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PubMed2002

CD28 costimulation mediates down-regulation of p27kip1 and cell cycle progression by activation of the PI3K/PKB signaling pathway in primary human T cells.

CD28 provides a costimulatory signal that cooperates with the TCR/CD3 complex to induce T cell activation, cytokine production, and clonal expansion. We have recently shown that CD28 directly regulates progression of T lymphocytes through the cell cycle. Although a number of signaling pathways have been linked to the TCR/CD3 and to CD28, it is not known how these two receptors cooperate to induce cell cycle progression. Here, using cell-permeable pharmacologic inhibitors of phosphatidylinositol 3-hydroxykinase (PI3K) and mitogen-activated protein kinase kinase (MEK1/2), we show that cell cycle progression of primary T lymphocytes requires simultaneous activation of PI3K- and MEK1/2-dependent pathways. Decreased abundance of cyclin-dependent kinase inhibitor p27(kip1), which requires simultaneous TCR/CD3 and CD28 ligation, was dependent upon both MEK and PI3K activity. Ligation of TCR/CD3, but not CD28 alone, resulted in activation of MEK targets extracellular signal-related kinase 1/2, whereas ligation of CD28 alone was sufficient for activation of PI3K target protein kinase B (PKB; c-Akt). CD28 ligation alone was also sufficient to mediate inactivating phosphorylation of PKB target glycogen synthase kinase-3 (GSK-3). Moreover, direct inactivation of GSK-3 by LiCl in the presence of anti-CD3, but not in the presence of anti-CD28, resulted in down-regulation of p27(kip1), hyperphosphorylation of retinoblastoma tumor suppressor gene product, and cellular proliferation. Thus, inactivation of the PI3K-PKB target GSK-3 could substitute for CD28 but not for CD3 signals. These results show that the PI3K-PKB pathway links CD28 to cell cycle progression and suggest that p27(kip1) integrates mitogenic MEK- and PI3K-dependent signals from TCR and CD28 in primary T lymphocytes.

CD28 Antigens↗

PubMed1994

ICAM-1 and LFA-3 enhance the ability of anti-CD3 mAb to stimulate interferon gamma production in interleukin-2-activated T cells.

Interleukin-2 (IL-2)-activated killer cells, also referred to as lymphokine-activated killer (LAK) cells, are stimulated by tumor cells to express cytotoxic activity and to also secrete cytokines such as interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). We previously reported that secretion of cytokines by IL-2-activated T cells (LAK-T cells) is dependent on the initial cross-linking of the T cell receptor (TCR)-CD3-molecular complex, but the cross-linking of accessory molecules, such as LFA-1, CD2, CD44 and CD45, on LAK-T cells can enhance this cytokine production. We have developed an approach involving interspecific gene transfer to define further the contributions of LFA-1 and CD2 to the activation of LAK-T cells. The genes for huICAM-1 (a ligand for LFA-1) and huLFA-3 (a ligand for CD2) were transfected singly and in combination into a null mouse melanoma background, and clonal populations of cells that stably express ICAM-1 and/or LFA-3 were derived. Expression of the introduced ICAM-1 and/or LFA-3 by transfected cells enhanced their ability to bind LAK-T cells; the LFA-1/ICAM-1-mediated binding was not further enhanced by activation with phorbol 12-myristate 13-acetate. ICAM-1- and/or LFA-3-transfected cells, in the presence of immobilized anti-CD3, exhibited a greater ability to stimulate IFN gamma secretion by LAK-T cells compared to the untransfected parental lines. This experimental system, which allows ICAM-1/LFA-1 and CD2/LFA-3 interactions to occur on the LAK-T cell at a site distal from the anti-CD3 signal, extends our understanding of LAK-T cell activation by establishing that both LFA-1/ICAM-1 and CD2/LFA-3 can mediate co-stimulation via adhesion and signaling events.

