Interactions between the tyrosine kinases p56lck,p59fyn and p50csk in CD4 signaling in T cells

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.     

Characterization of p59fyn-mediated signal transduction on T cell activation

Protein tyrosine kinase p59^fyn is associated with the TCR -CD3 complex and is suggested to play a role in T cell activation.To determine the molecular mechanism of p59^fyn-medlated signaltransduction in T cell activation, we established murine T cellhybridoma lines that expressed an elevated amount of wild-typeor mutant fyn. Clones that expressed high levels of normal p59^fynand active p59^fyn, encoded by wild-type and f-14 mutant fynrespectively, showed enhanced IL-2 production upon stimulationby anti-CD3 antibodies or natural antigen. On the other hand,clones that expressed kinase negative p59^fyn and p59^fyn withan SH2 (Src-homology 2) deletion encoded by t-1 mutant fyn showedlittle induction of IL-2 production upon stimulation. Thesedata suggest that p59^fyn is important in T cell signaling andthat the SH2 sequence plays a critical role in the reaction.Induction of tyrosine phosphorylatlon of multiple proteins uponantigenic stimulation was augmented similarly in the cells thatrespectively expressed wild-type and f-14 mutant fyn at elevatedlevels. The proteins that became highly tyrosine-phosphorylatedincluded phospholipase C (PLC-1), P95^vav, ZAP-70, the MAP kinase,CD3 and unidentified proteins of 120, 100 and 80 kDa. Tyrosinephosphorylation of the 120, 95 and 68 kDa proteins associatedwith PLC-1 was also observed in these cells upon stimulation.In contrast, only the 100 kDa protein and the MAP kinase wereincreasingly tyrosine phosphorylated in the antigen-stimulatedcells expressing t-1 fyn. These data suggest that PLC-1, PLC-1associated molecules, p95^vav, the 80 kDa protein, ZAP-70 andthe CD3 chain may be substrates of p59^fyn or of other tyrosinekJnases regulated by p59^fyn and be important in T cell signaling.     

Interleukin-7 activates p56lck and p59fyn,two tyrosine kinases associated with the p90 interleukin-7 receptor in primary human T cells

We have investigated signaling events associated with the cloned 90-kDa (p90) interleukin-7 receptor (IL-7R) to determine whether changes in the signaling pathways initiated by this molecule can explain the ability of T cells to proliferate to IL-7 following activation. Using in vitro kinase assays we find that the p90 IL-7R in both unstimulated and activated human T cells is physically associated with two molecules with intrinsic kinase activity. Western blotting analysis reveals these proteins to be the src kinase enzymes, p59^fyn and p56^lck. Binding of human recombinant IL-7 to the p90 IL-7R results in increased activity of both receptor-associated kinases in both resting and activated mature T cells. Thus, the signaling pathways initiated via the p90 IL-7R-associated src kinases are unlikely to be solely responsible for the proliferation of only activated T cells in response to IL-7. Additional signals, which may derive from other IL-7R-associated molecules such as the γc, are clearly required for IL-7-driven proliferation of activated primary T cells.     

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 p56^lck, p59^fyn 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:p56^lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolées, 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: p56^lck 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 p56^lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59^fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56^fyn 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.     

Interaction of human immunodeficiency virus glycoprotein 160 with CD4 in Jurkat cells increases p56lck autophosphorylation and kinase activity.

The tyrosine protein kinase p56lck, specifically expressed in lymphoid cells, undergoes modifications of its autophosphorylation and kinase activity when these cells are triggered by mAbs to the T cell determinants. The kinase activity and the autophosphorylation of p56lck were analysed following triggering Jurkat cells with the human immunodeficiency virus (HIV) glycoprotein gp160 which interacts with CD4: both the autophosphorylation and the kinase activity are increased within 1-5 min following addition of gp160, this increase is maximum at 5 min and is followed by a gradual return to the basal level within 2 h. Similar to observations made with anti-CD4 mAbs the increase in kinase activity of p56lck is not associated with changes in the gel mobility nor is it associated with T cell activation. Triggering of T cells with a combination of anti-CD3 mAbs which activate T cells but not p56lck and gp160 greatly potentiated the increase of p56lck autophosphorylation and kinase activity.     

