Immature thymocytes that fail to express TCRbeta and/or TCRgamma delta proteins die by apoptotic cell death in the CD44(-)CD25(-) (DN4) subset.

Pre-TCR/CD3 signals are essential for survival and maturation of (CD44(-)25(+)) DN3 thymocytes via the (CD44(-)25(-)) DN4 stage to CD4(+)CD8(+) (DP) cells, a process termed beta-selection. The exact developmental stages of apoptosis resulting from lack of pre-TCR/CD3 signals have so far not been determined. Here we analyzed apoptotic cell death in relation to expression of clonotypic TCR polypeptides and to cell cycle status in immature thymocyte subpopulations of wild type (wt) mice and of several strains of mice with compromised pre-TCR/CD3 signaling complexes. In wt mice or pre-TCR/CD3-deficient mice, apoptotic cells could not be detected among DN3 cells but accumulated in a subset of DN4 expressing CD69. Apoptotic CD69(+)DN4 cells were rare in wt mice and were found among DN4 cells that were negative or low for intracellular TCRbeta and negative for TCRgamma delta polypeptide chains. Apoptotic CD69(+)DN4 cells were abundant in pre-TCR/CD3 signaling-deficient mice in which most DN4 cells failed to express clonotypic TCR polypeptides. Survival of DN4 cells, but not maturation of DN3 cells to DN4, was found to depend on the expression of clonotypic TCR polypeptides in the same cell. The results suggest that thymocytes unsuccessful in alpha beta or in gamma delta lineage development die by apoptosis in the DN4 subset.     

Pre-TCR signaling regulates IL-7 receptor alpha expression promoting thymocyte survival at the transition from the double-negative to double-positive stage

The pre-T cell receptor (pre-TCR) and IL-7 receptor (IL-7R) are critical mediators of survival, proliferation and differentiation in immature thymocytes. Here we show that pre-TCR signaling directly maintains IL-7Ralpha expression as developing thymocytes undergo beta-selection. Inhibition of IL-7/IL-7R signaling in (CD44-CD25-) DN4 cells results in decreased generation of double-positive thymocytes due to increased death of rapidly proliferating beta-selected cells. Thus, we identify a mechanism by which pre-TCR signaling controls the selective survival of TCRbeta+ thymocytes, and define a further stage of T cell differentiation in which signaling from a TCR regulates the ability of that cell to respond to cytokine.     

Heterogeneity of the DN4 (CD44-CD25-) subset of CD4-CD8- double negative thymocytes; dependence on CD3 signaling

Recent studies have shown that apoptotic cell death associated with selection for thymocytes that express clonotypic TCRbeta or TCRgammadelta proteins takes place in the DN4 (CD44-CD25-) subset of CD4-CD8- double negative (DN) thymocytes. A detailed analysis of the DN4 subset is therefore of interest. Using intracellular (IC) staining for clonotypic TCR and CD3varepsilon proteins we find that DN4 cells consist of five subpopulations: TCRbetaIC(high)/CD3varepsilonIC(high)/TCRgammadeltaIC-, TCRbetaI-C-/CD3varepsilonIC(high)/TCRgammadeltaIC(+), TCRbetaIC(high)/CD3varepsilonIC(high)/TCRgammadeltaIC(+), TCRbetaIC(low)/CD3varepsilonIC(low)/TCRgammadeltaIC(-), and TCRbetaIC(-)/CD3varepsilonIC(-)/TCRgammadeltaIC(-). Expression levels of IC TCRbeta/CD3varepsilon, and of Thy1.2, CD2, and CD69 at the cell surface suggest that the TCRbetaIC(low)/CD3varepsilonIC(low)/TCRgammadeltaIC(-) subset harbors the direct precursors of DP cells, and is critical for life/death decisions in early thymic selection. TCRbeta/CD3varepsilon downregulation is less pronounced in DN4 and DP cells of mice deficient for CD3zeta or for p56(lck), suggesting that the dynamics of TCR protein regulation in the DN4 subset is dependent on CD3 signaling.     

The CD45 tyrosine phosphatase regulates CD3-induced signal transduction and T cell development in recombinase-deficient mice: restoration of pre-TCR function by active p56(lck).

