Proper T-cell activation and effector function are essential for effective immunity. T-cell antigen receptor (TCR) signals are known to regulate the outcome of differentiation, but the mechanisms remain unclear. Recent work from our lab demonstrates that the Src family protein tyrosine kinase, p56Lck, is able to specifically link TCR signals to activation of the Mitogen Activated Protein Kinase (MAPK) pathway through the function of its SH3 domain. The MAPK pathway is known to be involved in T-cell activation downstream of TCR ligation and has previously been implicated in T-helper type 2 (Th2) effector function. We have utilized an Lck SH3 mutant knock-in mouse line (Lck W97A) to investigate the potential role of this regulatory signaling mechanism in determining T-lymphocyte activation and effector function. Our results demonstrate that the Lck SH3 domain function is required for normal activation of T-lymphocytes following TCR stimulation as indicated by significantly reduced proliferation, IL-2 production, and CD69 induction in Lck W97A T-cells. Biochemical studies confirm that activation of the MAPK pathway is selectively altered in Lck W97A T-cells as P-ERK1/2 induction is significantly reduced but phospho-PLCg1 induction and calcium mobilization is unaffected. In vivo experiments demonstrate a specific and significantly impaired Th2 immunity in Lck W97A mice, with reduced serum levels of IgG1, IgE and IL-4 following immunization with DNP-KLH, or infection with the helminth Nippostrongylus brasiliensis. Th1 immunity does not appear differentially regulated in Lck W97A mice as serum levels of IgG3 and IgG2b are similar to WT following immunization with DNP-KLH, as well as serum levels of IFN-g1 following immunization with heat-killed Brucella abortus. In vitro differentiation studies confirm that Lck W97A T-lymphocytes are able to be directed to the Th2 phenotype, as indicated by intracellular staining for IL-4, with significantly increased levels of IFN-g under Th2 differentiating conditions compared to WT. These data indicate that the Lck SH3 domain regulates activation of T-lymphocytes by affecting MAPK pathway induction and demonstrate a novel and critical role for Lck in the regulation of Th2-type immunity. The Lck SH3 domain has also been implicated in the pathogenesis of Plasmodium, the causative agent of malaria. The role of the mosquito vector on malaria pathogenesis is not well understood. Initial studies examining the role of vector salivary gland proteins on cells of the innate immune system indicate that Anopheles stephensi saliva is able to enhance macrophage activation and phagocytosis as well as enhance macrophage Ag-presentation to T-lymphocytes in an in vitro model.

This thesis defines a novel role for the Herpes Simplex Virus (HSV) tegument protein, virion protein (VP) 11/12 as a modulator of host cell signalling. Studies aimed at examining infection induced lymphocyte inactivation, revealed that VP11/12 is tyrosine phosphorylated in three lymphocyte lineages (T cell, B cell and NK cell) following exposure to HSV-1 or HSV-2 infected fibroblasts. Tyrosine phosphorylation of VP11/12 was greater in lymphocytes compared to fibroblasts or epithelial cells and phosphorylation was enhanced by the lymphocyte specific Src family kinase (SFK) Lck during transfection- or infection-based assays. This suggested that VP11/12 is a substrate of Lck or a kinase activated by Lck. Lck is best known for initiating intracellular signalling downstream of the T cell receptor (TCR) and NK cell receptors. However, VP11/12 null HSV mutants retained the ability to block TCR signalling and NK cell cytotoxicity. Phosphorylation of VP11/12 occurred in the absence of any known Lck stimulus, like TCR ligation. Infection alone may activate Lck since Lck in infected Jurkat cells displayed features characteristic of activation: a reduced electrophoretic mobility in sodium dodecyl sulphate polyacrylamide gel and a marked increase in phosphorylation at the activation loop tyrosine (Y394). SFK substrates sometimes activate their cognate kinase through high affinity binding of the SFK Src homology (SH) 2 or SH3 domains. VP11/12 may serve this dual function since it interacts with Lck or Lck signalling complexes and is strictly required for Lck activation during infection. SFKs including Lck lie upstream of the canonical phosphoinositide 3-kinase (PI3K)-Akt pathway in signalling emanating from immune receptors, growth factor receptors and polyoma middle T antigen (MTAg). In HSV infection of Jurkat T cells and human embryonic lung fibroblasts, we find that VP11/12 interacts with PI3K either directly or indirectly and is required for infection induced activation of the PI3K-Akt signalling pathway. SFK activity is required for tyrosine phosphorylation of VP11/12, VP11/12-PI3K interactions, and Akt activation in infected fibroblasts. This data suggests that VP11/12 orchestrates signalling analogous to that of MTAg. In this model, VP11/12 activates SFKs to induce its own phosphorylation, subsequently allowing for interactions with PI3K and activation of Akt. / Virology

No description available.

