RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

Protein kinase C-theta-mediated signals enhance CD4+ T cell survival by up-regulating Bcl-xL

Productive engagement of TCR results in delivering signals required for T cell proliferation as well as T cell survival. Blocking TCR-mediated survival signals, T cells undergo apoptosis instead of proliferation upon TCR stimulation. During the activation process, T cells produce IL-2, which acts as an extrinsic survival factor. In addition, TCR stimulation results in up-regulation of Bcl-xL to enhance T cell survival intrinsically. We show in this study that protein kinase C (PKC)-theta is required for enhancing the survival of activated CD4+ T cells by up-regulating Bcl-xL. In response to TCR stimulation, CD4+ PKC-theta-/- T cells failed to up-regulate Bcl-xL, and underwent accelerated apoptosis via a caspase- and mitochondria-dependent pathway. Similar to PKC-theta-deficient primary CD4+ T cells, small interfering RNA-mediated knockdown of PKC-theta in Jurkat cells also resulted in apoptosis upon TCR stimulation. Forced expression of Bcl-xL was sufficient to inhibit apoptosis observed in PKC-theta knockdown cells. Furthermore, ectopic expression of PKC-theta stimulated a reporter gene driven by a mouse Bcl-xL promoter. Whereas an inactive form of PKC-theta or knockdown of endogenous PKC-theta led to inhibition of Bcl-xL reporter. PKC-theta-mediated activation of Bcl-xL reporter was inhibited by dominant-negative IkappaB kinase beta or dominant-negative AP-1. Thus, the PKC-theta-mediated signals may function not only in the initial activation of naive CD4+ T cells, but also in their survival during T cell activation by regulating Bcl-xL levels through NF-kappaB and AP-1 pathways.     

The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptor-mediated responses

EphB6 is the most recently identified member of the Eph receptor tyrosine kinase family. EphB6 is primarily expressed in thymocytes and a subpopulation of T cells, suggesting that it may be involved in regulation of T lymphocyte differentiation and functions. We show here that overexpression of EphB6 in Jurkat T cells and stimulation with the EphB6 ligand, ephrin-B1, results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for EphB6-specific signaling. Activation of the JNK pathway and the establishment of an IL-2/IL-2R autocrine loop have been shown to play a role in the negative selection of CD4(+)CD8(+) self-reacting thymocytes. In agreement, stimulation of murine thymocytes with ephrin-B1 not only blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an important role in regulating thymocyte differentiation and modulating responses of mature T cells.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

CD28 ligation costimulates cell death but not maturation of double-positive thymocytes due to defective ERK MAPK signaling

The differentiation of double-positive (DP) CD4(+)CD8(+) thymocytes to single-positive CD4(+) or CD8(+) T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.     

TCR signaling thresholds regulating T cell development and activation are dependent upon SHP-1

An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4-CD8- double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.     

Regulated costimulation in the thymus is critical for T cell development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Immunomodulatory effects of transforming growth factor-beta on T lymphocytes. Induction of CD8 expression in the CTLL-2 cell line and in normal thymocytes.

We investigated the role of transforming growth factor-beta 1 (TGF-beta) in regulation of T cell growth and differentiation. Treatment of CTLL-2 cells with TGF-beta inhibited IL-2-dependent proliferation and caused morphologic changes as well as increased adherence. A major change of phenotype in TGF-beta-treated cells was the de novo expression of CD8 alpha chain in 35% of cells, which required the continuous presence of TGF-beta. Of the CD8 alpha+ cells, 20 to 30% co-expressed CD8 beta chain. Increased CD8 expression occurred even in the total absence of cell growth, was not a consequence of growth inhibition, and was not a result of selective growth or survival of CD8+ cells. New RNA synthesis was required for TGF beta-induced CD8 alpha surface expression, inasmuch as this was prevented by treatment with actinomycin D. Northern blot analysis demonstrated that cells treated with IL-2 + TGF-beta rapidly accumulated mRNA encoding both chains of the CD8 dimer, to a level fourfold greater than control by 6 to 12 h. In contrast, the IL-2-dependent increases in IL-2R alpha, IL-2R beta, and Granzyme B mRNA levels in these cultures were profoundly inhibited by TGF-beta. When unfractionated murine thymocytes were stimulated with phorbol dibutyrate plus ionomycin and cultured with IL-2 + TGF-beta, an increase in CD8 alpha mRNA was seen and greater numbers of CD8+ cells with higher levels of CD8 alpha and CD8 beta surface expression resulted, as compared to controls treated with IL-2 alone. Furthermore, similar treatment of CD4-CD8-(double negative) thymocytes with TGF-beta induced de novo CD8 alpha expression by a substantial number of cells, and the majority of these CD8+ cells lacked TCR/CD3. These data suggest that TGF-beta has both positive and negative regulatory effects on the expression of gene products important for T lymphocyte differentiation and function.     

Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion. However, this stimulation is insufficient for extended T cell growth to multiple cytokines or differentiation into CTL or IFN-gamma-secreting effector T cells. IL-2 is required for these functions in part by regulation of cyclin D3 and granzyme B. Somewhat less efficiently, IL-4 stimulation of these transgenic T cells redundantly rescued many of these activities. These data demonstrate a fundamental requirement for IL-2 and perhaps other common gamma-chain-dependent cytokines to promote selective gene expression by Ag-activated T cells for their subsequent growth and differentiation into effector T lymphocytes.     

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.     

Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells.

The alpha beta T cell antigen receptor (TCR) that is expressed on most T lymphocytes is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER) and then transported to the plasma membrane. Expression of the TCR complex is quantitatively regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of surface alpha beta TCR complexes that are expressed on mature T cells. However, the molecular basis for low TCR expression in developing alpha beta T cells is unknown. In the present study we report the unexpected finding that assembly of nascent component chains into complete TCR alpha beta complexes is severely impaired in immature CD4+CD8+ thymocytes relative to their mature T cell progeny. In particular, the initial association of TCR alpha with TCR beta proteins, which occurs relatively efficiently in mature T cells, is markedly inefficient in immature CD4+CD8+ thymocytes, even for a matched pair of transgenic TCR alpha and TCR beta proteins. Inefficient formation of TCR alpha beta heterodimers in immature CD4+CD8+ thymocytes was found to result from the unique instability of nascent TCR alpha proteins within the ER of immature CD4+CD8+ thymocytes, with nascent TCR alpha proteins having a median survival time of only 15 min in CD4+CD8+ thymocytes, but > 75 min in mature T cells. Thus, these data demonstrate that stability of TCR alpha proteins within the ER is developmentally regulated and provide a molecular basis for quantitative differences in alpha beta TCR expression on immature and mature T cells. In addition, these results provide the first example of a receptor complex whose expression is quantitatively regulated during development by post-translational limitations on receptor assembly.