Perturbed homeostasis of peripheral T cells elicits decreased susceptibility to anti-CD3-induced apoptosis in prediabetic nonobese diabetic mice

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8(+) T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8(+) T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4(+)CD25(-)CD62L(+) but not CD4(+)CD25(+)CD62L(+) T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8(+) T cell AICD and increasing the number of CD4(+)CD25(+)CD62L(+) T cells in the periphery.     

Age-related synthesis of glucocorticoids in thymocytes

Glucocorticoids (GCs) are primarily synthesized in the adrenal glands but an ectopic production has also been reported in the brain, the gastrointestinal tract and in thymic epithelial cells (TEC). Here we show that thymocytes express genes encoding for all enzymes required for de novo GC synthesis and produce the hormone as demonstrated by both a GC specific reporter assay and a corticosterone specific ELISA assay. Interestingly, GC synthesis is detectable in cells from young mice (4 weeks) and thereafter increases during aging (14-22 weeks) together with an increased gene expression of the rate-limiting enzymes StAR and CYP11A1. Hormone production occurred at a thymocyte differentiation stage characterized by being double positive for the CD4 and CD8 surface markers but was found to be unrelated to CD69 expression, a marker for thymocytes undergoing positive selection. No GC synthesis was found in resting or anti-CD3 activated CD4 and CD8 positive T cells isolated from the spleen. Thymocyte-derived GC had an anti-proliferative effect on a GR-transfected cell line and induced apoptosis in thymocytes. The age- and differentiation stage-related GC synthesis in thymocytes may play a role in the involution process that the thymus gland undergoes.     

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.     

Inhibition of the anti-CD3-induced T cell proliferation by crosslinking of stimulatory antibodies in the presence of PMA and interleukin-2.

Anti-CD3 antibodies are directed to the nonpolymorphic part of the T cell receptor complex and may activate human peripheral T cells. Under some circumstances crosslinked anti-CD3 has been described to augment the proliferative response. Here we demonstrate that crosslinking of stimulatory anti-CD3 antibodies by anti-IgG in cell suspension abolishes their effect on proliferation of human resting peripheral T cells in the presence of PMA and/or IL-2. This effect was observed within a wide range of anti-CD3 concentrations (1 ng/ml to 1 microgram/ml) independent of the presence of monocytes. The inhibition was not due to the induction of cell death, since cells remained propidium iodide-negative after treatment. Protein-tyrosine phosphorylation after anti-CD3 crosslinking was more pronounced than in the presence of noncrosslinked anti-CD3. This indicates that the signal was transmitted after anti-CD3 crosslinking, however, it was unable to induce T cell proliferation. Reduced IL-2 receptor expression after anti-CD3 crosslinking and the inability of exogenous IL-2 to restore the proliferative response might indicate a reduced susceptibility to IL-2 as a reason for the described phenomenon.     

Possible involvement of cyclophilin B and caspase-activated deoxyribonuclease in the induction of chromosomal DNA degradation in TCR-stimulated thymocytes

TCR engagement of immature CD4(+)CD8(+) thymocytes induces clonal maturation (positive selection) as well as clonal deletion (negative selection) in the thymus. However, the cell death execution events of thymocytes during the negative selection process remain obscure. Using a cell-free system, we identified two different DNase activities in the cytosol of in vivo anti-TCR-stimulated murine thymocytes: one that induced chromosomal DNA fragmentation, which was inhibited by an inhibitor of caspase-activated DNase, and another that induced plasmid DNA degradation, which was not inhibited by an inhibitor of caspase-activated DNase. We purified the protein to homogeneity that induced plasmid DNA degradation from the cytosol of anti-CD3-stimulated thymocytes and found that it is identical with cyclophilin B (Cyp B), which was reported to locate in endoplasmic reticulum. Ab against Cyp B specifically inhibited the DNA degradation activity in the cytosol of anti-CD3-stimulated thymocytes. Furthermore, recombinant Cyp B induced DNA degradation of naked nuclei, but did not induce internucleosomal DNA fragmentation. Finally, we demonstrated that TCR engagement of a murine T cell line (EL4) with anti-CD3/CD28 resulted in the release of Cyp B from the microsome fraction to the cytosol/nuclear fraction. Our data strongly suggest that both active caspase-activated DNase and Cyp B may participate in the induction of chromosomal DNA degradation during cell death execution of TCR-stimulated thymocytes.     