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.

CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRbeta, and CCR7 expression. There were also increased thymic NK and gamma/delta T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Les lymphocytes T sont caractérisés par l’expression d’un récepteur à l’antigène des cellules T (TCR), soit le preTCR, soit le γδ TCR et le αβ TCR clonotypique, associé à un complexe de transduction formé du CD3 et de la chaîne ζ. La signalisation du complexe TCR:CD3:ζ est cruciale pour le développement des cellules T et pour leur activation spécifique par l'antigène. Ces signaux déclenchent la prolifération, la différentation, les fonctions effectrices et l’apoptose des cellules T. Les évènements proximaux de la signalisation du TCR:CD3:ζ impliquent des protéines tyrosine kinases (PTK) des familles CSK, SRC, SYK et TEC dont les acteurs principaux sont CSK (C-terminal SRC kinase), LCK (lymphocyte-specific protein tyrosine kinase), ZAP-70 (ζ chain-associated protein tyrosine kinase of 70 kDa) et ITK (interleukin-2-inducible T cell kinase). Après stimulation du complexe TCR:CD3:ζ, les PTKs sont activées et déclenchent une cascade de phosphorylation sur tyrosine dont la phosphorylation des motifs activateurs ITAM (immunoreceptor tyrosine-based activation motif) du CD3 et de la chaîne ζ, et la phosphorylation des protéines adaptrices. Celles-ci conduisent à l’assemblage du signalosome LAT:SLP-76, lequel contrôle en grande partie la diversité des signaux associés au complex TCR:CD3:ζ, dont le flux calcique, l’activation de la cascade des MAP kinases, l’activation des facteurs de transcription NF-κB, NFAT et AP-1 ainsi que la réorganisation du cytosquellette d’actine, l’adhésion cellulaire et la motilité. Pendant les cinq dernières décennies, le système immunitaire a été analysé extensivement in vitro et à l’aide de modèles animaux comme la souris. Par ailleurs, l’étude des déficits immunitaires héréditaires chez l’homme a permis aussi des avancées importantes et novatrices dans la compréhension du système immunitaire humain que les modèles animaux ne permettaient pas d’appréhender. Dans ma thèse de doctorat je rapporte le premier cas de déficit humain en LCK de transmission autosomique récessive et l’identification de nouvelles mutations autosomiques récessives provoquant un défaut humain de ZAP-70 et un défaut humain d’ITK. Je rends compte des phénotypes cliniques et immunologiques associés à ces immunodéficiences et je caractérise les défauts biochimiques de la signalisation TCR:CD3:ζ associés à ces déficits. Enfin, je compare mes observations avec les modèles murins déficients Lck-/-, Zap-70-/- et Itk-/- déjà bien établis. / T lymphocytes express either a preTCR, or a clonotypic γδ TCR or αβ TCR together with the CD3-complex and the associated ζ-chain. TCR:CD3:ζ-signalling is crucial for T cell development and antigen-specific activation including proliferation, differentiation, effector functions and apoptosis of mature T cells. Protein tyrosine kinase (PTK) cascades lie at the heart of proximal TCR:CD3:ζ-signalling. The CSK-, SRC-, SYK- and TEC-family members C-terminal SRC kinase (CSK), lymphocyte-specific protein tyrosine kinase (LCK), ζ-chain associated protein tyrosine kinase of 70 kDa (ZAP-70) and interleukin-2-inducible T cell kinase (ITK), respectively, are the major T cell players. After TCR:CD3:ζ-complex triggering, activation of PTKs results in tyrosine phosphorylation signals. These include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ-chains, and adaptor proteins that nucleate the proximal LAT:SLP-76-signalosome controlling almost all TCR:CD3:ζ-induced signalling events. These events initiate Ca2+-flux, activation of mitogen-activated protein kinases (MAPKs), activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB), activation of nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) as well as actin reorganization, cell-adhesion and motility. Throughout the last five decades, the immune system has been extensively investigated in vitro and in animal models such as the murine system. Additionally, studying and taking care of human primary immunodeficiency diseases (PIDs) has been seminal for our understanding of the human immune system as animal models not always recapitulate the subtleties found in men. In my doctoral thesis I report the first case of autosomal recessive human LCK-deficiency, a novel autosomal recessive mutation leading to human ZAP-70-deficiency and a novel autosomal recessive mutation leading to human ITK-deficiency. I provide detailed clinical, immunological and biochemical analyses especially of TCR:CD3:ζ-signalling and compare my findings to the well-established Lck-/-, Zap-70-/- and Itk-/- murine models.

La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse du VIH-1 en modulant les voies de signalisation de la cellule hôte. La signalisation par le TcR est essentielle à la sélection positive pour générer les cellules simples positives (SP) CD4+ et simples positives (SP) CD8+, processus largement dépendant de l’activité de la Src kinase Lck et de son habileté à lier la queue cytoplasmique des corécepteurs CD4 et CD8. Nous avons précédemment trouvé que l’expression de Nef dans le VIH ou VIS peut induire une sévère déplétion des thymocytes et une baisse d’expression du corécepteur CD4 à la membrane. Nous avons également montré que Nef bloque la génération des thymocytes doubles positifs (DP) CD4+ CD8+ en plus d’altérer la transition des cellules DP vers CD4+ SP. Par contre, ce phénotype est récupérable par plusieurs approches dont le croisement d’une souris transgéniques exprimant Nef avec une souris exprimant la forme constitutivement active de Lck Y505F. Les résultats indiquent que la maturation des cellules CD4+ est altérée par le dysfonctionnement de la signalisation CD4-Lck. Toutefois, les mécanismes moléculaires par lesquels Nef contribue au bloc de la génération des cellules CD4+ dans le thymus demeurent très imprécis. Dans cette étude, en utilisant des approches biochimiques et de microscopie confocale, nous avons trouvé que les thymocytes transgéniques Nef+ expriment plus de Lck que les thymocytes Nef-. Malgré cette augmentation, une partie significative de Lck est incapable d’atteindre la membrane plasmique. Cette fraction était significativement accumulée dans un compartiment intracellulaire des thymocytes transgéniques exprimant Nef. Également, en utilisant la technique d’essai kinase in vitro, nous avons trouvé que l’activité kinase de Lck est significativement augmentée dans les thymocytes transgéniques mais demeure stable suite à une stimulation par un α-CD3ε + α-CD4. Également, comparativement aux thymocytes Nef-, la kinase Lck dans les thymocytes transgéniques était résistante à la dégradation suite à une stimulation. En examinant le statut de c-Cbl, le principal régulateur négatif de Lck, nous avons montré que c-Cbl colocalise faiblement avec Lck, malgré son hyperphosphorylation constitutive. Ceci pourrait expliquer l’échec de la dégradation de Lck. En plus, nous avons trouvé que suite à une stimulation par un α-CD3ε + α-CD4, la phosphorylation de Zap-70 en tyrosine 493 par Lck est diminuée, résultant d’une importante baisse de l’activité kinase de Zap-70 et d’un bloc des premiers évènements de la voie de signalisation par le TcR. Ces données indiquent que la signalisation CD4-Lck est interrompue par la présence de Nef. / HIV-1 Nef protein plays an essential role in the HIV-1 pathogenesis by modulating the host signaling transduction pathways. TcR signalling is important for the thymic selection process to CD4 and CD8 single positive T cells and is greatly