Activation of CD45-deficient T cell clones by lectin mitogens but not anti-Thy-1

CD45, the leukocyte-common antigen, Is a transmembrane proteintyrosine phosphatase uniquely expressed by cells of hematopoletlcorigin. We have developed CD4⁺ and CD8⁺ T cell clones that aredeficient in the expression of CD45 and have previously shownthat these cells fall to proliferate in response to antigenor cross-linked CD3. These studies have now been extended toshow that stimulation with antl-Thy-1, a mltogenlc signal forthe CD4⁺CD45⁺ and CD8⁺CD45⁺ T cells, falls to induce proliferationin the CD45^– T cells. Examination of the CD8⁺CD45^–T cells correlates antl-Thy-1 unresponslveness with a failureto increase in tyrosine phosphorylatlon. Furthermore, stimulationof CD8⁺CD45⁺ T cells with antl-Thy-1 results in an increasein p56^ick activity but not in CD8⁺CD45^– T cells. In contrastto the results with antl-Thy-1, both the CD4^+– CD45 andCD8⁺CD45^– T cells respond to treatment with lectin mitogens,concanavalln A or phytohemagglutlnln. Lectin-lnduced proliferationwas inhibited by the addition of cyclosporln A. Treatment ofCD45^– T cells with PMA and lonomycln also results in proliferationindicating that activation of protein kinase C in conjunctionwith an increase in intracellular calcium rescues the defectcafsed by CD45 deficiency. The data suggest that CD45 Is requiredfor the activation of tyrosine kinase activity Immediate orprior to transmembrane signaling.     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

Defective p56Lck activity in T cells from an adult patient with idiopathic CD4+ lymphocytopenia

Idiopathic CD4(+) lymphocytopenia (ICL) is defined by a stable loss of CD4(+) T cells in the absence of any known cause of immune deficiency. This syndrome is still of undetermined origin. It affects adult patients, some of them displaying opportunistic infections similar to HIV-infected subjects. The hypothesis that the cellular immune defect may be due to biochemical failures of the CD3-TCR pathway is investigated here in a patient associating a severe selective CD4(+) lymphocytopenia with an increased CD8(+) T cell count discovered in the course of a cryptococcal meningitidis. A 40% reduction of T cell proliferation to CD3-TCR stimulation is observed only in the CD4(+) subpopulation. The early CD3-induced protein tyrosine phosphorylations are conserved in both CD4(+) and CD8(+) subsets, and the levels of the T cell protein tyrosine kinases p56(Lck), p59(Fyn) and ZAP-70 are normal. However, we find a 50% reduction of p56(Lck) kinase activity in the patient's T cells compared to a healthy control donor. p59(Fyn) activity does not appear to be altered. Nevertheless, we do not find any genetic abnormality of p56(Lck). These results thus suggest that a defect of an unknown protein regulating p56(Lck) activity takes place in this patient's T cells. Taken together, these findings reveal p56(Lck) alteration in ICL and confirm the critical role of this kinase in the maintenance of the peripheral CD4(+) T cell subpopulation.     

CD4+ T cell-independent maintenance and expansion of memory CD8+ T cells derived from in vitro dendritic cell activation

CD4+ T cells are essential for the maintenance of CD8+ memory T (Tm) cells following acute infection, but the importance of CD4+ T cells for the maintenance and expansion of CD8+ Tm cells to non-infectious antigens remains mostly unknown. Here, we showed that ovalbumin (OVA)-specific CD8+ Tm cell precursors derived from in vitro stimulation of TCR transgenic OT I CD8+ T cells with OVA protein-pulsed bone marrow-derived dendritic cells (DCOVA) can give rise to functional CD8+ Tm cells after adoptively transferred into mice. These CD8+ Tm cells can be maintained and remain fully functional in CD4+ T cell-absent environments in vivo. Furthermore, CD4+ T cells are not essential for the expansion of these CD8+ Tm cells. Finally, these in vitro DCOVA-activated CD8+ Tm cells maintained in CD4-deficient mice are also able to confer fully protective immunity against a later challenge of OVA-expressing tumor cells. Collectively, these findings demonstrate that in contrast to acute infections, maintenance and expansion of CD8+ Tm cells after priming with OVA protein-pulsed dendritic cells are independent of CD4+ T cells.     

