Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.     

Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication

Nitric oxide (NO) has been implicated in certain immunopathogenetic mechanisms during the course of infection with human immunodeficiency virus (HIV). We have evaluated the levels of NO release and lymphocyte apoptosis in peripheral blood mononuclear cell (PBMC) cultures from HIV-1 infected subjects and healthy controls. We have also examined these 2 parameters in parallel cultures maintained under conditions where either NO synthesis was inhibited or high level of NO was present. Nitrite contents in culture supernatants were measured as the stable end products of the released NO. Levels of spontaneous apoptosis and activation-induced cell death (AICD) by anti-CD3 or by phytohemagglutinin were evaluated using flow cytometry. Additional experiments were also aimed at addressing a potential link between NO synthesis and HIV-1 replication in human monocyte-derived macrophages (MDMs). Acutely infected MDMs with HIV-1Bal were maintained in culture, without any additional activation signal, for a period of 14 days. Nitrites in the supernatants and mRNA accumulation of the inducible NO synthase (iNOS) in infected cells were assessed over the whole culture period. In addition, the effect of blocking NO synthesis during and after infection of MDMs, using an inhibitor of NO, was evaluated on the level of viral replication as measured by the presence of P24 antigen in the supernatants. Similarly, the effect on HIV replication of high NO levels in MDM cultures, supplied by a donor of NO during the 24 h period of infection, was also studied. We conclude that no elevation in NO release could be detected in PBMC cultures from HIV-1 infected subjects and that modulation of NO content may slightly regulate the level of spontaneous lymphocyte apoptosis but not that of AICD. Infection of MDMs with HIV-1 does not seem to induce detectable NO release or iNOS mRNA accumulation. Similarly, neither inhibition of NO synthesis nor the presence of high NO levels during the infection period could modify the outcome of virus replication in macrophages.     

Production of monoclonal antibody to CD4 antigen and development of reagent for CD4+ lymphocyte enumeration

A hybridoma secreting monoclonal antibody (mAb) specific to CD4 protein was generated. This monoclonal antibody, named MT4, was proved to be specific to CD4 protein as it reacted with CD4-DNA transfected COS cells, CD4+ cell lines and CD4+ lymphocytes. Furthermore, MT4 mAb inhibited the binding of standard CD4 monoclonal antibodies to CD4 proteins on CD4+ cells. To develop a home made reagent for CD4+ lymphocyte determination by flow cytometry, fluorescein isothiocyanate (FITC) was conjugated to MT4 mAb. To evaluate the developed reagent, 30 HIV infected and 30 healthy individuals were determined for CD4+ lymphocytes by using both a commercial Simultest reagent kit and home made FITC labeled MT4 mAb simultaneously. The study has shown that both percentages and absolute CD4+ lymphocyte counts obtained from both reagents were equivalent. The correlation coefficient for regression analysis was 0.995 and 0.996 for percentages and absolute CD4+ lymphocyte counts, respectively. The results suggest that home made FITC labeled MT4 reagent is an acceptable alternative reagent for monitoring CD4+ lymphocytes in blood samples by flow cytometry.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

The effect of in vitro human immunodeficiency virus infection on dendritic-cell differentiation and function