The pre-TCR complex regulates the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes during T cell development. In CD45(-/-) mice there is an accumulation of DN cells, suggesting a possible role for CD45 in pre-TCR signaling. We therefore crossed CD45(-/-) with Rag-1(-/-) mice to investigate the signaling functions of the CD3 complex in DN thymocytes. Remarkably, treatment of Rag-1(-/-)/CD45(-/-) mice with a CD3 mAb caused maturation to the DP stage at only 3% of the level measured in Rag-1(-/-) mice. Furthermore, ligation of the CD3 complex on Rag-1(-/-) /CD45(-/-) thymocytes in vitro induced less tyrosine phosphorylation in specific proteins when compared to Rag-1(-/-) thymocytes. CD45(-/-) mice were also crossed with pLGFA mice expressing a constitutively active form of the lck tyrosine kinase which restored the DN to DP transition to near normal levels. Our results are consistent with a model in which CD45-activated p56(lck) is critical for pre-TCR signal transduction.     

Hedgehog signaling regulates differentiation from double-negative to double-positive thymocyte

The hedgehog (Hh) signaling pathway is involved in the development of many tissues. Here we show that sonic hedgehog (Shh) is involved in thymocyte development. Our data suggest that termination of Hh signaling is necessary for differentiation from CD4-CD8-double-negative (DN) to CD4+CD8+ double-positive (DP) thymocyte. Shh is produced by the thymic stroma, and Patched and Smoothened (Smo), the transmembrane receptors for Shh, are expressed in DN thymocytes. A neutralizing monoclonal antibody against Shh increases differentiation of DN to DP thymocytes, and Shh protein arrests thymocyte differentiation at the CD25+ DN stage, after T cell receptor beta (TCRbeta) gene rearrangement. We show that one consequence of pre-TCR signaling is downregulation of Smo, allowing DN thymocytes to proliferate and differentiate.     

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.     

Unexpectedly late expression of intracellular CD3epsilon and TCR gammadelta proteins during adult thymus development.

During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3epsilon, TCR beta and TCR gammadelta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3epsilon(+) and icTCR gammadelta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gammadelta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alphabeta lineage divergence will be discussed.     

Extensive proliferation of T cell lineage-restricted progenitors in the thymus: an essential process for clonal expression of diverse T cell receptor beta chains

For clonal diversification of TCR, a large number of T cell progenitors are required in which highly diverse TCRbeta chains are accommodated individually. In the present study, we examined the proliferative potential of thymic progenitors that have been defined to be T cell lineage restricted. We show that the earliest fetal thymus (FT) cells from Rag2(-/-) mice, when cultured individually in a thymic organ culture system, produced 150-1,800 CD25(+) cells. Since differentiation and proliferation of Rag2(-/-) thymocytes are arrested at the stage of TCRbeta chain gene rearrangement, the observed proliferation was considered to represent the proliferative potential of progenitors prior to the TCRbeta rearrangement. A comparable level of proliferation was revealed to occur by analyzing the Dbeta-Jbeta rearrangement profiles of T cells generated from single progenitors in the earliest population of FT from normal mice. The proliferative potential of progenitors declined along with the progression of developmental stages. Such an extensive proliferation of progenitors after the restriction to the T cell lineage may be an essential process ensuring the clonal diversification of TCRbeta chains.     

Double-negative thymocyte subsets in CD3′ chain-deficient mice: Absence of HSA+CD44−CD25− cells

Double-negative (DN) thymocyte subsets were examined in mice deficient in the CD3′ chain (ζ −/−). The HSA ⁺CD44⁻CD25⁻ subset was found to be missing, and DN thymocytes seemed to differentiate directly from HSA⁺CD25⁺CD44⁻cells to double-positive (DP) cells. When fetal thymic ontogeny was examined, we found a marked difference between ζ −/− embryos and heterozygous littermates from embryonic day 17.5, in terms of CD25, CD4 and CD8 expression, and thymus size. The ζ −/− thymocytes failed to down-regulate CD25 and to expand exponentially. The cell cycle status of adult thymocyte subsets indicated that although the HSA ⁺CD25⁻CD44⁻ subset was missing, the CD25⁺ DN population contained normal numbers of cycling cells, and the CD25⁺ DP cells (which were not detectable in normal mice) contained 5–10% cells in G2/M + S. Taken together these data suggest that the CD3′ chain might have a specific role in the control of proliferation of DN thymocytes during T cell development. Our data clearly show that one can dissociate the signal for a CD25⁺ DN cell to differentiate (which occurs in the absence of CD3′), from a signal to proliferate and from loss of cell surface CD25.     