Antibodies, Monoclonal↗

PubMed1997

The protein tyrosine kinase p56(lck) regulates the serine-base exchange activity in Jurkat T cells.

Different classes of protein kinase inhibitors for protein kinase C, cAMP-dependent protein kinase or protein tyrosine kinases have been studied for their effect on phospholipid metabolism. The results show that among the compounds studied, only 4'-aminohydroxyflavone (AHF), previously described as a specific inhibitor of the protein tyrosine kinase p56(lck), markedly increased phosphatidylserine synthesis in Jurkat T cells. The biosyntheses of phosphatidylcholine and phosphatidylethanolamine were not affected. Also, the synthesis of phospholipids from tritium-labeled fatty acid as precursor was left unchanged by the p56(lck) inhibitor. The decreased phosphatidylserine synthesis induced when triggering the CD3-TCR complex was impaired by AHF, suggesting that p56(lck) could be implicated in the regulation of the serine-base exchange enzyme system. Direct evidence for the participation of p56(lck) in the regulation of the serine-base exchange enzyme system was obtained by using p56(lck)-deficient Jurkat cells (J.CaM 1.6) in which the basal base exchange activity was markedly increased and on the other hand AHF had no effect. In addition, transfection of J.Cam 1.6 cells with p56(lck)-cDNA allowed recovery of the AHF activity.

CD3 Complex↗

PubMed2005

Polarized development of memory cell-like IFN-gamma-producing cells in the absence of TCR zeta-chain.

TCR/CD3 complex-mediated signals play critical roles in regulating CD4(+) Th cell differentiation. In this report, we have examined the in vivo role of a key TCR/CD3 complex molecule zeta-chain in regulating the differentiation of Th cells. We have studied T cells from zeta-chain-deficient mice (zetaKO mice), zeta-chain-bearing mice (zeta(+) mice), and from zetaKO mice expressing a FcRgamma chain transgene (FcRgammaTG, zetaKO mice). Our results demonstrated that, compared with those of control mice, CD4(+) T cells and not CD8(+) T cells from zetaKO mice were polarized into IFN-gamma-producing cells. Some of these IFN-gamma-producing cells could also secrete IL-10. Interestingly, zetaKO mouse T cells produced IFN-gamma even after they were cultured in a Th2 condition. Our studies to determine the molecular mechanisms underlying the polarized IFN-gamma production revealed that the expression level of STAT4 and T-bet were up-regulated in freshly isolated T cells from zetaKO mice. Further studies showed that noncultured zetaKO mice CD4(+) T cells and thymocytes bore a unique memory cell-like CD44(high), CD62L(low/neg) phenotype. Altogether, these results suggest that, in the absence of the zeta-chain, CD4(+) T cells develop as polarized IFN-gamma-producing cells that bear a memory cell-like phenotype. The zeta-chain-bearing T cells may produce a large amount of IFN-gamma only after they are cultured in a condition favoring Th1 cell differentiation. This study may provide important implications for the down-regulation of zeta-chain in T cells of patients bearing a variety of tumors, chronic inflammatory and infectious diseases.

Animals↗

PubMed1991

Immobilized anti-CD3-induced T cell growth: comparison of the frequency of responding cells within various T cell subsets.