dependent on the activity of Src kinase Lck and its ability to bind to CD4 and CD8 cytoplasmic tail. We previously found that expression of HIV or SIV Nef can induce severe thymocytes depletion and downregulation of CD4 expression in Nef+ mice. We also recently showed that Nef blocks generation of double positive thymocytes and impairs DP to CD4+ SP T cells transition. The reversal of this phenotype was accomplished by several approaches, among them by crossing Nef+ mice with mice expressing constitutively active Lck Y505F. These results imply that the maturation of CD4+ T cells is disrupted due to impairment of Lck-mediated CD4 receptor signaling. However, the molecular mechanisms by which Nef contributes to the impairment in thymic CD4 generation remains largely unclear. In this study, using confocal microscopy and biochemical approaches, we found that Nef+ thymocytes express more Lck than the Nef- control. Despite of this increase, a significant portion of Lck molecules were unable to reach to the plasma membrane. It was significantly accumulated in the intracellular endosomal compartment of the Nef+ thymocytes. Moreover, using IVKA we found that the activity of Lck is significantly increased in Nef+ thymocytes but was not further increased upon stimulation by α-CD3ε, α-CD4 or α-CD3ε + α-CD4. Moreover, compared to Nef- controls, Lck kinase in Nef+ thymocytes was resistant to degradation upon stimulation. Examining the status of c-Cbl, the main negative regulator of Lck, showed that c-Cbl localized with Lck poorly, despite his constitutive hyperphosphorylation. This explains the failure of Lck degradation. In addition, we found that upon stimulation, Zap-70 phosphorylation at tyrosine 493 by Lck is decreased, resulting by a decrease of Zap-70 kinase activity and TcR proximal event block. These data indicate that CD4-Lck signaling was interrupted by the presence of Nef.

Selection of the T cell repertoire in the thymus is governed by the need to create a repertoire of peripheral T cells that can respond to any foreign antigen in the context of self-major histocompatibility complex (MHC), while enforcing central tolerance to self-antigens. Perturbations in signalling molecules, that reduce the affinity of thymic selection, can lead to the production of a peripheral repertoire with increased autoimmunity, as has been shown for mutations in the Zap-70 kinase. Upstream of Zap-70 is Lck, the most proximal tyrosine kinase required for T cell receptor (TCR) triggering upon TCR engagement by peptide:MHC. In order to study how Lck influences T cell activation, a transgenic mouse model (LckVA), in which Lck is expressed constitutively from a T cell specific transgene and mice have very low expression of Lck (~5% of WT) in both the thymus and periphery, was used. It has been shown that Lck is critical for successful T cell development, yet the results of this thesis show that even 5% of WT levels of Lck are sufficient for selection of thymic T cells on both polyclonal and F5 TCR transgenic backgrounds. Previous studies utilising mice expressing an inducible Lck transgene, which also had reduced Lck expression in the periphery, showed Lck to be critical in determining the activation threshold of T cells. In contrast, peripheral T cells in LckVA mice had similar activation thresholds to wild type T cells, as measured by in vitro upregulation of early activation markers. Further analysis of LckVA peripheral T cells revealed differential influences of low expression of Lck on downstream signalling pathways upon TCR engagement. For example, ERK signalling was impaired, while calcium flux and proliferation were enhanced in LckVA T cells. Finally, LckVA T cells were altered in their ability to differentiate, showing enhanced production of cytokines and retaining the capacity to form memory cells.