Possible involvement of glial cell line-derived neurotrophic factor and its receptor,GFRalpha1, in survival and maturation of thymocytes

The glial cell line-derived neurotrophic factor (GDNF) and its receptors (GFR) play important roles in the promotion of survival and differentiation of central and peripheral neuronal populations. We show that GFRalpha1, a component of GDNF receptor, was expressed in thymocytes at an early stage of thymocyte-development and was involved in the survival of thymocyte precursors. GFRalpha1and GDNF were expressed in thymus, but not in spleen or lymph nodes in adult mice. During embryonic thymocyte development, GFRalpha1 was predominantly expressed on thymocytes from days 14.5 to 16.5 of gestation, and thereafter its expression gradually declined. In adult thymus, GFRalpha1 was expressed only on CD4(-)CD8(-) double-negative (DN) thymocytes, but not on CD4(+)CD8(+) double-positive or single-positive thymocytes. It was strongly expressed on RAG2(-/-) thymocytes arrested at the DN stage, and ist expression was reduced during their differentiation after in vivo anti-CD3 antibody stimulation. Additionally, fetal thymocyte precursors grew in serum-free medium of the fetal thymus organ culture system in the presence of recombinant GDNF (rGDNF), while the cells without rGDNF died. These results suggested that GDNF/GFRalpha1 are involved in the survival of both the nervous system and DN immature thymocytes.     

A critical lineage-nonspecific role for pTalpha in mediating allelic and isotypic exclusion in TCRbeta-transgenic mice

Although it is well established that early expression of TCRbeta transgenes in the thymus leads to efficient inhibition of both endogenous TCRbeta and TCRgamma rearrangement (also known as allelic and "isotypic" exclusion, respectively) the role of pTalpha in these processes remains controversial. Here, we have systematically re-evaluated this issue using three independent strains of TCRbeta-transgenic mice that differ widely in transgene expression levels, and a sensitive intracellular staining assay that detects endogenous TCRVbeta expression in individual immature thymocytes. In the absence of pTalpha, both allelic and isotypic exclusion were reversed in all three TCRbeta-transgenic strains, clearly demonstrating a general requirement for pre-TCR signaling in the inhibition of endogenous TCRbeta and TCRgamma rearrangement. Both allelic and isotypic exclusion were pTalpha dose dependent when transgenic TCRbeta levels were subphysiological. Moreover, pTalpha-dependent allelic and isotypic exclusion occurred in both alphabeta and gammadelta T cell lineages, indicating that pre-TCR signaling can potentially be functional in gammadelta precursors. Finally, levels of endogenous RAG1 and RAG2 were not down-regulated in TCRbeta-transgenic immature thymocytes undergoing allelic or isotypic exclusion. Collectively, our data reveal a critical but lineage-nonspecific role for pTalpha in mediating both allelic and isotypic exclusion in TCRbeta-transgenic mice.     

Induction of CD8 molecules on thymic gamma/delta T cells in vitro is dependent upon alpha/beta T cells.

Thymocytes differentiate upon interactions with microenvironmental components, but the precise role of different stromal cells, or other T cells, in early differentiative events remains unclear. Here we have analyzed the in vitro differentiation of double-negative (DN) thymocytes from young adult mice. We demonstrate that a substantial proportion of DN thymocytes differentiate into CD8+ gamma/delta T cells upon stimulation with concanavalin A and recombinant interleukin 2. However, if alpha/beta T cells are excluded from the initial population of DN thymocytes, the CD8+ gamma/delta T cells do not appear in the cultures. These results suggest a role for T-T cell interactions in thymic differentiative events, and provide evidence for physiological interactions between the alpha/beta and gamma/delta T cell compartments within the thymus.     