Human T cells can be divided into subsets based on the expression of CD29, CD45RA, CD45RO, LFA-3, or CD11a. It has been suggested that the subset of CD4+ T cells that expresses high densities of CD29, CD11a, CD45RO, and LFA-3 contains "memory" T cells, whereas the subset of cells that expresses CD45RA contains "naive" T cells. In order to obtain a more complete picture of the functional capacities of human naive and memory CD4+ and CD8+ T cell subsets, highly purified T cells were activated with a uniform stimulus and responses were examined in bulk cultures and under limiting dilution conditions. T cell activation was achieved with an immobilized mAb to the CD3 molecular complex, 64.1. In bulk cultures, immobilized 64.1 stimulated a vigorous response. Moreover, the number of cells entering the cell cycle, the magnitude of the [3H]thymidine incorporation, and the growth of the cells over 6 days in culture by naive and memory CD4+ T cells was comparable. To delineate the frequency of responsive cells in each subset more precisely, cells were cultured with immobilized 64.1 at limiting dilution and the precursor frequency of responding cells was assessed by examining wells microscopically for visible growth. Immobilized 64.1 was able to induce some T cells from each subset to grow in the complete absence of AC, when exogenous IL2 was present. The number of responding CD4+ and CD8+ cells was comparable. The percentage of naive cells responding in each population was approximately three times greater than the frequency of memory cells. IL4 could also support the growth of immobilized 64.1-activated CD4+ T cells, but the frequency of responding cells was much lower than that supported by IL2. The vast majority of the IL-4 responsive CD4+ cells resided within the naive cell subset. The data indicate that the response of CD4+ and CD8+ naive and memory T cell subsets to immobilized anti-CD3 depends on the density of responding cells. Naive T cells have an enhanced capacity to grow when cultured in the absence of other T cells or accessory cells. This ability may facilitate their expansion during primary immune responses.

Antibodies, Monoclonal↗

PubMed1988

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3.

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.

Adult↗

PubMed1988

Comparative effects of tumor necrosis factor-alpha and IL-1 beta on mitogen-induced T cell activation.

The effect of rTNF-alpha on human T cell function was examined and compared with that of rIL-1 beta by assessing the ability of each cytokine to support mitogen-induced proliferation, IL-2 production, and IL-2R expression. TNF-alpha and IL-1 beta each enhanced DNA synthesis induced by PHA or immobilized mAb to the CD3 molecular complex. In addition, each cytokine increased the number of cells entering the G1 phase of the cell cycle and augmented IL-2R expression. The combination of optimal concentrations of these factors supported these responses to a greater extent than either cytokine alone, suggesting that T cell responsiveness is independently regulated by the action of at least two separate monocyte derived cytokines. Whereas TNF-alpha had little effect, IL-1 beta augmented IL-2 mRNA expression and IL-2 production by mitogen-stimulated cells. Furthermore, IL-1 beta enhanced proliferation with increasing length of culture. Whereas TNF-alpha also enhanced proliferation late in culture, it was less effective in this regard than IL-1 beta. Thus, IL-1 beta and TNF-alpha augment mitogen-induced T cell proliferation by increasing the number of cells initially activated and by promoting subsequent cell cycle progression. They differ, however, in their capacity to promote IL-2 mRNA and IL-2 production and therefore ongoing T cell proliferation.

Antibodies, Monoclonal↗

PubMed1996

Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association.

Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).

Amino Acid Sequence↗

PubMed2001

T cells in mice expressing a transgenic human TCR beta chain get positively selected but cannot be activated in the periphery by signaling through TCR.

TCR-CD3 complex-mediated signaling is crucial for both developmental selection and antigenic activation of T cells. We report that mice expressing a recombined human TCRbeta chain (Tg), which have normal development of T cells, mounted very weak responses to immunization with protein antigens as well as the HA307-319 peptide recognized by the human T cell clone HA1.7 from which the transgene is derived. An anti-CD3epsilon mAb triggered equivalent proliferation from Tg and non-Tg T cells, but an anti-human TCRbeta mAb induced proliferation poorly in Tg T cells in contrast to human T cells or HA1.7. In Tg mice, T cells expressing endogenous TCR were CD44(high), whereas most transgene-expressing T cells remained CD44(low), suggesting that transgene-expressing cells are not activated in the periphery to participate in immune responses. However, anti-human TCRbeta could induce some activation markers on T cells and cross-linking of the Tg TCR by plate-coated anti-human TCRbeta efficiently induced T cell proliferation. Human TCRbeta-mediated Tg T cell activation could be rescued by exogenous IL-2, as well as by the calcium ionophore A23187, but not by phorbol esters. Thus, this human TCRbeta chain functions efficiently for positive selection of mouse T cells, but not for their peripheral activation, probably because of a lack of oligomerization leading to defects in signaling for calcium flux and IL-2 induction. The data thus suggest an early point of separation of signaling pathways between positive selection and peripheral activation of T cells.

