Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.     

Activation of the Ras-related GTPase Rap1 by thymocyte TCR engagement and during selection

Signals mediated by activation of the small GTPase Ras play an essential role both in thymocyte development and in TCR-mediated activation of mature T cells. Given the critical requirement of Ras signaling pathways in thymocyte development, and recent indications that Rap1 may negatively regulate Ras-dependent signaling pathways, we examined the possible involvement of Rap1 in thymocyte TCR signaling. We find that Rap1 and proposed regulators of Rap1 (the proto-oncogene product Cbl, Crk family adaptor proteins, and the Rap1 guanine nucleotide exchange factor C3G) are expressed at equivalent levels in both double-negative and double-positive murine thymocytes. Rap1 was transiently activated following TCR stimulation of both total thymocytes and purified double-positive thymocytes, and this activation correlated with tyrosine phosphorylation of Cbl and Cbl association with CrkL. TCR-dependent Rap1 activation was enhanced by co-stimulation through CD28 and could be mimicked by treatment of thymocytes with phorbol ester and calcium. In contrast to mature peripheral T lymphocytes, Rap1 stimulation by CD3 ligation in thymocytes did not require intracellular calcium mobilization. Intriguingly, we found a clear elevation of activated Rap1 in thymocytes undergoing positive selection, suggesting a functional role for Rap1 in thymocyte development and selection.     

Characterization of the subset of immature thymocytes which can undergo rapid in vitro differentiation

Recently, we reported that thymocytes expressing the CD8 molecule on their surface can give rise to CD4+CD8+ double-positive and CD4+ single-positive progeny following intrathymic transfer into an irradiated host mouse. Thymcoytes expressing a high density of CD8, referred to as CD8hi, and those expressing a low density of the molecule, CD8lo, were both able to differentiate in vivo. In this study we examined the ability of these CD8+ thymocytes populations and of CD4-CD8- double-negative thymocytes to change their phenotype during brief in vitro culture. CD8+ thymocytes were prepared by anti-CD4 plus complement lysis followed by positive selection of the survivors on anti-CD8-coated plates. After 16 h of culture, greater than 60% of CD8+ thymocytes became double-positive. Both CD8hi and CD8lo cells were able to show this in vitro change: about 30% of the former and about 80% of the latter became double-positive. In contrast to this, double-negative thymocytes which had been depleted of cells expressing low densities of CD8 did not show such a phenotypic conversion in vitro. Further panning experiments suggested that all of the CD8+ thymocytes actually express a low surface density of the CD4 molecule which is undetectable in our cytofluorometric assays.     

Raf regulates positive selection

T cell development is regulated by extracellular signals that mediate cellular proliferation and differentiation via specific signal transduction pathways. To determine the importance of the mitogen-activated protein kinase (MAP kinase) pathway in thymocyte development, we analyzed transgenic mice expressing dominant negative Raf (DN Raf) and a constitutively active v-Raf under the control of the p56^lck proximal promoter. DN Raf had a profound effect on T cell receptor (TCR)-mediated signaling events as assessed by the inhibition of mitogen-induced proliferation of thymocytes in vitro. Overall thymocyte numbers were decreased by at most twofold from nontransgenic littermates. Positive selection was inhibited in DN Raf transgenic mice, as evidenced by both reduced numbers of mature thymocytes and a decrease in CD8⁺ thymocytes in female mice doubly transgenic for DN-Raf and a class I-restricted H-Y TCR. In contrast, the differentiation of double-positive thymocytes to single-positive thymocytes was enhanced in H-YTCR transgenic mice expressing constitutively active Raf (v-Raf). Thus, Raf regulates positive selection in the thymus.     

CD6: expression during development,apoptosis and selection of human and mouse thymocytes

CD6, a 130-kDa surface glycoprotein, is expressed primarily on cells of T lineage. A co-stimulatory role for CD6 in mature T cells has been shown, but the function of CD6 during thymocyte development is unknown. Since CD6 ligands are expressed on thymic epithelium, their interactions with CD6 could be important in thymic selection. In this report we show that CD6 is developmentally regulated in human and mouse thymocytes, and further demonstrate that increase in the level of CD6 expression correlates with expression of the selection marker CD69. We also show that activation via CD2 induces CD6 expression on mature human thymocytes and on a subset of immature human thymocytes that are resistant to apoptosis. In human and mouse thymocytes that express heterogeneous TCR, CD6 increases occur as double-positive thymocytes are selected to a single-positive stage. In contrast, in thymocytes from TCR transgenic mice, CD6 is barely increased following selection, suggesting that as functional avidity increases, requirements for CD6-dependent co-stimulation decrease. Taken together, these results indicate that during thymic development CD6-dependent signals may contribute both to thymocyte survival, and to the overall functional avidity of selection in both man and mouse.     