Role of CTLA-4 in the activation of single- and double-positive thymocytes

CTLA-4, a homologue of CD28, is a negative regulator of T cell activation in the periphery and is transiently expressed on the cell surface after T cell activation. However, the role of CTLA-4 in T cell activation in the thymus is not clear. This investigation was initiated to determine the role of CTLA-4 in the activation of CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes using fetal thymic organ cultures (FTOC) of MHC class II-restricted, OVA(323-339)-restricted TCR transgenic mice (DO11.10). We found that treatment of the FTOC with anti-CTLA-4-blocking Ab during activation with OVA(323-339) increased the proportion and number of DP thymocytes, but decreased the proportion and number of SP thymocytes compared with OVA(323-339)-stimulated FTOC without anti-CTLA-4 Ab treatment. In addition, anti-CTLA-4 Ab treatment inhibited OVA(323-339)-induced expression of the early activation marker, CD69, in DP thymocytes, but increased CD69 in SP thymocytes. Similarly, CTLA-4 blockage decreased phosphorylation of ERK in DP thymocytes by Ag-specific TCR engagement, but increased phosphorylation of ERK in SP thymocytes. CTLA-4 blockage inhibited deletion of DP thymocytes treated with a high dose of OVA(323-339), whereas CTLA-4 blockage did not inhibit deletion of DP thymocytes treated with a low dose of OVA(323-339). We conclude that CTLA-4 positively regulates the activation of DP thymocytes, resulting in their deletion, whereas blocking CTLA-4 suppresses the activation of DP thymocytes, leading to inhibition of DP thymocyte deletion. In contrast, CTLA-4 negatively regulates the activation of SP thymocytes.     

Absence of programmed death receptor 1 alters thymic development and enhances generation of CD4/CD8 double-negative TCR-transgenic T cells

Programmed death receptor 1 (PD-1) is expressed on thymocytes in addition to activated lymphocyte cells. Its ligation is thought to negatively regulate T cell activation, and PD-1(-/-) mice develop autoimmunity. To study the role of PD-1 on the development and function of a monoclonal CD8(+) T cell population, 2C TCR-transgenic/recombination-activating gene 2(-/-)/PD-1(-/-) mice were generated. Unexpectedly, approximately 30% of peripheral T cells in these mice were CD4/CD8 double negative (DN). Although the DN cells were not activated by Ag-expressing APCs, they functioned normally in response to anti-CD3/anti-CD28. These cells had a naive surface phenotype and lacked expression of NK1.1, B220, and gammadelta TCR; and the majority did not up-regulate CD8alphaalpha expression upon activation, arguing that they are not predominantly diverted gammadelta-lineage cells. The thymus was studied in detail to infer the mechanism of generation of DN peripheral T cells. Total thymus cellularity was reduced in 2C TCR-transgenic/recombination-activating gene 2(-/-)/PD-1(-/-) mice, and a relative increase in DN cells and decrease in double-positive (DP) cells were observed. Increased annexin V(+) cells among the DP population argued for augmented negative selection in PD-1(-/-) mice. In addition, an increased fraction of the DN thymocytes was HSA negative, suggesting that they had undergone positive selection. This possibility was supported by decreased emergence of DN PD-1(-/-) 2C cells in H-2(k) bone marrow chimera recipients. Our results are consistent with a model in which absence of PD-1 leads to greater negative selection of strongly interacting DP cells as well as increased emergence of DN alphabeta peripheral T cells.     

In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes.

Some thymocytes, upon activation via the TCR complex in vitro, undergo apoptotic cell death. In this report, we examine the cell death induced in the thymus after administration of anti-CD3 or anti-TCR antibodies. We found that shortly after antibody injection, cortical thymocytes undergo apoptosis as characterized by morphologic changes and DNA fragmentation. Anti-CD3 administration led to depletion of nearly all CD4+CD8+ thymocytes, and approximately 50% of CD4+CD8- thymocytes. This depletion predominantly affected cells bearing low levels of CD3, although some depletion also occurred among cells expressing intermediate and high levels. Administration of an anti-TCR antibody also induced apoptosis, but affected significantly fewer thymocytes than anti-CD3. This effect was probably not due to different binding affinities for the two antibodies, because both antibodies show similar dose response effects in an in vitro model of activation-induced apoptosis. This work demonstrates that findings on activation-induced apoptosis in vitro can be extended to the in vivo situation, and further, that the activation of cortical thymocytes, in situ, results in apoptosis and removal of the activated cells. The possible relationships between this activation-induced cell death in immature thymocytes and the process of negative selection of autoreactive T cells is discussed.     