Animals↗

PubMed1994

The anti-CD6 mAb, IOR-T1, defined a new epitope on the human CD6 molecule that induces greater responsiveness in T cell receptor/CD3-mediated T cell proliferation.

The T lymphocyte cell surface molecule, CD6, has been shown in a number of studies to play an important role in T cell activation. Its physiological ligand or function is still unknown. A panel of five anti-CD6 mAbs was used in the present study to investigate the structure-function relationship of this molecule. Cross-blocking assays indicate that three different epitopes were defined on the CD6 molecule by these mAbs. One of these epitopes defined by the mAb, IOR-T1, is insensitive to thiol-reducing agents, such as dithiothreitol and 2-mercaptoethanol. Of the other two epitopes, one was defined by 2H1 and the other was shared by three other mAbs, T12, 6D3, and Dako-CD6. All the CD6 mAbs at optimal concentration exhibit equal potency in enhancing T cell proliferation mediated through the T cell receptor/CD3 complex by optimal concentration of the anti-CD3 mAb, OKT3 (100 ng/ml). Simultaneous cross-linking of both the anti-CD6 mAbs and OKT3 is essential for the synergistic effect. When suboptimal concentrations of OKT3 (1 ng/ml) were used (no detectable cell proliferation), the synergistic effect of the anti-CD6 mAbs was still evident but with a differential effect. The epitope defined by IOR-T1 consistently induced greater T cell responsiveness under these conditions. Our results suggest that the CD6 molecule may play an important role in T cell activation, and that signals through an epitope of stable conformation appear to be of importance when antigen levels are low or interacting with low-avidity antigen receptors.

Antibodies, Monoclonal↗

PubMed1998

Characterisation of a monoclonal antibody recognising the CD3epsilon chain of the bovine T cell receptor complex.

We describe the characterisation of a monoclonal antibody (mAb), designated MM1A, which reacts with an antigen molecule on the surface of bovine alphabeta and gammadeltaTcR+ T cells. The mAb immunoprecipitated a series of polypeptides of 21 kDa, 22 kDa, 32 kDa, 36 kDa and 44 kDa which is consistent with it recognising the TcR/CD3 complex. COS cells, transfected with a cDNA encoding the bovine CD3epsilon chain, reacted with mAb MM1A indicating that the epitope recognised is on the epsilon chain of the complex and confirming that the mAb recognised bovine CD3.

Animals↗

PubMed1996

Functions of TCR and pre-TCR subunits: lessons from gene ablation.

Recent gene-targeting experiments have highlighted the existence of checkpoints that ensure that alpha beta T cells do not complete intrathymic differentiation if they have not attained certain landmark events. These 'proofreading' mechanisms operate by way of the pre-TCR and TCR complexes, which are sequentially expressed during T-cell development. These complexes are likely to signal via their associated CD3 subunits. By activating intracellular effectors, the CD3 subunits probably modulate gene expression profiles and drive the maturing alpha beta T cells through a precise developmental sequence.

Animals↗

PubMed1996

Involvement of Src-homology-2-domain-containing protein-tyrosine phosphatase 2 in T cell activation.