Gene expression analysis of thymocyte selection in vivo

Self versus non-self discrimination is a key feature of immunorecognition. Through TCR-activated apoptotic mechanisms, autoreactive thymocytes are purged at the CD4(+)CD8(+) double-positive (DP) precursor stage prior to maturation to CD4(+) or CD8(+) single-positive (SP) thymocytes. To investigate this selection process in vivo, gene expression analysis by oligonucleotide array was performed in TCR transgenic mice. In total, 244 differentially expressed DP thymocyte genes induced or repressed by TCR triggering in vivo were identified. Genes involved in the biological processes of apoptosis, DNA recombination, antigen processing and adhesion are coordinately engaged. Moreover, analysis of gene expression in thymocyte subsets revealed that TCR ligand-induced expression profiles vary according to their developmental stage, with 48 genes showing DP preference and nine showing SP thymocyte preference. Finally, our data suggest that both the extrinsic and the intrinsic apoptosis pathways are operating in thymic selection.     

Double-positive thymocytes resistant to antigen-MHC-induced negative selection lack active caspase

Thymocytes bearing autoreactive TCR are eliminated from the organism by a process termed negative selection. The molecular basis of this deletion has been recently shown to be a consequence of TCR-triggered activation of a caspase by certain peptide-MHC ligands in the immature CD4+CD8+ double-positive (DP) thymocyte subpopulation. Of note, the numerically minor TCRhigh DP thymocyte subpopulation, unlike the major TCRlow DP subset, is resistant to negative selection. Despite exposure to cognate peptide, TCRhigh DP thymocytes mature into single-positive thymocytes and are exported into the periphery. Here we investigated the mechanism by which these thymocytes escape negative selection. Using a cytochemical assay in conjunction with a caspase-specific affinity ligand, we demonstrate that the resistance of the TCRhigh DP thymocytes to negative selection correlates with the disappearance of TCR-triggered caspase activity in these cells. Thus thymocytes which have presumably begun the positive selection process inactivate the thymic caspase pathway and are no longer susceptible to negative selection.      

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.     

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.     

CD45 enhances positive selection and is expressed at a high level in large, cycling, positively selected CD4+CD8+ thymocytes.

T-cell development is arrested at the CD4+CD8+ (DP; double-positive) stage of thymocyte development in CD45 null mice. However, the mechanism by which CD45 participates in the positive selection of T cells remains to be investigated. In this report we describe a DP thymocyte population that associates positive selection with expression of high levels of CD45, CD4 and CD8. DP thymocytes of this phenotype are large, cycling cells and represent approximately 20% of DP thymocytes in normal mice. In mice expressing a transgenic T-cell receptor (TCR) specific for the male antigen presented by H-2Db (H-Y TCR), the up-regulation of TCR, CD5 and CD69 in this large DP population occurred in a major histocompatibility complex (MHC)-restricted manner. To investigate further the role of CD45 in positive selection, we determined whether thymocytes that expressed a transgenic CD45RO molecule under the control of the proximal lck promoter can influence the positive selection of T cells in H-Y TCR transgenic mice. It was found that in female H-Y TCR transgenic mice, MHC-restricted positive selection of CD4- CD8+ H-Y TCR+ thymocytes was enhanced by increased CD45RO expression. Thus, CD45 increases the efficacy of positive selection of CD4- CD8+ thymocytes that express H-Y TCR.     

Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes

Apoptosis appears to play a major role in the differentiationand selection of T and B lymphocytes, but the mechanisms ofclonal deletion of T cells in thymus are not well understood.We have prepared an anti-human Fas IgM mAb with associated apoptosis-inducingactivity in Fas antigen-positive target cells including humanT cells. We analyzed the expression of apoptosis antigen Fason human thymocytes by cytofluorometry showing low, but significantamounts of Fas antigen on double-negative and double-positiveundifferentiated thymocytes. On the contrary, most of the differentiatedthymocytes (single-positive or CD3-brightest) expressed undetectablelevels of Fas antigen. About 1-2% of thymocytes expressed highamounts of Fas antigen, and these cells, which were CD3-bright,were CD4-bright and CD8-low at the stage of late double-positivelineage. Immunohlstologlcal analysis shows these Fas-brightcells on the edge of the medulla. Stimulation through the TCRcomplex was shown to induce the expression of Fas antigen onthymocytes at the late double-positive stage and prolonged stimulationthrough the TCR complex rendered the Fas-bright thymocytes sensitiveto apoptosis-induclng activity of anti-Fas. To show the involvementof the Fas system in the negative selectlon/clonal deletionof thymocytes, we organ-cultured human thymus in the presenceof the superantigen, staphylococcus enterotoxin B (SEB), andnew antagonistic anti-Fas mAb, which can inhibit the apoptosis-induclngactivity of the original anti-Fas mAb. The SEB-reactJve TCRcomplex on thymocytes was at first down-regulated by SEB, thenthe SEB-reactlve clone was deleted by apoptosis, which was inhibitedby an antagonistic anti-Fas mAb. Thus, Fas antigen is shownto be involved in the negative selection/clonal deletion ofsuperantlgen-reactive thymocytes.     

