CD4-mediated Signaling Research Articles

The inositol 5-phosphatase SHIP-1 and adaptors Dok-1 and 2 play central roles in CD4-mediated inhibitory signaling

CD4 functions to enhance the sensitivity of T cells to antigenic peptide/MHC class II. However, if aggregated in isolation, e.g. in the absence of T cell receptor (TCR), CD4 can transduce yet undefined signals that lead to T cell unresponsiveness to antigen and apoptosis. In Human Immunodeficiency Virus-1 (HIV-1) disease, CD4+ T cell loss can result from gp120-induced CD4 signaling in uninfected cells. We show here that CD4 aggregation leads to Lck-dependent phosphorylation of the RasGAP adaptors Downstream of kinase-1/2 (Dok-1/2) and the inositol 5-phosphatase-1 (SHIP-1) and association of the two molecules. Studies using SHIP-1 shRNA, knockout mice and decoy inhibitors further indicate that CD4-mediated inhibition of TCR-mediated T cell activation is SHIP-1 and Dok-1/2 dependent, and involves SHIP-1 hydrolysis of Phosphatidylinositol 3,4,5-trisphosophate (PI(3,4,5)P3) needed for TCR signaling. Our studies provide evidence for a novel mechanism by which ill-timed CD4-mediated signals activated by ligands such as HIV-1 gp120 lead to disarmament of the immune system.

HIV-1 gp120 induces anergy in naive T lymphocytes through CD4-independent protein kinase-A-mediated signaling.

The ability of the envelope glycoprotein gp120 [human immunodeficiency virus (HIV) env] to induce intracellular signals is thought to contribute to HIV-1 pathogenesis. In the present study, we found that the exposure of CD4+ CD45RA+ naive T cells to HIVenv results in a long-lasting hyporesponsiveness to antigen stimulation. This phenomenon is not dependent on CD4-mediated signals and also can be generated by the exposure of naive T cell to soluble CD4-HIVenv complexes. The analysis of the proximal signaling reveals that HIVenv does not activate Lck as well as the mitogen-activated protein kinase intermediate cascade. Conversely, the envelope glycoprotein stimulates the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity and induces the progressive accumulation of the phosphorylated form of the cAMP-responsive element binding. Of note, the ligation of CXCR4 by stromal cell-derived factor-1alpha but not the engagement of CD4 by monoclonal antibody stimulates the PKA activity and induces a long-lasting hyporesponsivity state in naive CD4+ lymphocytes. The pretreatment of lymphocytes with H89, a cell-permeable PKA inhibitor, prevents the induction of anergy. These findings reveal a novel mechanism by which HIVenv may modulate the processes of clonal expansion, homeostatic proliferation, and terminal differentiation of the naive T lymphocyte subset.

T cell receptor-independent CD4 signalling: CD4–MHC class II interactions regulate intracellular calcium and cyclic AMP

CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II β2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP.

CD4-Mediated Signals Induce T Cell Dysfunction In Vivo

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.

The effects of CD3, CD4 and CD28 signaling on lymphocytes during human immunodeficiency virus-1 infection.

We investigated the effects of early human immunodeficiency virus-1 infection (HIV-1) on CD4- and CD28-mediated co-signaling of the T cell receptor (TCR)/CD3 complex in peripheral blood lymphocytes (PBL). CD4 ligation either alone or in conjunction with TCR occupancy resulted in abrogated signaling shown by impaired co-association of the tyrosine kinase ZAP-70 with the CD3-zeta chains in virally infected PBL. In addition, down-regulation of CD4-associated TCR signaling resulted in diminished tyrosine phosphorylation of mitogen-activated protein kinase (MAPK), a serine threonine kinase which is critically involved in the regulation of transcription factors. Furthermore, these aberrant CD4-driven signals rendered HIV-1-infected PBL susceptible to activation-induced cell death. By contrast, cross-linking of the TCR/CD3 complex with the CD28 receptor improved tyrosine phosphorylation of MAPK and salvaged infected PBL from activation-induced cell death. Our data demonstrate the importance of appropriate CD3, CD4 and CD28 co-stimulatory function to prevent apoptosis. The CD4-mediated signaling defects of the TCR could contribute to the loss of immunocompetent cells during HIV-1 infection via activation-induced cell death, whereas stimulation through the CD28 pathway could reverse these detrimental effects.

Binding of human immunodeficiency virus type 1 to CD4 induces association of Lck and Raf-1 and activates Raf-1 by a Ras-independent pathway.

