Lck activity controls CD4/CD8 T cell lineage commitment

Thymocytes carrying MHC class I-restricted TCRs differentiate into CD8 T cells, while those recognizing MHC class II become CD4 T cells. The mechanisms underlying how MHC class recognition, coreceptor expression, and effector function are coordinated are not well understood. Since the tyrosine kinase Lck binds with more affinity to CD4 than CD8, it has been proposed as a candidate to mediate this process. By using transgenic mice with altered Lck activity, we show that thymocytes carrying a class II-restricted TCR develop into functional CD8 T cells when Lck activity is reduced. Conversely, thymocytes carrying a class I-restricted TCR develop into functional CD4 T cells when Lck activity is increased. These results directly show that quantitative differences in the Lck signal control the CD4/CD8 lineage decision.     

CD45 ectodomain controls interaction with GEMs and Lck activity for optimal TCR signaling

The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.     

HIV-1 transgenic rat CD4+ T cells develop decreased CD28 responsiveness and suboptimal Lck tyrosine dephosphorylation following activation

Impaired CD4+ T cell responses, resulting in dysregulated T-helper 1 (Th1) effector and memory responses, are a common result of HIV-1 infection. These defects are often preceded by decreased expression and function of the alpha/beta T cell receptor (TCR)-CD3 complex and of co-stimulatory molecules including CD28, resulting in altered T cell proliferation, cytokine secretion and cell survival. We have previously shown that HIV Tg rats have defective development of T cell effector function and generation of specific effector/memory T cell subsets. Here we identify abnormalities in activated HIV-1 Tg rat CD4+ T cells that include decreased pY505 dephosphorylation of Lck (required for Lck activation), decreased CD28 function, reduced expression of the anti-apoptotic molecule Bcl-xL, decreased secretion of the mitogenic lympokine interleukin-2 (IL-2) and increased activation induced apoptosis. These events likely lead to defects in antigen-specific signaling and may help explain the disruption of Th1 responses and the generation of specific effector/memory subsets in transgenic CD4+ T cells.     

The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation

The src‐family kinases p56^lck (Lck) and p59^fyn (Fyn) are expressed in T cells and are among the first signaling molecules to be activated downstream of the T cell receptor (TCR). Evidence is emerging that although closely related, these signaling molecules have discrete functions during development, maintenance and activation of peripheral T cells. For example, during thymopoiesis Lck is uniquely able to provide all the signals required for pre‐TCRβ selection, although Fyn can substitute for a subset of these. Positive selection of CD4 single‐positive (SP) cells is also critically dependent on the expression of Lck but not Fyn, while differentiation of CD8 SP cells proceeds relatively efficiently in the absence of Lck. In naïve peripheral T cells either Lck or Fyn can transmit TCR‐mediated survival signals, and yet only Lck is able to trigger TCR‐mediated expansion signals under conditions of lymphopenia. Stimulation of naïve T cells by antigenic stimuli is also severely compromised in the absence of Lck, but more subtly impaired by the absence of Fyn. We discuss recent experiments addressing how these two src‐kinase family members interface with downstream signaling pathways to regulate these diverse aspects of T cell behavior.     

The activation of Csk by CD4 interferes with TCR-mediated activatory signaling

CD4-Lck recruitment to TCR/CD3, as well as Lck activation is essential for T cell activation. Indeed, the blockage of CD4-Lck recruitment to TCR during antigen recognition exerts a drastic inhibitory effect on T cell activation by interfering with both early and late phases of T cell signaling. In the present work, we report a novel inhibitory mechanism by which CD4 can shut down proximal T cell-activating signals. Indeed, we show that upon ligation of CD4 by antibodies the inhibitory kinase, p50(csk), is strongly induced and prolonged during the time. In contrast, p50(csk) was not activated when TCR and CD4 were properly engaged by their ligands. We also demonstrate that anti-CD4 treatment stimulated Csk kinase associated to the membrane adapter, PAG/Cbp, without affecting the total amount of Csk bound to PAG/Cbp. As a consequence, early tyrosine phosphorylation events as well as downstream signaling pathways leading to IL-2 gene expression induced by TCR were inhibited in anti-CD4 pretreated cells. We suggest a new model to explain the activation of negative signals by CD4 molecule.     

Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells

C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h. No alterations in surface expression of CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4-5-fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2-3-fold higher Lck activity. In Jurkat cells, basal levels of zeta chain phosphorylation were elevated, and spontaneous NFAT-AP-1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and zeta chains compared to control. TCR-induced activation of NFAT-AP-1 and TCR/CD28-stimulated IL-2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR-stimulated responses.     

Targeting of CD45 protein tyrosine phosphatase activity to lipid microdomains on the T cell surface inhibits TCR signaling

CD45, a transmembrane protein tyrosine phosphatase (PTP), can either positively or negatively regulate Src-family protein tyrosine kinase (PTK) activity in vivo. It is proposed that TCR-initiated signaling requires the segregation of PTP activities from the engaged TCR, based upon the differential membrane compartmentalization on the T cell surface. To test the importance of CD45 exclusion from lipid microdomains for proper TCR signaling, a chimeric molecule was generated by fusing the CD45 cytoplasmic region, which contains the PTP domains, to the amino-terminal 12 amino acids of Lck, which target Lck to lipid microdomains. Using 3A9 T lymphocyte hybridoma (3A9H) cells whose TCR recognizes hen egg-white lysozyme (HEL), Lck-CD45 expression resulted in its targeting to lipid microdomains. The 3A9H cells expressing Lck-CD45 were reduced in their responses to HEL or co-cross-linking of CD3 and CD4, as assessed by IL-2 production and Ca(2+) mobilization. Src-family PTK activity associated with lipid microdomains was also decreased. These results suggest that the segregation of CD45 from proximal TCR signaling components is necessary for TCR signaling and that the targeting of CD45 PTP activity to lipid microdomains on the T cell surface results in decreased sensitivity of TCR-mediated signaling.     

T cell costimulation by the hepatitis C virus envelope protein E2 binding to CD81 is mediated by Lck

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.     

Differential T cell receptor-mediated signaling in naive and memory CD4 T cells

Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3ζ subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3ζ in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72^syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.     

CD40 on NOD CD4 T cells contributes to their activation and pathogenicity

Our goals in this study were to investigate conditions under which T cells from NOD mice express CD40 and to determine how CD40 on autoreactive CD4 T cells contributes to their pathogenicity in T1D. Using CD40-positive diabetogenic T cell clones and CD4 T cells from NOD mice, we examined expression of CD40 upon activation through the TCR and costimulation through either CD28 or CD40. Our results indicate that CD40 expression is increased upon activation with antigen/MHC and that activation of NOD CD4 T cells through TCR/CD40 rapidly induced CD40 expression. Furthermore, CD40 costimulation promoted T cell proliferation to the same extent as costimulation through TCR/CD28. Importantly, costimulation of CD4 T cells through CD40 also interfered with T cell homeostasis by altering regulation of CTLA-4 expression. Through CD40–CD154 blocking studies, we demonstrated that signaling between T cells through CD40 and its ligand contributes to activation of pathogenic T cells and that blocking CD40 on T cells abrogates their ability to transfer diabetes. Thus, costimulation through CD40 on NOD T cells contributes to their pathogenicity by providing additional pathways for activation and by inhibiting upregulation of CTLA-4 during T cell activation.     

CD40 on NOD CD4 T cells contributes to their activation and pathogenicity

Our goals in this study were to investigate conditions under which T cells from NOD mice express CD40 and to determine how CD40 on autoreactive CD4 T cells contributes to their pathogenicity in T1D. Using CD40-positive diabetogenic T cell clones and CD4 T cells from NOD mice, we examined expression of CD40 upon activation through the TCR and costimulation through either CD28 or CD40. Our results indicate that CD40 expression is increased upon activation with antigen/MHC and that activation of NOD CD4 T cells through TCR/CD40 rapidly induced CD40 expression. Furthermore, CD40 costimulation promoted T cell proliferation to the same extent as costimulation through TCR/CD28. Importantly, costimulation of CD4 T cells through CD40 also interfered with T cell homeostasis by altering regulation of CTLA-4 expression. Through CD40-CD154 blocking studies, we demonstrated that signaling between T cells through CD40 and its ligand contributes to activation of pathogenic T cells and that blocking CD40 on T cells abrogates their ability to transfer diabetes. Thus, costimulation through CD40 on NOD T cells contributes to their pathogenicity by providing additional pathways for activation and by inhibiting upregulation of CTLA-4 during T cell activation.     