Ephrin-B1 is critical in T-cell development

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. In this study, we investigated the role of EFNB1 and the Ephs it interacts with (collectively called EFNB1 receptors) in mouse T-cell development. In the thymus, CD8 single positive (SP) and CD4CD8 double positive (DP) cells expressed high levels of EFNB1 and EFNB1 receptors, whereas CD4 SP cells had moderate expression of both. Soluble EFNB1-Fc in fetal thymus organ culture caused significant subpopulation ratio skew, with increased CD4 SP and CD8 SP and decreased DP percentage, while the cellularity of the thymus remained constant. Moreover, in EFNB1-treated fetal thymus organ culture, CD117(+), CD25(+), DP, CD4 SP, and CD8 SP cells all had significantly enhanced proliferation history, according to bromodeoxyuridine uptake. In vitro culture of isolated thymocytes revealed that EFNB1-Fc on solid-phase protected thymocytes from anti-CD3-induced apoptosis, with concomitant augmentation of several antiapoptotic factors, particularly in CD4 SP and CD8 SP cells; on the other hand, soluble EFNB1-Fc promoted anti-CD3-induced apoptosis, as was the case in vivo. This study reveals that EFNB1 and EFNB1 receptors are critical in thymocyte development.     

组蛋白去乙酰化酶3通过抑制AICD维持T细胞自稳

为探讨组蛋白去乙酰化酶3(HDAC3)在T细胞自稳中的作用,采用LoxP-cd4cre酶系统在胸腺CD4⁺CD8⁺双阳性T细胞(DP)中敲除hdac3基因．hdac3基因敲除小鼠不影响T细胞在胸腺中的发育,但导致外周T细胞显著降低,而且,hdac3基因敲除的外周T细胞主要以活化/效应/记忆表型为主．机制分析表明,hdac3基因敲除的外周T细胞凋亡增加并伴随细胞增殖加速,同时,Fas 和Fas配体阳性细胞比率以及Fas配体的表达显著增加．体外TCR活化不影响正常外周T细胞的凋亡,但导致hdac3基因敲除的外周T细胞凋亡显著增加．实验结果表明,HDAC3通过抑制活化诱导的细胞凋亡维持外周T细胞自稳．     

Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex

Development of tolerance to self Ag occurs during a negative cell selection process in the thymus. This selection process is thought to involve interactions between Ag-specific thymocyte receptors and self Ag presented by the MHC proteins on accessory cells, resulting in deletion of potentially harmful self-reactive precursors. However, the mechanisms underlying this clonal deletion have not been identified. In confirmation of previous findings (C. A. Smith, G. T. Williams, R. Kingston, E. J. Jenkins, and J. J. T. Owen, 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181), we have found that an anti-CD3 antibody stimulated DNA fragmentation, characteristic of a suicide mechanism known as apoptosis or programmed cell death (PCD), in suspensions of human thymocytes. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin. Although the magnitude and duration of the Ca2+ increase were similar to those observed in response to Con A, the mitogen did not stimulate DNA fragmentation or cell death. Phorbol ester prevented Ca2+-dependent DNA fragmentation and cell killing in response to anti-CD3 or other agents that stimulated PCD, suggesting that activation of protein kinase C abrogated cell suicide. Disappearance of CD4+CD8+ immature thymocytes was generally observed in response to all agents that stimulated PCD, whereas mature PBL were insensitive to stimulation of PCD. Our results suggest that antibody-mediated stimulation of immature thymocytes via the TCR complex results in Ca2+-dependent, endonuclease-mediated cell killing, depending on the activation status of protein kinase C.     