T cell receptor-negative thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation pathway

In order to investigate the role of T cell receptor (TcR) expression in thymocyte maturation, we have analyzed thymocytes from C.B-17/SCID mice, which are unable to productively rearrange their antigen receptor genes and fail to express TcR. Despite this defect, SCID thymocytes are functional as they produce lymphokines and proliferate in response to a variety of stimuli. Phenotypic analysis revealed that thymocyte populations from young adult SCID mice resemble thymocyte populations from normal embryonic mice in that they are large, Thy-1.2+, CD4-, CD8-, TcR- and enriched in CD5lo, IL2R+ and Pgp1+ cells. However, other TcR- populations normally present in adult mice (i.e., CD4-CD8+ cells and CD4+CD8+ cells) are absent from the thymus of TcR- adult SCID mice. To understand the basis of the developmental arrest of TcR- SCID thymocytes at the CD4-CD8- stage of differentiation, we analyzed thymi from the occasional "leaky" SCID mouse which possesses small numbers of TcR+ thymocytes. We found that the presence of TcR+ cells within a SCID thymus was invariably associated with the presence of CD4+ and/or CD8+ SCID thymocytes. Interestingly, however, the CD4+/CD8+ SCID thymocytes were not themselves necessarily TcR+. That is, emergence of SCID thymocytes expressing CD4/CD8 was tightly linked to the presence of TcR+ cells within that SCID thymus, but the SCID thymocytes that expressed CD4/CD8 were not necessarily the same cells that expressed TcR. Finally, we found that the introduction into TcR- SCID mice of normal bone marrow cells that give rise to TcR+ cells within the SCID thymus promoted the differentiation of SCID thymocytes into CD4-CD8+ and CD4+CD8+ TcR- cells. These data indicate that TcR+ cells within the thymic milieu provide critical signals which promote entry of CD4-CD8-TcR- precursor T cells into the CD4/CD8 differentiation pathway. When applied to differentiation of normal thymocytes, these findings may imply a critical role for early appearing CD4-CD8- TcR (gamma/delta)+ cells in initiating normal thymic ontogeny.     

Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation

Precursor CD4-CD8- (DN) thymocytes rearrange their TCR-beta genes, and only those which succeed in beta-selection subsequently expand and differentiate into immature CD4+CD8+ (DP) thymocytes. The cell subsets corresponding to the successive steps of this transition can be defined in terms of CD44 and CD25 expression. We partially synchronized the differentiation process by eliminating cycling cells with the anti-mitotic agent demecolcine. Using in vivo pulse labeling with bromodeoxyuridine, we determined the order of entry into DNA synthesis of the different DN and transitory (CD4-/lo CD8+) cell subsets. Two independent proliferation phases were identified. The first cells to enter the cell cycle were CD44-CD25lo, and CD4/CD8/TCR-/BrdU four-color staining showed that they all expressed a low density of the TCR-beta chain, an element of the pre-TCR (the TCR-alpha locus is still in germ-line configuration at this stage). Cycling of CD44+CD25+ cells was detected later, and no starting point was observed at the CD44-CD25hi stage. CD8 expression was immediately detectable in cycling cells, but they took 24 h to reach the DP stage. The study of TCR-Calpha-deficient mice showed that beta gene rearrangement occurred once proliferation had ceased at the DP stage, and that it had no influence on the DN-DP transition. These data show that precursor thymocytes undergo two independent waves of expansion, and that the second wave is restricted to cells capable of pre-TCR expression.     

Early TCRalpha expression generates TCRalphagamma complexes that signal the DN-to-DP transition and impair development

Clonotypic T cell receptor (TCR) genes undergo ordered rearrangement and expression in the thymus with the result that TCRalpha and TCRgamma proteins are not expressed in the same cell at the same time. Such "TCRalpha/gamma exclusion" is a feature of normal thymocyte differentiation, but it is abrogated in TCR-transgenic mice, which prematurely express transgenic TCRalpha proteins in early double-negative (DN) thymocytes. We report here that early expression of TCRalpha proteins results in the formation of TCRalphagamma complexes that efficiently signal the differentiation of DN into double-positive thymocytes independently of pre-TCR and TCRbeta expression. Thus, abrogation of TCRalpha/gamma exclusion by early TCRalpha expression results in the formation of isotypically mixed TCRalphagamma complexes whose in vivo signals circumvent TCRbeta selection and redirect thymocyte development along an aberrant developmental pathway.     