TCR-induced downregulation of protein tyrosine phosphatase PEST augments secondary T cell responses

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4(+) and CD8(+) T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4(+) and CD8(+) primary human T cells upon T cell activation. This was also true in mouse CD4(+) T cells, but less striking in mouse CD8(+) T cells. PTP-PEST reintroduced into Jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-kappaB elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

CD4+ T cell hyper-responsiveness in CD45 transgenic mice is independent of isoform

The CD45 tyrosine phosphatase is required for T cell development and function by virtue of its role as a positive regulator of src family kinase activity. In addition, recent data have highlighted that CD45 also acts as a negative regulator of Lck function by dephosphorylation of critical tyrosine residues. Lck functionality and TCR responsiveness are elevated in transgenic mice expressing the CD45RO isoform at 'intermediate' (10-40% of wild type) levels, indicating that the expression level of CD45 is critical in determining the sensitivity of T cells to TCR stimulation. However, it is unclear whether such a phenotype is specific for the CD45RO isoform, typically expressed by activated T cells. In the present work, the roles of three isoforms of CD45, RO, RB and RABC, in thymocyte development, T cell responses and TCR signalling pathways were directly compared. The data demonstrate that expression of CD45RB or CD45RABC at intermediate levels also results in CD4(+) T cell hyper-reactivity, as previously published for CD45RO. These data emphasize the dual functions of CD45 as both a positive and a negative regulators of TCR signalling irrespective of specific isoform expression.     

HIV envelope-directed signaling aberrancies and cell death of CD4+ T cells in the absence of TCR co-stimulation

HIV-1 infection in CD4₊ T cells initiates a viral cytopathiceffect (CPE) that is dependent on the activation of intracellularprotein tyrosine kinases (PTK). PTK in T cells are also activatedduring the course of TCR or CD4 receptor engagement and themanner of receptor engagement may generate signals leading eitherto cell proliferation, tolerance induction (anergy) or programmedcell death (PCD). We have identified PTK triggered during theinteraction of cells stably expressing surface HIV envelope(gp120/gp41; HIVenv) and CD4⁺ T cells, which leads to extensiveand rapid individual cell death. We have found that this killingis accompanied by tyrosine phosphorylation and activation ofthe CD4-associated p56^ick kinase, and by activation of a secondmember of the src family of PTK, p59^fyn kinase, normally associatedwith T cell stimulation through the TCR. Interestingly, in contrastwith normal T cell signaling, the subunit of the TCR failsto become tyrosine-phosphorylated during signaling accompanyingHIV-directed cell killing. Downstream activation of the ZAP-70PTK also does not occur. Unlike T cell apoptosis triggered bysoluble HIVenv glycoproteins, which requires co-stimulationof CD4 and the antigen-specific TCR, T cell killing by membrane-associatedHIVenv does not require TCR co-stimulation, because aberrantsignaling and cell death are triggered by CD4+ but TCR^–cell lines. These results are the first report where dual activationof the Lck and Fyn PTK does not result in normal downstreamsignaling through the ZAP PTK. We suggest by analogy to SCIDresulting from ZAP-70 mutations, that the dissociation of upstreamPTK activation from ZAP-70 signaling contributes to T cell depletionby HIV and to the development of AIDS.     

Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H

We have recently described the A6H antigen as a novel 120-140 kDa molecule which is co-expressed on human peripheral blood T cells and renal cell carcinoma cells. Engagement of the A6H antigen results in co-stimulation of CD4+ T cells but it remained unknown how cross-talk between the A6H antigen and the TCR-CD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis. In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests that A6H ligation is sufficient for triggering some of the early events in T cell activation, whereas full activation of the T cell, characterized by proliferation and cytokine production, requires co-ligation of the TCR-CD3 complex.     

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.