Activation of resting T lymphocytes by ligands to the complex of T cell antigen receptor (TCR) and CD3 is initiated by a series of critical tyrosine phosphorylation and dephosphorylation events. Protein-tyrosine kinases of the Syk, Src and Csk families and the CD45 protein-tyrosine phosphatase (PTPase) are known to be involved in these early biochemical reactions. We have found that one of the two T-cell-expressed SH2-domain-containing PTPases, SHPTP2, is rapidly phosphorylated on tyrosine upon addition of anti-CD3 mAbs. This response was absent in cells lacking the Src family kinase Lck. Concomitantly with tyrosine phosphorylation, SHPTP2 co-immunoprecipitated with two unphosphorylated cellular proteins; phosphatidylinositol 3-kinase p85 and Grb2. Binding of SHPTP2 to Grb2 occurred through the SH2 domain of Grb2, while the association between SHPTP2 and p85 seemed to be mediated through Grb2 as an intermediate. In addition, many other molecules associate with Grb2 and may thereby become juxtaposed to SHPTP2. Our results indicate that SHPTP2 participates actively at an early stage in TCR signaling and that its phosphorylation on tyrosine may direct a Grb2-dependent association with selected substrates.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.

The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.

Antibodies, Monoclonal↗

PubMed1991

Structure of the T cell antigen receptor (TCR): two CD3 epsilon subunits in a functional TCR/CD3 complex.

Transgenic mice carrying and expressing the human CD3 epsilon gene incorporate the corresponding protein product into T cell receptor (TCR)/CD3 complexes on thymocyte and T cell surfaces. The chimeric antigen receptors allow normal T cell development and selection of repertoires in vivo and are able to transduce activation signals in vitro. We have exploited the ability to distinguish mouse (m) and human (h)CD3 epsilon chains to analyze the stoichiometry of CD3 epsilon in transgenic mouse TCRs. Immunoprecipitation and fluorescence resonance energy transfer experiments demonstrate that such TCRs can contain both h- and mCD3 epsilon chains, implying that more than one CD3 epsilon subunit occurs per TCR. Antigen comodulation studies are consistent with a stochastic use of h- or mCD3 epsilon during receptor assembly, and further suggest a structure for the TCR/CD3 complex with two CD3 epsilon chains. The determination of CD3 epsilon subunit stoichiometry, together with existing biochemical data, allows the generation of a minimal model for the structure of the TCR and illustrates the potential value of the transgenic approach to the analysis of complex receptors.

Animals↗

PubMed1997

Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

Animals↗

PubMed1993

Restoration of early thymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon.

Thymic repertoire selection requires the expression of the alpha beta CD3 T-cell receptor (TCR) together with the coreceptors CD4 and CD8. The appearance of CD4 and CD8 on thymocytes is the hallmark of a complex maturation step, accompanied by downregulation of the interleukin 2 receptor (IL-2R) alpha chain, arrest of rearrangement (i.e., allelic exclusion) of the TCR beta-chain locus, a burst of cell divisions, and reduction in cell size. This maturation step is inhibited in TCR beta-chain-deficient mouse strains and may depend on surface expression of an immature TCR complex containing CD3 and TCR beta chains but no TCR alpha chain. Here we show that the CD4+8+ double-positive (DP) stage can be induced by treatment of fetal thymic organ cultures with anti-CD3 epsilon monoclonal antibodies in several TCR beta-chain-deficient mouse strains: severe combined immunodeficient (scid) mice, mice carrying a mutation in the recombination activating gene 1 (Rag-1), or mice carrying a deletion in the TCR beta-chain locus itself. These findings suggest that CD3 epsilon is expressed on the thymocyte surface independent of and prior to the TCR beta chain. The data are consistent with the notion that in wild-type mice the DP stage is induced by transmembrane signaling through an immature CD3-TCR beta-chain complex, which can be bypassed by crosslinking of CD3 epsilon alone.

Animals↗

PubMed1996

CD4-dependent and -independent association of protein tyrosine kinases to the T cell receptor/CD3 complex of CD4+ mouse T lymphocytes.

Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56lck, p59fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolés, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4+ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4+ T lymphocytes. Our results show that, in normal CD4+ T lymphocytes, as in CD4+ T cell lines, there is a stable and readily detectable association between CD4:p56lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4+ lymphocytes have detectable amounts of p56lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4+ T cells and CD4+ T cell lines in kinase association to the TcR/CD3 complex.

Animals↗