La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse du VIH-1 en modulant les voies de signalisation de la cellule hôte. La signalisation par le TcR est essentielle à la sélection positive pour générer les cellules simples positives (SP) CD4+ et simples positives (SP) CD8+, processus largement dépendant de l’activité de la Src kinase Lck et de son habileté à lier la queue cytoplasmique des corécepteurs CD4 et CD8. Nous avons précédemment trouvé que l’expression de Nef dans le VIH ou VIS peut induire une sévère déplétion des thymocytes et une baisse d’expression du corécepteur CD4 à la membrane. Nous avons également montré que Nef bloque la génération des thymocytes doubles positifs (DP) CD4+ CD8+ en plus d’altérer la transition des cellules DP vers CD4+ SP. Par contre, ce phénotype est récupérable par plusieurs approches dont le croisement d’une souris transgéniques exprimant Nef avec une souris exprimant la forme constitutivement active de Lck Y505F. Les résultats indiquent que la maturation des cellules CD4+ est altérée par le dysfonctionnement de la signalisation CD4-Lck. Toutefois, les mécanismes moléculaires par lesquels Nef contribue au bloc de la génération des cellules CD4+ dans le thymus demeurent très imprécis. Dans cette étude, en utilisant des approches biochimiques et de microscopie confocale, nous avons trouvé que les thymocytes transgéniques Nef+ expriment plus de Lck que les thymocytes Nef-. Malgré cette augmentation, une partie significative de Lck est incapable d’atteindre la membrane plasmique. Cette fraction était significativement accumulée dans un compartiment intracellulaire des thymocytes transgéniques exprimant Nef. Également, en utilisant la technique d’essai kinase in vitro, nous avons trouvé que l’activité kinase de Lck est significativement augmentée dans les thymocytes transgéniques mais demeure stable suite à une stimulation par un α-CD3ε + α-CD4. Également, comparativement aux thymocytes Nef-, la kinase Lck dans les thymocytes transgéniques était résistante à la dégradation suite à une stimulation. En examinant le statut de c-Cbl, le principal régulateur négatif de Lck, nous avons montré que c-Cbl colocalise faiblement avec Lck, malgré son hyperphosphorylation constitutive. Ceci pourrait expliquer l’échec de la dégradation de Lck. En plus, nous avons trouvé que suite à une stimulation par un α-CD3ε + α-CD4, la phosphorylation de Zap-70 en tyrosine 493 par Lck est diminuée, résultant d’une importante baisse de l’activité kinase de Zap-70 et d’un bloc des premiers évènements de la voie de signalisation par le TcR. Ces données indiquent que la signalisation CD4-Lck est interrompue par la présence de Nef. / HIV-1 Nef protein plays an essential role in the HIV-1 pathogenesis by modulating the host signaling transduction pathways. TcR signalling is important for the thymic selection process to CD4 and CD8 single positive T cells and is greatly dependent on the activity of Src kinase Lck and its ability to bind to CD4 and CD8 cytoplasmic tail. We previously found that expression of HIV or SIV Nef can induce severe thymocytes depletion and downregulation of CD4 expression in Nef+ mice. We also recently showed that Nef blocks generation of double positive thymocytes and impairs DP to CD4+ SP T cells transition. The reversal of this phenotype was accomplished by several approaches, among them by crossing Nef+ mice with mice expressing constitutively active Lck Y505F. These results imply that the maturation of CD4+ T cells is disrupted due to impairment of Lck-mediated CD4 receptor signaling. However, the molecular mechanisms by which Nef contributes to the impairment in thymic CD4 generation remains largely unclear. In this study, using confocal microscopy and biochemical approaches, we found that Nef+ thymocytes express more Lck than the Nef- control. Despite of this increase, a significant portion of Lck molecules were unable to reach to the plasma membrane. It was significantly accumulated in the intracellular endosomal compartment of the Nef+ thymocytes. Moreover, using IVKA we found that the activity of Lck is significantly increased in Nef+ thymocytes but was not further increased upon stimulation by α-CD3ε, α-CD4 or α-CD3ε + α-CD4. Moreover, compared to Nef- controls, Lck kinase in Nef+ thymocytes was resistant to degradation upon stimulation. Examining the status of c-Cbl, the main negative regulator of Lck, showed that c-Cbl localized with Lck poorly, despite his constitutive hyperphosphorylation. This explains the failure of Lck degradation. In addition, we found that upon stimulation, Zap-70 phosphorylation at tyrosine 493 by Lck is decreased, resulting by a decrease of Zap-70 kinase activity and TcR proximal event block. These data indicate that CD4-Lck signaling was interrupted by the presence of Nef.