Early activation events render T cells susceptible to HIV-1-induced syncytia formation. Role of protein kinase C.

In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.     

HIV-infected humans, but not chimpanzees, have circulating cytotoxic T lymphocytes that lyse uninfected CD4+ cells

It has been suggested that autoimmune phenomena contribute to the depletion of CD4+ T cells and the development of AIDS in HIV-1 infected humans based, in part, on observations that some HIV-1-infected humans have autoantibodies reactive with Ag expressed on uninfected CD4+ cells. In this study, 11 of 14 asymptomatic HIV-1-infected homosexuals and hemophiliacs, but none of 17 uninfected homosexuals or heterosexuals, were found to have cytotoxic lymphocytes in blood that can lyse uninfected CD4+ T cells from humans and chimpanzees but not human B lymphoblastoid cells or mouse T cells. The cytotoxic PBL were concluded to be CTL rather than NK cells, with the phenotype being CD3+, TCR-1 alpha beta+, CD8+, CD4-, CD16- based on findings that PBL-mediated lysis of uninfected CD4+ cells was 1) blocked by a mAb to CD3, which inhibits CTL but not NK activity; 2) diminished by treatment of PBL with a mAb to CD8 and C, but not by treatment with mAb to CD4 or CD16 and C; and 3) blocked by mAb WT31 directed against the TCR-1 alpha beta. In contrast, PBL from HIV-1-infected chimpanzees, which to date have not developed AIDS, lacked detectable CTL lytic for uninfected CD4+ cells.     

CD8+ T cells from HIV-1-infected individuals inhibit acute infection by human and primate immunodeficiency viruses.

T lymphocytes expressing the CD8 surface antigen block HIV replication in CD4+ peripheral blood cells from HIV-infected individuals. We report here that CD4+ cells from HIV seronegative donors, when infected in vitro with HIV, also do not replicate virus when cocultured with CD8+ T cells from HIV-infected individuals. CD8+ cells from HIV-uninfected donors did not show this effect on virus replication. HLA-restriction of the antiviral response was not observed, and virus-containing cells were not eliminated from culture. The antiviral activity was broadly cross-reactive, as CD8+ cells from individuals infected only with HIV-1 suppressed the replication of diverse strains of HIV-1 and HIV-2, as well as the simian immunodeficiency virus. This ability of CD8+ cells to control HIV replication could play an important role in the maintenance of an asymptomatic state in HIV-infected individuals.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.     

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.     

What is the role of autophagy in HIV-1 infection?

HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.     

Importance of membrane fusion mediated by human immunodeficiency virus envelope glycoproteins for lysis of primary CD4-positive T cells

In established T-cell lines, the membrane-fusing capacity of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins mediates cytopathic effects, both syncytium formation and single-cell lysis. Furthermore, changes in the HIV-1 envelope glycoproteins are responsible for the increased CD4(+) T-cell-depleting ability observed in infected monkeys upon in vivo passage of simian-human immunodeficiency virus (SHIV) chimeras. In this study, a panel of SHIV envelope glycoproteins and their mutant counterparts defective in membrane-fusing capacity were expressed in primary human CD4(+) T cells. Compared with controls, all of the functional HIV-1 envelope glycoproteins induced cell death in primary CD4(+) T-cell cultures, whereas the membrane fusion-defective mutants did not. Death occurred almost exclusively in envelope glycoprotein-expressing cells and not in bystander cells. Under standard culture conditions, most dying cells underwent lysis as single cells. When the cells were cultured at high density to promote syncytium formation, the envelope glycoproteins of the passaged, pathogenic SHIVs induced more syncytia than those of the respective parental SHIV. These results demonstrate that the HIV-1 envelope glycoproteins induce the death of primary CD4(+) T lymphocytes by membrane fusion-dependent processes.     

Lysis of cells infected with HIV-1 by human lymphocytes targeted with monoclonal antibody heteroconjugates

The present study was undertaken to determine whether human PBL can be specifically focused to lyse cells infected with HIV-1 by mAb heteroconjugates that can bridge target and effector cells. A mAb directed against the central portion of HIV-1 glycoprotein gp110 was chemically cross-linked to a mAb directed against the CD3/TCR complex or to a mAb directed against the CD16 Fc gamma-R expressed on large granular lymphocytes (LGL). HIV-1-infected cells, but not uninfected cells, were found to be lysed to a greater extent by PBL in the presence of the gp110 X CD3 or the gp110 X CD16 antibody heteroconjugate than in the presence of the single antibodies or a mixture of the mAb comprising the heteroconjugates. Pretreatment of PBL with anti-CD3 or IL-2 augments their ability to lyse HIV-1-infected cells in the presence of the heteroconjugates. Lysis by anti-CD3-activated PBL in the presence of the gp110 X CD3 heteroconjugate was found to be mediated by CD8+-enriched T cells, whereas lysis by IL-2-treated PBL in the presence of the gp110 X CD16 heteroconjugate is mediated by PBL enriched for CD16+ cells, which are primarily LGL. Furthermore, PBL from asymptomatic, HIV-1-infected seropositive donors were found to be functional in lysing HIV-1-infected cells in the presence of the antibody heteroconjugates. Such antibody heteroconjugates, which can target T cells or LGL to lyse HIV-1-infected cells, may be of prophylactic or therapeutic value in HIV-1-infected individuals.     