We have analyzed CD4-mediated signaling during the early stages of human immunodeficiency virus type 1 (HIV-1) infection. Binding of purified HIV-1 virions or recombinant HIV-1 glycoprotein gp120 to CD4 receptors resulted in association and tyrosine phosphorylation and activation of tyrosine kinase Lck and serine/threonine kinase Raf-1. The association between Lck and Raf-1 was mediated by stimulation of the CD4 receptors, since it was abolished by preincubation of the virus with soluble CD4 and was not detected in CD4-negative A201 T cells. However, the Lck-Raf-1 association was restored in A201 cells permanently transfected with human CD4 cDNA and stimulated with anti-CD4 antibodies. In addition, a catalytically active Lck was required for the association of Lck and Raf-1. Surprisingly, the CD4-mediated signaling, induced by the HIV-1 binding, did not result in stimulation of the Ras GTP-binding activity or its association with Raf-1, indicating that the signaling pathway generated by the HIV-1 binding is not identical to the classical Ras/Raf-1 pathway. Furthermore, overexpression of activated Raf-1 in Jurkat T cells stimulated the HIV long terminal repeat promoter activity and significantly enhanced HIV-1 replication. This suggests that the Lck-Raf-1 pathway, rapidly stimulated by the binding of HIV-1 or gp120 to CD4 receptors, may play an essential role in the transcriptional activation of the integrated HIV-1 provirus as well as in its pathogenicity.

Disruption of Intrathymic CD4-1a Interactions on Immature CD4+CD8+ Thymocytes Results in Diminished TCR Expression on Mature CD8+ T Cell Progeny

Cell surface TCR expression by developing thymocytes is actively regulated during ontogeny. Whereas most immature CD4+CD8+ thymocytes express low levels of TCRαβ, mature CD4+ and CD8+ thymocytes express significantly higher levels of surface TCR. Low TCR expression on CD4+CD8+ thymocytes is due, at least in part, to inhibitory signals generated by CD4 interactions with MHC class II ligands in the thymus. In the present study we wished to determine whether levels of TCR expressed on mature thymocytes were also influenced by CD4-Ia interactions that had occurred on their CD4+CD8+ precursors. To do so, we examined TCR expression on mature CD8+ T cells from animals in which CD4-ligand interactions had been disrupted experimentally by in vivo administration of anti-CD4 mAb or by targeted disruption of an MHC class II gene. We found that TCR expression was significantly diminished on all mature CD8+ T cells from both anti-CD4 mAb-treated mice and MHC class II-deficient animals. These results demonstrate that TCR expression by mature CD8+ T cells, as well as that of immature CD4+CD8+ thymocytes, is regulated by CD4-mediated signals acting on CD4+CD8+ thymocytes. Because the effects of disrupting CD4-Ia interactions on TCR expression by CD8+ T cells were independent of TCR specificity, these findings directly support the concept that CD4-CD8+ T cells arise from precursor CD4+CD8+ cells.

Deletion of T lymphocytes in human CD4 transgenic mice induced by HIV-gp120 and gp120-specific antibodies from AIDS patients.

CD4, a T cell receptor for major histocompatibility complex class II antigen, is a key regulator of immunological reactivities. When engaged together with the T cell antigen receptor, CD4 enhances immune reactions, whereas when ligated independently of the antigen receptor CD4 inhibits the activation of T cells or initiates their deletion. CD4 serves also as a receptor for the human immunodeficiency virus (HIV), which binds the receptor with high avidity through its envelope molecule, gp120. Studies in tissue culture have shown that its affinity to CD4 gives the virus opportunities to utilize CD4-mediated signaling and to manipulate immunocytes. We show here in human CD4 transgenic mice that appropriately cross-linked HIV envelope protein causes massive deletion of HIV-reactive T cells in vivo.

Tyrosine kinase activity of CD4-associated p56lck may not be required for CD4-dependent T-cell activation.

The lymphoid-specific tyrosine kinase p56lck (Lck) is critical for the development and activation of T lymphocytes, and Lck kinase activity has been implicated in both T-cell antigen receptor/CD3- and CD4-mediated signaling. CD4-dependent T-cell activation has been demonstrated to be dependent upon the association of CD4 with Lck. To examine the role of the kinase activity of Lck in CD4-dependent T-cell activation, we have generated several kinase-deficient mutants of Lck. When transfected into CD4+ murine T-cell hybridoma cells, these mutants cause approximately 90% diminution in CD4-associated Lck kinase activity. Specifically, upon CD4 crosslinking there is decreased Lck autophosphorylation and decreased phosphorylation of an exogenous substrate. When CD4 is crosslinked to the T-cell antigen receptor-CD3 complex, decreased phosphorylation of associated substrates is also observed. In spite of this striking inhibition of Lck kinase function, cells expressing the kinase-deficient mutants demonstrate normal or enhanced CD4-dependent antigen responsiveness. These data demonstrate that the level of Lck kinase activity does not correlate with its CD4-associated function and suggest that the kinase activity of Lck may not be required for CD4-mediated signaling.

Inhibition of T Cell Receptor Expression and Function in Immature CD4 + cD8 + Cells by CD4

Most immature CD4+CD8+ thymocytes express only a small number of T cell receptor (TCR) molecules on their surface, and the TCR molecules they do express are only marginally capable of transducing intracellular signals. TCR expression and function was not intrinsically low in immature CD4+CD8+ thymocytes, but was found to be actively inhibited by CD4-mediated signals. Indeed, release of CD4+CD8+ thymocytes from CD4-mediated signals resulted in significant increases in both TCR expression and signaling function. These results suggest that, in CD4+CD8+ cells developing in the thymus, increased TCR expression and function requires release from CD4-mediated inhibition.