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An assay which measures the secretion of two lymphokines, granulocyte-macrophage colony-stimulating factor and interleukin 3 (IL-3), by single T cells activated with an anti-TCR antibody, F23.1, was used to analyze the effects of anti-CD4 antibodies on antigen-independent T cell activation. Single cells of a CD4+F23.1+ clone were micromanipulated into wells to which F23.1 had been immobilized, and their lymphokine secretion was measured 24 hr later. The frequency of lymphokine-secreting cells was consistently reduced up to 10-fold in the presence of soluble anti-CD4 antibody (GK1.5) but only up to 2.5-fold by an antibody to the cell adhesion molecule, LFA-1. In both bulk and single-cell cultures, responses to suboptimal concentrations of F23.1 were more susceptible to inhibition by GK1.5 than responses to optimal F23.1. The failure of GK1.5 to inhibit IL-2-stimulated lymphokine synthesis in bulk cultures suggested that CD4 ligation did not deliver a negative signal to the clone. By contrast, when either anti-CD4 or anti-LFA-1 was immobilized on the same surface as F23.1, the frequency of lymphokine-secreting cells could be increased up to 10-fold. It is concluded that anti-CD4 antibodies can act directly on the responding T cell to affect TCR-dependent activation, in the absence of interaction with antigen-presenting cells or any other cell type.     

Sustained linked stimulation via CD3 and CD4 is required for the IL-4-independent development of IL-4 synthesizing CD4+ T cells

Previous work has shown that CD4 engagement can promote the development of interleukin-4-producing cells from naive CD4+ T cells activated with anti-CD3 antibody and interleukin-2 in the absence of other exogenous signals, including interleukin-4 itself. When CD44low CD4+ T cells were activated with immobilized anti-CD3 antibody and interleukin-2, they proliferated and produced interferon-gamma but not interleukin-4. Co-immobilization of antibodies to CD3 and CD4 enhanced cell recoveries and induced interleukin-4 as well as interferon-gamma synthesis. Here we show that these effects of CD4 ligation were not observed when anti-CD4 antibody was replaced with another CD4 ligand, interleukin-16, or when the anti-CD3 and anti-CD4 antibodies were spatially separated by immobilization on different beads. Removal of the anti-CD4 antibodies within the first three days of stimulation also prevented the development of detectable interleukin-4-producing cells. The data suggest that interleukin-4-independent priming of interleukin-4-producing cells in this system requires sustained stimulation via both the T cell receptor and CD4 with close physical association between the ligands for these two receptors.     

Development of murine lupus in CD4-depleted NZB/NZW mice. Sustained inhibition of residual CD4+ T cells is required to suppress autoimmunity.

Chronic administration of anti-CD4 mAb prevents autoimmune disease in NZB/NZW F1 (B/W) mice. This may be due either to CD4 cell depletion or to inhibition of CD4 cell function. To evaluate the relative importance of these mechanisms, we devised a system in which the consequences of cell depletion could be analyzed independent of the inhibitory effects of chronic mAb therapy. This was accomplished by performing adult thymectomy before mAb administration. Specifically, female B/W mice underwent thymectomy or sham thymectomy at age 6 wk, followed at age 3 mo by a short course of either anti-CD4 (2 mg/wk for 3 wk) or saline. Treatment with anti-CD4 depleted 90% of circulating CD4 cells, but a small subpopulation (10%) of CD4 cells was refractory to depletion. In non-thymectomized mice, the CD4 population gradually reconstituted after cessation of therapy. In contrast, in thymectomized mice, recovery of CD4 cells was prevented by the absence of the thymus. Despite the striking reduction in CD4 cells in thymectomized mice, severe autoimmune disease developed, with autoantibody levels, proteinuria, and mortality comparable with non-thymectomized, nondepleted controls. The unexpected development of lupus nephritis in thymectomized, CD4-depleted B/W mice suggested that the thymus might be required to achieve the benefits of therapy with anti-CD4. To exclude this possibility, we demonstrated that chronic therapy with anti-CD4 prevents autoimmunity in thymectomized B/W mice. These findings imply that: 1) substantial depletion of CD4 T cells is not sufficient to suppress autoimmunity; 2) suppression of autoimmunity requires sustained functional inhibition of CD4 T cells; and 3) a small subpopulation of CD4 cells that is refractory to depletion by anti-CD4 is sufficient to promote the full expression of murine lupus in B/W mice.     