Delineation of human thymocytes with or without functional potential by CD1-specific antibodies

Hematopoietic precursors lacking T cell antigen receptors (TCR-CD3-) and CD4 and CD8 surface markers (i.e. double-negative thymocytes) give rise to functionally mature T lymphocytes. Yet their major progeny are immunologically unresponsive thymocytes in spite of having acquired TCR-CD3 and CD4-CD8. Because only mature thymocytes migrate to peripheral lymphoid organs and most thymocytes die in situ, the knowledge of the events associated with functional maturation in the double-negative thymocyte progeny is a fundamental question in T cell development. We reasoned that a clue to trace the fate of early human thymocytes may perhaps come from the study of the developmental acquisition of CD1 antigen, currently used to define better the functionally inert CD4+8+ (double-positive) stage and absent in mature, medullary thymocytes and peripheral T cells. By using antibodies specific for CD1 (HTA 1/T6) we show here that a large fraction of double-negative thymocytes also express CD1. CD1+3-, CD1+3+, CD1-3+, and CD1-3- subsets all exist. The CD1+3- subset generates CD1+3-4-8+ precursors of CD1+ double-positive cells. A large portion of the CD1+3+ subset bears TCR gamma delta-CD3 complexes. The CD1- subsets are responsive in assays of function, in which they can be stimulated to use the interleukin 2 pathway of proliferation and to mediate cytotoxicity. In contrast, all CD1+ thymocytes behave as functionally inert cells. Thus, the CD1 surface marker delineates human thymocyte precursors and their products which lack, or possess, functional potential in vitro, on both alpha beta and gamma delta lineages.     

Accelerated transition from the double-positive to single-positive thymocytes in G alpha i2-deficient mice

Deletion of alpha i2 subunit of heterotrimeric G proteins induces a 2-4-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes as compared with wild-type littermates, but how G alpha i2 is involved in thymocyte development is unknown. To determine a role for G alpha i2 in a specific developmental stage of thymocyte differentiation, we studied the ontogeny of thymocytes in G alpha i2-deficient mice. Our data show that an accelerated transition from the double-positive (DP) to SP thymocytes, rather than impairment in thymic emigration, accounts for a high proportion of the SP thymocytes in the absence of G alpha i2. Lack of G alpha i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The augmented response may be the reason behind the expedited transition from the DP to SP thymocytes in the animal. In accordance with this, effects of G alpha i2 deficiency on CD8 or CD4 SP thymocyte differentiation required engagement of the TCRs with either MHC class I or MHC class II molecule. The abnormal thymocyte development resulted in an increase in positive selection, altered usage of TCR Vbeta gene, aberrant development of CD4+ CD25+ T regulatory cells and untimely thymic involution, the contribution of which to colitis development in the animal is discussed. These findings reveal a previously unappreciated role for G alpha i2 protein in clonal selection and functionality of thymocytes.     

Selective elimination of double-positive immature thymocytes by a thymic epithelial cell line

A cloned epithelial cell line, TEL-2, has been established from the stroma tissues of normal mouse thymus. Incubation of mouse thymocytes on TEL-2 cells resulted in the selective elimination of double-positive (CD4+CD8+) cells from the culture, whereas single-positive (CD4+CD8- or CD4-CD8+) thymocytes remaining in the culture were concentrated in non-integrated cell population. The CD3- or CD3 low-positive thymocytes were also eliminated by the TEL-2 cells from the culture, followed by the concentration of CD3 high-positive cells in the culture. Only intact viable thymocytes were integrated into TEL-2 cells. Electron microscopic examination showed that the integrated cells into TEL-2 cytoplasm were gradually degenerated. Mature single-positive T cells, mature B cells or double-negative thymocytes were not integrated into TEL-2 cells. The TEL-2 cell may provide information on the mechanism of selective disappearance of double-positive immature cells from the thymus.     