A CD3- subset of CD4-8+ thymocytes: a rapidly cycling intermediate in the generation of CD4+8+ cells

T lymphocytes with the surface phenotype CD4+8- and CD4-8+ are considered to be representative of functionally mature cells. We show here that adult murine thymus contains a subpopulation of CD4-8+ cells that differ from CD4-8+ cells found in the periphery in that they do not express the T cell receptor-associated CD3 molecular complex. Such CD3-4-8+ thymocytes are cortisone sensitive and rapidly cycling in situ. Furthermore, in contrast to mature T cells, most CD3-4-8+ thymocytes express low levels of CD5 and high levels of the B2A2 antigen. CD3-4-8+ thymocytes fail to respond to a variety of mitogenic stimuli in vitro but do give rise upon short-term culture to CD4+8+ cells. It is suggested that CD3-4-8+ thymocytes represent a transitional stage of thymus differentiation between the CD4-8- and CD4+8+ compartments.     

Interferons alpha/beta inhibit IL-7-induced proliferation of CD4- CD8- CD3- CD44+ CD25+ thymocytes, but do not inhibit that of CD4- CD8- CD3- CD44- CD25- thymocytes.

Type 1 interferons (IFN-alpha/beta) have recently been shown to inhibit interleukin-7 (IL-7)-induced growth and survival of early B-lineage cells. The CD3- CD4- CD8- (triple negative; TN) thymocytes from normal mice strongly proliferated upon stimulation with IL-7 in suspension, culture. Such an IL-7-induced proliferation was suppressed by the addition of IFN-alpha/beta, but a fraction of the TN thymocytes still showed proliferation. The IL-7-induced growth of TN thymocytes from acid mice, which lack the CD44- CD25- subpopulation, was completely inhibited by the addition of IFN-alpha/beta. The IL-7 induced proliferation of CD4- CD8- thymocytes from T-cell receptor (TCR) transgenic mice, the majority of which are CD3+ CD44- CD25-, was resistant to IFN-alpha/beta-mediated suppression. In fetal thymus organ cultures (FTOC), the addition of IL-7 greatly increased the population of CD4- CD8- CD44+ CD25+ thymocytes and IFN-alpha/beta inhibited this IL-7-driven expansion. In contrast, the addition of IL-7 markedly decreased the percentages of CD4- CD8- CD3- CD44- CD25- cells, and IFN-alpha/beta reversed the effect and increased the subpopulations of CD44- CD25+ and CD44- CD25-. Finally, IFN-beta mRNA was found to be expressed in the thymus. The data suggest that type I interferons inhibit IL-7-driven proliferation of TN thymocytes, but do not block the normal differentiation process.     

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.     

CD45 can act as a negative regulator for the transition from early to late CD4+ CD8+ thymocytes

The differentiation process from CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) stage is accompanied by vigorous proliferation. The resulting DP cells contain a sizable proportion of large cycling cells, but most DP cells are small resting cells. To explore the molecular mechanisms which regulate cell proliferation of DP thymocytes prior to further development, we used TCR-transgenic (Tg) mice with non-selecting MHC (Tg-Neut), which contain almost exclusively DP thymocytes that are not subject to either positive or negative selection. In Tg-Neut, the thymus contained DP cells of relatively large size, which showed higher extracellular signal-regulated kinase activity and enhanced responsiveness to mitogen compared to small DP cells. This indicates that all the large DP cells in the thymus are not positively selected and that they possess proliferative potential. When Tg-Neut mice were backcrossed with CD45 knockout mice (CD454-/- Tg-Neut), the thymus showed an increase of large DP cells and cycling cells, but a decrease of apoptotic cells. Furthermore, Bcl-2 expression and Jun N-terminal kinase activity, which are associated with resistance to apoptosis, were enhanced. These observations suggest that thymocyte proliferation in the DP stage is suppressed by a CD45-related process with regulation of mitogen-activated protein kinase and Bcl-2 unless DP cells receive TCR-mediated signals.