Development of a transient CD4(+)CD8(+) T cell subset in the cervical lymph nodes following intratracheal instillation with an adenovirus vector

Past studies have described the serendipitous appearance of peripheral CD4(+)CD8(+) double-positive (DP) T cells in both humans and nonhuman primates usually following a viral infection or resulting from a malignancy. However, understanding the role of DP T cells has been hampered by the lack of their reproducible generation. Herein, we describe DP T cells produced after a single intratracheal or intranasal dose of recombinant adenovirus 2 or 5 vector into mice. In a time-dependent fashion, DP T cells localized only in the deep cervical lymph nodes but not in the lungs or in any of the respiratory lymph nodes. These DP T cells were TCR(alpha)beta(+) and CD8(alpha)beta(+), but not TCR(gamma)delta(+) nor CD8(alpha)alpha(+), suggesting that these cells are unrelated to intestinally derived DP T cells. Upon co-stimulation with anti-CD3 and anti-CD28, DP T cells showed increased expression of VLA-1, VLA-2, and CD69, and were more effective than CD4(+) T cells in T helper cell activity, as evidenced by increased IgA, IgG, and IgM production. Such co-stimulation also favored the production of IFN-gamma and IL-10 where CD4(+) T cells were more inclined to produce IFN-gamma and IL-2.     

T cell activation via the T cell receptor: a comparison between WT31 (defining alpha/beta TcR)-induced and anti-CD3-induced activation of human T lymphocytes.

The T cell receptor (TcR) heterodimer of alpha/beta glycoprotein is noncovalently associated with CD3 glycoprotein forming TcR/CD3 complex. The TcR have been shown to recognize antigen, and CD3 antigen is responsible for signal transduction. In this study we compared the effects of WT31 (defining alpha/beta TcR) monoclonal antibody (MoAb) and anti-CD3 MoAb on various steps of human T cell activation. Both antibodies depolarized plasma membranes, increased cell volume, induced IL-2 production and the expression of IL-2 receptors (CD25 antigen) and induced DNA synthesis. Furthermore, the two antibodies showed no synergistic effect on any of these parameters. However, both MoAb showed synergism with phorbol ester (PMA). WT31-induced T cell activation was Ca(2+)-dependent because the addition of EGTA to the medium inhibited DNA synthesis and CD25 antigen expression. The blockers of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and staurosporin, in a dose-dependent manner inhibited WT31-induced DNA synthesis. Cholera toxin but not the pertussis toxin inhibited WT31-induced T cell activation, suggesting involvement of G protein in WT31-induced T cell activation. These data indicate that WT31 antibody activates human T cells by a pathway that is similar to that of anti-CD3-induced T cell activation.     

Antibody binding to CD5 (Tp67) and Tp44 T cell surface molecules: effects on cyclic nucleotides, cytoplasmic free calcium, and cAMP-mediated suppression

T cells can be activated to proliferate by antibodies to the T cell antigen receptor or the molecularly associated CD3 complex if monocytes are present. We have shown previously that monoclonal antibodies to the human T cell differentiation antigens CD5 (Tp67) and Tp44 each augment and prolong proliferative responses of anti-CD3-activated T cells, even in the absence of monocytes. Here we show that the functional and biochemical mechanisms of CD5 and Tp44 signal transmission are distinct. T cell proliferation is suppressed by agents that increase the concentration of intracellular cAMP. We found that antibody binding to the Tp44 surface molecule overcomes this suppression, whereas antibody binding to CD5 does not, indicating that ligand-Tp44 interaction changes T cell sensitivity to cAMP-mediated growth inhibition. The ability of anti-CD3, anti-Tp44, and anti-CD5 monoclonal antibodies to directly alter cyclic nucleotide levels in the Jurkat T cell line was examined. Anti-CD3 alone caused a rapid four- to sixfold increase in cAMP levels, but did not affect cGMP levels. However, anti-Tp44 and anti-CD5 each caused a rapid three- to fourfold increase in cGMP levels without affecting cAMP levels. In other experiments, cytoplasmic free calcium levels were measured in resting T cells after CD5 or Tp44 stimulation by using the dye indo-1 and flow cytometry. This sensitive method showed that anti-CD5 alone caused an increase in cytoplasmic calcium free levels within 3 min of antibody addition, whereas anti-Tp44 had no effect. Finally, anti-Tp44 and IL 1 each augmented proliferation of phorbol ester-stimulated lymphocytes, whereas anti-CD5 did not. The effects of IL 1 and Tp44 could be further distinguished in that the effect of anti-Tp44 was resistant to inhibition by dBcAMP whereas IL 1 was not. These data suggest that the receptor function of both Tp44 and CD5 involves changes in cyclic nucleotides levels, and that the mechanism by which anti-Tp44 and anti-CD5 antibodies affect T cell proliferative responses may be related to their selective effects on cGMP levels and cytoplasmic calcium concentrations.     

Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.