-TCR-mediated selection.     

Constitutive Notch signalling promotes CD4 CD8 thymocyte differentiation in the absence of the pre-TCR complex, by mimicking pre-TCR signals

Notch1 signalling is essential for the commitment of multipotent lymphocyte precursors towards the alphabeta T-cell lineage and plays an important role in regulating beta-selection in CD4(-)CD8(-) double-negative (DN) thymocytes. However, the role played by Notch in promoting the development of CD4(+)CD8(+) double-positive (DP) thymocytes is poorly characterized. Here, we demonstrate that the introduction of a constitutively active Notch1 (ICN1) construct into RAG(-/-) lymphocyte precursors resulted in the generation of DP thymocytes in in vitro T-cell culture systems. Notably, developmental rescue was dependent not only on the presence of an intact Notch1 RAM domain but also on Delta-like signals, as ICN1-induced DP development in RAG(-/-) thymocytes occurred within an intact thymus or in OP9-DL1 co-cultures, but not in OP9-control co-cultures. Interestingly, ICN1 expression in SLP-76(-/-) precursors resulted in only a minimal developmental rescue to the immature CD8(+) single-positive stage, suggesting that Notch is utilizing the same signalling pathway as the pre-TCR complex. In support of this, ICN1 introduction resulted in the activation of the ERK-MAPK-signalling cascade in RAG(-/-) thymocytes. Taken together, these studies demonstrate that constitutive Notch signalling can bypass beta-selection during early T-cell development by inducing pre-TCR-like signals within a T-cell-promoting environment.     

Co-ordinate expression of the pre-T-cell receptor complex and a novel immature thymocyte-specific antigen, IMT-1, during thymocyte development

Previously we described a monoclonal antibody (mAb) that reacted with a cell-surface antigen, immature thymocyte antigen-1 (IMT-1), which is expressed on thymocytes of late CD4- CD8- (double negative) to early CD4+ CD8+ (double positive) differentiation stages. In this study, we investigated the expression of IMT-1 on various cell lineages in thymus as well as in peripheral lymphoid organs. We found that IMT-1 is expressed on T-cell receptor (TCR)-betalo and TCR-deltalo thymocytes, but not on TCR-betahi, TCR-deltahi or natural killer (NK)1.1+ thymocytes, or on peripheral alpha beta or gamma delta T cells. We also investigated the kinetics of expression of IMT-1 during fetal thymocyte development and compared it with the expression of the pre-TCR complex, comprising CD3, pre-TCR-alpha (pTalpha) and TCR-beta. We found that expression of both was similar, starting at day 14.5 of gestation, peaking on day 16.5 and gradually decreasing thereafter. Furthermore, the expression of both IMT-1 and pTalpha was drastically reduced when DN thymocytes in recombination activating gene (RAG)-2-/- mice were challenged in vivo with anti-CD3 mAb. These results indicate that IMT-1 is expressed on not only immature thymocytes of alpha beta T-cell lineage but also on those of gamma delta T-cell lineage, and that the expression of IMT-1 and the pre-TCR complex is co-ordinately regulated during the alpha beta lineage thymocyte development.     

Lineage choices in the developing thymus: choosing the T and NKT pathways

Thymic development proceeds through several defined stages that generate not only alpha beta and gamma delta T cells but can produce dendritic cells and B cells. The earliest thymocytes exist in the CD4(-)CD8(-) double negative compartment within a heterogeneous fraction termed DN1. Recent progress has identified several candidate populations that may be the bone fide T-cell progenitor population. The potential roles of these populations, which include hematopoietic stem cells, early lymphocyte precursors, common lymphoid progenitors, and early T lineage progenitors are being elucidated. The alpha beta T-cell lineage consists of distinct subsets, one of which is NKT cells. The developmental relationship of NKT cells to conventional T cells has been controversial. Recent work has shown that these cells are probably derived from CD4(+)CD8(+) thymocytes. The discovery and application of CD1d tetramers has made it possible to more fully describe NKT-cell development.     

Regulation of cytokine receptor signaling by SOCS1

Summary: The suppressor of cytokine signaling (SOCS) family of proteins is a novel class of negative feedback regulators of cytokine receptor signaling. SOCS1 is rapidly induced following stimulation by several type I and type II cytokines, and it attenuates their signaling by its ability to bind and inhibit all four of the Janus family of intracellular tyrosine kinases (JAKs). Studies from our own and other laboratories have documented another important function of SOCS1 in facilitating ubiquitination of protein substrates and their subsequent proteasomal degradation. SOCS1 also functions as a potential tumor suppressor by inhibiting several hematopoietic oncogenes. In addition to these negative regulatory functions, we have recently shown a positive regulatory role for SOCS1 in increasing the stability of major histocompatibility complex (MHC) class II proteins by preventing their degradation. These findings illustrate multiple roles for SOCS1 in cytokine receptor signaling, and provide groundwork for detailed analysis of the role of SOCS1 in pre‐T cell receptor (TCR) and TCR signaling, and regulation of T helper (Th)1 and Th2 differentiation.