HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.     

Requirements for modulation of the CD4 molecule in response to phorbol myristate acetate. Role of the cytoplasmic domain

CD4 (T4) is a 60 kD glycoprotein expressed on a subset of T lymphocytes. CD4 augments T cell responses to suboptimal Ag stimulation. In addition, the CD4 molecule is the receptor for HIV-1. CD4 is phosphorylated on serine residues within the cytoplasmic domain and its cell surface expression is decreased in response to PMA, APC bearing the appropriate Ag or HIV infection. The kinetics of CD4 phosphorylation and modulation are similar, suggesting that the two events may be related. L3T4, the murine CD4 equivalent, is not modulated from the surface of mature, peripheral T cells in response to PMA. The difference in the ability to modulate L3T4 and CD4 in response to PMA may be due to differences between the two molecules or to differences between the cells in which they are expressed. To further define the requirements for CD4 modulation, we used retroviral vectors to transfer the cDNA for CD4 and various mutants of CD4 into two murine T cell hybridomas that express L3T4. One of these hybridomas, By155.16, does not modulate L3T4 in response to PMA and the other, 5D5.63, does modulate L3T4 in response to PMA. When expressed by these hybridomas CD4 is not modulated from the surface of By155.16 and is modulated from the surface of 5D5.63 in response to PMA. In both of these hybridomas, CD4 is phosphorylated on serine residues in response to PMA. A mutant form of CD4, CD4 delta, was constructed in which the majority of the cytoplasmic domain was deleted. When expressed in 5D5.63, CD4 delta was not modulated in response to PMA. Replacing the cytoplasmic domain of CD4 with that of the human IL-2 receptor did not reconstitute the ability of CD4 to be modulated. These results suggest that the inability to modulate L3T4 from the surface of murine peripheral T cells is due to features of the cell and not the molecule. Furthermore, the cytoplasmic domain of CD4 is required for its modulation from the cell surface in response to PMA.     

Apoptosis mediated through CD45 is independent of its phosphatase activity and association with leukocyte phosphatase-associated phosphoprotein

Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.     

The requirements for Fas-associated death domain signaling in mature T cell activation and survival

Fas-associated death domain (FADD) is a death domain containing cytoplasmic adapter molecule required for the induction of apoptosis by death receptors. Paradoxically, FADD also plays a crucial role in the development and proliferation of T cells. Using T cells from mice expressing a dominant negative form of FADD (FADDdd), activation with anti-TCR Ab and costimulation or exogenous cytokines is profoundly diminished. This is also seen in wild-type primary T cells transduced with the same transgene, demonstrating that FADD signaling is required in normally differentiated T cells. The defective proliferation does not appear to be related to the early events associated with TCR stimulation. Rather, with a block in FADD signaling, stimulated T cells exhibit a high rate of cell death corresponding to the initiation of cell division. Although CD4 T cells exhibit a moderate deficiency, this effect is most profound in CD8 T cells. In vivo, the extent of this defective accumulation is most apparent; lymphocytic choriomenigitis virus-infected FADDdd-expressing mice completely fail to mount an Ag-specific response. These results show that, in a highly regulated fashion, FADD, and most likely caspases, can transduce either a signal for survival or one that leads directly to apoptosis and that the balance between these opposing outcomes is crucial to adaptive immunity.     

CD4 phosphorylation partially reverses Nef down-regulation of CD4

HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (approximately 95%), whereas that induced by PMA was partial (approximately 40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.     

Tim-3 negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection

Cytotoxic CD8(+) T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection. These Tim-3 expressing T cells are dysfunctional in terms of their capacities to proliferate or to produce cytokines. In this study, we evaluated the effect of Tim-3 expression on the cytotoxic capabilities of CD8(+) T cells in the context of HIV infection. We investigated the cytotoxic capacity of Tim-3 expressing T cells by examining 1) the ability of Tim-3(+) CD8(+) T cells to make perforin and 2) the direct ability of Tim-3(+) CD8(+) T cells to kill autologous HIV infected CD4(+) target cells. Surprisingly, Tim-3(+) CD8(+) T cells maintain higher levels of perforin, which was mainly in a granule-associated (stored) conformation, as well as express high levels of T-bet. However, these cells were also defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8(+) T cells from chronic progressors by increasing; a) their degranulation capacity, b) their ability to release perforin, c) their ability to target activated granzyme B to HIV antigen expressing CD4(+) T cells and d) their ability to suppress HIV infection of CD4(+) T cells. In this latter effect, blocking the Tim-3 pathway enhances the cytotoxcity of CD8(+) T cells from chronic progressors to the level very close to that of T cells from viral controllers. Thus, the Tim-3 receptor, in addition to acting as a terminator for cytokine producing and proliferative functions of CTLs, can also down-regulate the CD8(+) T cell cytotoxic function through inhibition of degranulation and perforin and granzyme secretion.     

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.     

Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.     