Few infected CD4+ T cells but a high proportion of replication-competent provirus copies in asymptomatic human immunodeficiency virus type 1 infection.

The virus load in CD4+ T cells from six asymptomatic human immunodeficiency virus type 1 (HIV-1)-infected individuals was determined by limiting-dilution analysis with a sensitive virus isolation procedure and the polymerase chain reaction (PCR). Both methods allowed detection of one HIV-1-infected cell among 10(5) uninfected cells. The number of provirus-containing CD4+ T cells was found to be 1 per 4,000 to 150,000 (median, 1 per 29,000), as determined by virus isolation and 1 per 2,500 to 26,000 (median, 1 per 12,000), as determined by PCR. Infected cells contained an average of 1 to 2 provirus copies, and a high proportion of the provirus copies (1 in 1 to 1 in 6; median, 1 in 2) were replication competent. The results suggest that only a few CD4+ T cells are likely to be lost as a direct consequence of the presence of HIV-1 in infected cells in asymptomatic individuals and that additional mechanisms may contribute to the depletion of CD4+ T cells observed in vivo.     

High major histocompatibility complex-unrestricted lysis of simian immunodeficiency virus envelope-expressing cells predisposes macaques to rapid AIDS progression

Before the development of virus-specific immune responses, peripheral blood mononuclear cells (PBMC) from uninfected rhesus monkeys and human beings have the capacity to lyse target cells expressing simian immunodeficiency virus (SIV) or human immunodeficiency virus-1 (HIV) envelope (gp130 and gp120) antigens. Lysis by naive effector cells does not require major histocompatibility complex (MHC)-restricted antigen presentation, is equally effective for allogeneic and xenogeneic targets, and is designated MHC-unrestricted (UR) lysis. UR lysis is not sensitive to EGTA and does not require de novo RNA or protein synthesis. Several kinds of envelope-expressing targets, including cells that poorly express MHC class I antigens, can be lysed. CD4(+) effectors are responsible for most of the lytic activity. High lysis is correlated with high expression of HIV or SIV envelope, specifically, the central one-third of the gp130 molecule, and lysis is completely inhibited by a monoclonal antibody against envelope. Our work extends observations of human lymphocytes expressing HIV gp120 to the SIV/rhesus monkey model for AIDS. Additionally, we address the relevance of UR lysis in vivo. A survey of PBMC from 56 uninfected rhesus monkeys indicates that 59% of the individuals had peak UR lytic activity above 15% specific lysis. Eleven of these monkeys were subsequently infected with SIV. Animals with UR lytic activity above 15% specific lysis were predisposed to more rapid disease progression than animals with low UR lytic activity, suggesting a strong correlation between this form of innate immunity and disease progression to AIDS.     

Autophagy and CD4+ T lymphocyte destruction by HIV-1

The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.     

The anti-idiotypic antibody 1F7 selectively inhibits cytotoxic T cells activated in HIV-1 infection

Circulating CD8+ T lymphocyte numbers rise substantially following infection with HIV-1. This expanded CD8+ T cell population includes HIV-specific CTL and CTL that kill activated uninfected CD4+ lymphocytes. Experimental, epidemiological and clinical evidence supports the possibility that expansion of CD8+ CTL contributes to CD4+ T cell depletion and disease progression in human HIV infection. Therefore, modulation of CD8+ T cell numbers or of certain CD8+ CTL activated in HIV-infected individuals may be beneficial. It was found that 1F7, a mAb against an idiotype common to anti-HIV and anti-simian immunodeficiency virus (SIV) antibodies, selectively inhibited both anti-HIV CTL and CTL against uninfected CD4+ T cells. Alloantigen-specific CTL and NK cells from either HIV-infected individuals or controls were unaffected by 1F7. Prolonged incubation of CD8+ T cells from HIV-infected individuals with 1F7 induces apoptosis, which was shown to be reflected functionally in reduced total CTL activity and in especially reduced CTL activity against uninfected CD4+ lymphocytes. The selective reactivity of 1F7 with certain CD8+ CTL could be applied towards the modulation of CD8+ T cell responses involved in AIDS pathogenesis.     