Abnormal CD45RC expression and elevated CD45 protein tyrosine phosphatase activity in LEC rat peripheral CD4+ T cells

LEG rats are known to show a maturational arrest in the development of CD4⁺8⁺ to CD4⁺8^? cells in the thymus. Despite the blockade of maturation of CD4⁺8^? thymocytes, CD4⁺ T cells were observed in peripheral lymphoid organs, and these cells exhibit a defect in interleukin-2 (IL-2) production upon concanavalin A (Con A) stimulation. Although peripheral CD4⁺ cells in normal rat highly expressed CD45RC (CD45RC^high), the level of CD45RC expression was low (CD45RC^low) in LEC rat peripheral CD4⁺ cells. However, CD4⁺ cells from both strains highly expressed CD45 when those cells were stained by pan-CD45 mAb, suggesting that LEC rat CD4⁺ cells are deficient in expression of the CD45RC isoform, but not of CD45 molecules. When backcross rats from (F344 × LEC)F₁ × LEC were examined, the phenotype for CD45 expression pattern in CD4⁺ cells was clearly correlated with IL-2 production level in response to Con A stimulation. Thus, CD45RC^low cells exhibit a defect in IL-2 production, while CD45RC^high cells show normal IL-2 production. Protein tyrosine phosphatase (PTPase) activity in the membrane fraction of LEC rat CD4⁺ cells was threefold higher than that of normal rat CD4⁺ cells. Con A stimulation led to an increase in tyrosine phosphorylation levels, especially 100- and 40-kDa proteins, in normal rat CD4⁺ cells. In LEC rat CD4⁺ cells, however, the level of tyrosine phosphorylation in those proteins were very low. These results suggest that an elevated CD45 PTPase activity is responsive for a defect in IL-2 production in LEC rat peripheral CD4⁺ T cells.     

Human T lymphocyte activation induces tyrosine phosphorylation of α-tubulin and its association with the SH2 domain of the p59fyn protein tyrosine kinase

A glutathione-S-transferase-src-homology domain 2 (GST-SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59^fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, α- and β-tubulin were identified by N-terminal sequencing. Further analysis established that α-tubulin exists as a tyrosine-phosphorylated protein in Jurkat cells, where it interacts with p59^fyn, but not with p56^lck. By contrast, in untransformed resting human T lymphocytes α-tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine-phosphorylated α-tubulin that is able to interact with the fyn-SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.     

Natural CD4 CD25(+) regulatory T cells control the burst of superantigen-induced cytokine production: the role of IL-10.

In normal mice a subpopulation of CD4 T cells constitutively expresses the IL-2 receptor alpha chain (CD25). This natural CD4 CD25(+) subset is thymus-born, constitutively expresses IL-10 mRNA,does not produce IL-2 and is resistant to apoptosis. These cells behave as regulatory T cells in the control of self-tolerance, inflammatory reactions and T cell homeostasis. The mechanisms by which natural CD4 CD25(+) cells control the immune response is unclear. We examined CD25-deficient mice, which over-express various cytokines, including proinflammatory molecules, after bacterial superantigen stimulation in vivo. We observed that this abnormal cytokine production could be controlled by the injection of natural CD4 CD25(+) T cells and that IL-10 production is needed, as CD4 CD25(+) T cells from IL-10 knockout mice do not correct cytokine over-production in vivo. As the circulating IL-10 produced by CD25-deficient mice was ineffective, we deduced that the key source of IL-10 was the regulatory T cell population. IL-10 is also involved in the control of cytokine production by normal T cells. However, the target of IL-10 in this control is undefined. Whether it acts directly on the effector T cells or on the regulatory CD4 CD25(+) T cells themselves to induce their functional maturation has to be clarified.     

A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo

Immune-stimulating complexes (ISCOMS) are adjuvant vectors which are unusual in being able to prime both CD4(+) and CD8(+) T cells by parenteral and mucosal routes. However, their mode of action is unclear and to define better the cellular interactions involved we have studied the ability of ISCOMS containing ovalbumin (OVA) to prime TCR transgenic CD4(+) or CD8(+) T cells in vivo. Immunization with OVA ISCOMS caused activation and clonal expansion of CD4(+) and CD8(+) T cells in the T cell areas of the draining lymph nodes, followed by the migration of both CD4(+) and CD8(+) T cells into the B cell follicle. The T cells were primed to proliferate and secrete IFN-gamma after re-stimulation in vitro with the appropriate OVA peptide and CD8(+) T cell priming occurred in the absence of CD4(+) T cells. Increasing the number of dendritic cells (DC) in vivo with flt3 ligand augmented the expansion and activation of the OVA-specific T cells, particularly CD8(+) T cells. These studies indicate DC play a central role in the priming of both CD4(+) and CD8(+) T cells in vivo, and suggest that an ability to target DC may allow ISCOMS to be powerful vaccine vectors for stimulating protective immunity.