Two discrete events, human T-cell leukemia virus type I Tax oncoprotein expression and a separate stress stimulus, are required for induction of apoptosis in T-cells

Apoptosis, or programmed cell death, is a key event in biologic homeostasis but is also involved in the pathogenesis of many human diseases including human immunodeficiency virus (HIV) infection. Although multiple mechanisms contribute to the gradual T cell decline that occurs in HIV-infected patients, programmed cell death of uninfected bystander T lymphocytes, including CD4+ and CD8+ T cells, is an important event leading to immunodeficiency. The HIV envelope glycoproteins (Env) play a crucial role in transducing this apoptotic signal after binding to its receptors, the CD4 molecule and a coreceptor, essentially CCR5 and CXCR4. Depending on Env presentation, the receptor involved and the complexity of target cell contact, apoptosis induction is related to death receptor and/or mitochondria-dependent pathways. This review summarizes current knowledge of Env-mediated cell death leading to T cell depletion and clinical complications and covers the sometimes conflicting studies that address the possible mechanisms of T cell death.     

Molecular and Cellular Analysis of Human Immunodeficiency Virus-Induced Apoptosis in Lymphoblastoid T-Cell-Line-Expressing Wild-Type and Mutated CD4 Receptors

We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39–48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56^lck (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild-type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56^lck or NF-κB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56^lck signaling, and may involve a critical region for CD4 dimerization.     

Reduced monomeric CD4 is the preferred receptor for HIV

CD4 is a co-receptor for binding of T cells to antigen-presenting cells and the primary receptor for the human immunodeficiency virus type 1 (HIV). CD4 exists in three different forms on the cell surface defined by the state of the domain 2 cysteine residues: an oxidized monomer, a reduced monomer, and a covalent dimer linked through the domain 2 cysteines. The disulfide-linked dimer is the preferred immune co-receptor. The form of CD4 that is preferred by HIV was examined in this study. HIV entry and envelope-mediated cell-cell fusion were tested using cells expressing comparable levels of wild-type or disulfide bond mutant CD4 in which the domain 2 cysteines were mutated to alanine. Eliminating the domain 2 disulfide bond increased entry of HIV reporter viruses and enhanced HIV envelope-mediated cell-cell fusion 2-4-fold. These observations suggest that HIV enters susceptible cells preferably through monomeric reduced CD4, whereas dimeric CD4 is the preferred receptor for binding to antigen-presenting cells. Cleavage of the domain 2 disulfide bond is possibly involved in the conformational change in CD4 associated with fusion of the HIV and cell membranes.     

HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells

Cell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here, we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.     

TRAIL deficiency does not rescue impaired CD8+ T cell memory generated in the absence of CD4+ T cell help

Ag-specific CD8(+) T cells immunized in the absence of CD4(+) T cell help, so-called "unhelped" CD8(+) T cells, are defective in function and survival. We investigated the role of the proapoptotic molecule TRAIL in this defect. We first demonstrate that TRAIL does not contribute to the CD8(+) T cell response to Listeria monocytogenes strain expressing OVA (LmOVA) in the presence of CD4(+) T cells. Secondly, we generated mice doubly deficient in CD4(+) T cells and TRAIL and analyzed their CD8(+) T cell response to LmOVA. Memory CD8(+) T cells in double-deficient mice waned over time and were not protective against rechallenge, similar to their TRAIL-sufficient unhelped counterparts. To avoid the effects of CD4(+) T cell deficiency during memory maintenance, and to address whether TRAIL plays a role in the early programming of the CD8(+) T cell response, we performed experiments using heterologous prime and early boost immunizations. We did not observe activation-induced cell death of unhelped CD8(+) T cells when mice were infected with followed vaccinia virus expressing OVA 9 days later by LmOVA infection. Furthermore, primary immunization of CD4(+) T cell-deficient mice with cell-associated Ag followed by LmOVA infection did not reveal a role for TRAIL-mediated activation-induced cell death. Overall, our results suggest that CD4(+) T cell help for the CD8(+) T cell response is not contingent on the silencing of TRAIL expression and prevention of TRAIL-mediated apoptosis.     

Several CD4 domains can play a role in human immunodeficiency virus infection in cells.

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.     

A tailless fas-FADD death-effector domain chimera is sufficient to execute Fas function in T cells but not B cells of MRL-lpr/lpr mice

The Fas receptor delivers signals crucial for lymphocyte apoptosis through its cytoplasmic death domain. Several Fas cytoplasmic-associated proteins have been reported and studied in cell lines. So far, only Fas-associated death domain protein (FADD), another death domain-containing molecule has been shown to be essential for Fas signals in vivo. FADD is thought to function by recruiting caspase-8 through its death-effector domain. To test whether FADD is sufficient to deliver Fas signals, we generated transgenic mice expressing a chimera comprised of the Fas extracellular domain and FADD death-effector domain. Expression of this protein in lymphocytes of Fas-deficient MRL-lpr/lpr mice completely diminishes their T cell but not their B cell abnormalities. These results suggest that FADD alone is sufficient for initiation of Fas signaling in primary T cells, but other pathways may operate in B cells.