The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells

mAb 60.3 and IB4 to CD18, the common beta-subunit of the human leukocytic cell adhesion molecule family, efficiently inhibit syncytium formation induced by the interaction of HIV type 1 (HIV-1)-infected monocytoid cells and CD4+ T cells. The antibodies also interfere with cellfree HIV-1 infection of U-937 clone 16 cells. Virus-induced aggregation of these cells and the subsequent syncytia formation leading to massive cell death are efficiently blocked, and the number of infected cells remains at a very low level, 2 to 5%, for the entire culture period. However, anti-CD18 mAb do not inhibit binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4. The results indicate participation of CD18, or of the protein complex CD11a-c/CD18, in addition to CD4, in the infection and cytopathic effect of HIV-1. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.     

Inhibition of T cell antigen receptor-dependent phosphorylation of CD4 in human immunodeficiency virus type 1 infected cells.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.     

Coexistence of fusion receptors for human T-cell leukemia virus type-I (HTLV-I) and human immunodeficiency virus type-1 (HIV-1) on MOLT-4 cells.

Human immunodeficiency virus type-1 (HIV-1) and human T-cell leukemia virus type-I (HTLV-I) have a similar tropism for target cell types, especially for CD4+ T cells. In this study, we provide evidence that receptors of these two viruses exist independently on the target cell. We established an HTLV-I-producing CD8+ T cell line (ILT-8M2) with a remarkable cell fusion capacity. When cocultured with MOLT-4 cells, ILT-8M2 cells induced giant syncytia more efficiently than any other tested HTLV-I-producer cell lines. In contrast to other HTLV-I-producers, ILT-8M2 cells were minimally susceptible to cytopathic effects of HIV-1 due to very low expression of CD4, although they were able to be persistently infected by HIV-1. The indicator MOLT-4 cells are known to respond well to HIV-1-induced cell fusion, but they lose this ability if they become persistently infected with HIV-1 because of the reduction of CD4 receptor expression. ILT-8M2 was, however, still capable of inducing syncytia with the MOLT-4 cells persistently infected by HIV-1 (MOLT-4/IIIB). This syncytium formation was dependent on the HTLV-I-envelope, as it was inhibited by HTLV-I-positive human sera or a monoclonal antibody to HTLV-I gp46 but not by monoclonal antibodies to HIV-1 gp120 or CD4. Moreover, ILT-8M2 cells persistently infected by HIV-1 (ILT-8M2/IIIB) induced both HTLV-I- and HIV-1-mediated syncytia with uninfected MOLT-4 cells. These results suggest that HTLV-I induces cell fusion utilizing receptors on the target cells independent of HIV-1-receptors.     

Stilbene disulfonic acids. CD4 antagonists that block human immunodeficiency virus type-1 growth at multiple stages of the virus life cycle

The stilbene disulfonic acids 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid and, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid bound the variable-1 immunoglobulin-like domain of CD4 on JM cells. The interaction blocked the binding of the anti-CD4 monoclonal antibody OKT4A and the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1). DIDS inhibited the acute infection of CD4+ cells by HIV-1 with a potency (IC50 approximately 30 microM) similar to that which blocked gp120 binding (IC50 approximately 20 microM) to the cellular antigen. Pretreating uninfected CD4+ C8166 cells with DIDS blocked their fusion with chronically infected gp120+ cells. DIDS covalently and selectively modified lysine 90 of soluble CD4 and abolished the gp120-binding and antiviral properties of the recombinant protein. When added to cells productively infected with HIV-1, DIDS blocked virus growth and cleared cultures of syncytia without inhibiting cellular proliferation. The stilbene disulfonic acids are a novel class of site-specific CD4 antagonists that block multiple CD4-dependent events associated with acute and established HIV-1 infections.     

Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins.

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.     

CD4-independent infection by human immunodeficiency virus type 1 after phenotypic mixing with human T-cell leukemia viruses.

Although human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome and related disorders, it has been suggested that viral cofactors may accelerate the progression of the disease. We present evidence that human T lymphoid cells productively coinfected by HIV type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) or HTLV-II generate a progeny of phenotypically mixed viral particles that allow the penetration of HIV-1 into previously nonsusceptible CD4- human cells, including mature CD8+ T lymphocytes, B lymphoid cells, epithelial cells, and skeletal muscle cells. The infection is independent of the major HIV-1 receptor, (i.e., the CD4 glycoprotein) since OKT4a, a neutralizing anti-CD4 monoclonal antibody, fails to block the penetration of HIV-1. Similarly, infection is not inhibited by monoclonal antibody M77, directed toward the neutralizing loop of the gp120 envelope glycoprotein of HIV-1. In contrast, pretreatment of the virus stock with HTLV-I-neutralizing human serum completely abolishes the penetration of phenotypically mixed HIV-1 into CD4- cells. These results suggest that HTLV-I or HTLV-II may increase the pathogenicity of HIV-1 by broadening the spectrum of its cellular tropism and, thus, favoring its spread within the organism of coinfected hosts.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.