Enhancement of CD3-mediated thymocyte apoptosis by the cross-linkage of heat-stable antigen.

Heat-stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4-CD8- double-negative and CD4+CD8+ double-positive thymocytes but not CD4+ or CD8+ single-positive thymocytes. Effects of anti-HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single-cell suspension culture system. Immobilized R13 enhanced the CD3-mediated DNA fragmentation and killing of thymocytes but not the dexamethasone-induced or phorbol myristate acetate-induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3-mediated apoptosis when HSA and T-cell receptor (TCR)/CD3 were co-cross-linked by a cross-reactive secondary antibody. Even without the cross-reactive secondary antibody, soluble R13 enhanced CD3-mediated apoptosis, although a greater than 100-fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3-mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation-induced apoptosis of thymocytes and the involvement of HSA in negative selection.     

Protein phosphatase subunit G5PR that regulates the JNK-mediated apoptosis signal is essential for the survival of CD4 and CD8 double-positive thymocytes

Early T lineage cells are selected in the thymus by the specific recognition of peptide components presented by MHC molecules on the surface of thymic epithelial cells and dendritic cells. As a potential regulator of the apoptotic and survival signals, the protein phosphatase 2A-component G5PR regulates Bim phosphorylation in B-cells. Here, we studied whether G5PR is involved in the regulation of the similar apoptotic pathway for cell survival during the selection of thymocytes. T-cell-specific G5PR knockout (G5pr(-/-)) mice displayed thymic atrophy, significant reduction in thymocyte numbers, particularly a 10-fold decrease in the number of CD4 and CD8 double-positive (DP) thymocytes and few mature single-positive (SP) cells. G5pr(-/-) thymocytes exhibited normal potential of proliferation and differentiation during the transition from double-negative (DN) to DP stage, but significantly increased susceptibility to apoptosis at the DP stage. G5PR deficiency did not affect on Bim activation in thymocytes, but caused hyper-activation of JNK and Caspase-3 with augmented Fas ligand (FasL) expression, indicating that G5PR regulates the thymocyte unique apoptotic signal involved in JNK-mediated Caspase-3 activation but not in Bim activation. G5PR is essential for the survival of DP cells during thymocyte development.     

Degree of ERK activation influences both positive and negative thymocyte selection

Considerable evidence suggests that the ERK pathway is required for positive but not negative thymocyte selection. Here, we report that ERK is highly activated in double-positive (DP) thymocytes expressing an MHC class I-restricted TCR (P14) in response to negatively selecting conditions, whereas ligands that trigger positive selection induced weaker ERK activation. Biochemical evidence also shows that death by neglect is associated with a further reduction in ERK activation. These findings are consistent with the affinity / avidity model of thymocyte selection. To further examine the role of ERK in negative selection we used the MEK-1 inhitibor, PD98059, a specific pharmacological inhibitor of the ERK pathway. Biochemical data demonstrated a reduction of ERK activity by PD98059 in the presence of the negatively selecting ligand. Analysis of P14 TCR-transgenic fetal thymic lobes cultured with PD98059 under negatively selecting conditions showed impaired clonal deletion of DP thymocytes and a concomitant increase in positive selection of functional mature, TCR(hi) transgenic T cells. This demonstrates that altering ERK activity switched negative to positive selection. Contrary to previous reports that show an exclusive role for ERK signaling in positive selection, our data demonstrate that negative selection is also sensitive to the degree of ERK activation.     

Double-positive T cell receptorhigh thymocytes are resistant to peptide/major histocompatibility complex ligand-induced negative selection

To investigate negative selection events during intrathymic ontogeny, we established T cell receptor (TCR)-transgenic mice [N15tg/RAG-2^?/? (H-2^b)] expressing a single TCR specific for vesicular stomatitis virus nuclear octapeptide N_52–59 (VSV8) in the context of the major histocompatibility complex (MHC) class I molecule, K^b. Administration of VSV8 in vivo induced apoptosis in less than 4 h, deleting the majority of immature double-positive (DP) thymocytes by 24 h. In contrast, DP TCR^high as well as single-positive (SP) thymocytes were refractory to this death process. Moreover, DP TCR^high cells differentiated into SP thymocytes in vitro and in vivo, maturing into functional cytotoxic T lymphocytes upon intrathymic transfer to β RAG 2^?/? recipients. Hence, negative selection processes involving MHC-bound peptide ligands are operative only prior to the late DP thymocyte stage in this MHC class I-restricted TCR transgene system.