CD1a+ dendritic cells (DC) differentiate from a major population of nonadherent CD13(hi)lin- cells that appear when human cord blood CD34+ hematopoietic progenitor cells are cultured with stem-cell factor, granulocyte/macrophage (MA) colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha) for 5 days. CD13hilin- cells, which also comprise MA and granulocyte precursors, are CD4+ and can thus be targets of human immunodeficiency virus (HIV). Low replication was noted when these day 5 cells were infected with lymphotropic HIV-1LA1 (p24: < or = 4 ng/mL on day 8 postinfection [PI]), while high virus production occurred with MA-tropic HIV-1Ba-L, HIV-1Ada, or HIV-1-m-n. (p24: 50 to > or = 1,000 ng/mL). Strong cytopathicity (CPE) was then observed in nonadherent cells as in adherent MA. However, FACS analysis on day 7 PI showed that HIV did not affect differentiation of DC that survived CPE: apart from CD4 downmodulation related to HIV production, overall expression of CD40, CD80, and CD86 costimulatory molecules, and of HLA-DR, was unchanged relative to controls. At that time, the capacity of DC from HIV-infected cultures to stimulate the mixed leukocyte reaction was only altered less than 10-fold. Immunocytochemistry on day 7 PI showed that most HIV-infected cells were included in syncytia that were stained by anti-CD1a, anti-S100, and anti-CD14 antibodies, indicating that syncytia consisted of DC and cells of the MA lineage. Polymerase chain reaction analysis of FACS-sorted CD1a+ cells confirmed that they harbored then HIV DNA. Viral DNA was also detected in CD1a+ DC from noninfected cultures that had been exposed to HIV only after sorting. Therefore, we examined whether in infected cultures DC precursors were infected at the onset or if virus spread later from other infected cells to differentiated DC. This was answered by showing that, 24 hours postexposure to HIV, viral DNA was preferentially detected in day 5 sorted CD13hilin- versus CD13hilin- cells, and that it was found in the CD1a+ progeny of CD13(hi)lin- cells 48 hours later. In addition, HIV replication did not affect myeloid clonogenic progenitors in day 0 to day 7 PI cultures, although viral DNA was detected in colony-forming unit-granulocyte/macrophage (CFU-GM)/CFU-M colonies derived from day 3 and 7 PI cultures. Thus, precursors of DC and their progeny are susceptible to HIV in vitro, but, apart from CPE, the effect of virus production on DC differentiation or function is limited.     

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.     

Differential susceptibility to HIV-GP120-sensitized apoptosis in CD4+ T-cell clones with different T-helper phenotypes: role of CD95/CD95L interactions

The susceptibility of Th1 and Th2 cell clones to apoptosis following HIV-gp120/CD4 cross-linking and TCR activation was investigated. We show that only Th1 clones are susceptible to HIV-gp120-sensitized apoptosis, although both types of clones express similar levels of CD4 and bind similar amounts of recombinant gp120. Both types of clones, however, undergo apoptosis induced by CD95 cross-linking with agonistic monoclonal antibody (MoAb). Apoptosis induced by gp120 in the Th1 clones is inhibited by either an anti-CD95 neutralizing MoAb or an anti-CD95L neutralizing MoAb as well as by a specific interleukin-1 beta converting enzyme (ICE) inhibitor. When triggered to apoptosis by gp120, Th1 but not Th2 clones express both cell-associated and soluble CD95L. The CD95L produced by Th1 clones induces cell death, inhibitable by anti-CD95 neutralizing MoAb, of CD95 positive Jurkat cells. These data suggest that, like activation-induced apoptosis, HIV-gp120 sensitized apoptosis in Th1 clones occurs via CD95/CD95L interaction and that lack or insufficient production of CD95L is responsible, at least in part, for the resistance of Th2 clones to such apoptosis.     

Experimental study of the possible implications of some apoptosis mechanisms in the pathogenesis of HIV infection. 1. In vitro apoptotic death of peripheral blood lymphocytes

The human immunodeficiency virus (HIV) infection produces a gradual depletion of T-helper CD4+ lymphocytes as, surprisingly, a consequence of apoptosis of the uninfected lymphocytes. We suggested that this is the result of the action exerted by HIV inductors of apoptosis (for example, gp 120) in the absence of viral apoptosis suppressor, which confers protection to the infected cell. We intended to demonstrate this hypothesis within the framework of a complex study regarding the apoptosis mechanisms in HIV infection. We started this study by setting up an apoptosis model on HIV-negative peripheral blood lymphocytes (PBLs)* cultivated in vitro in the presence of dexamethasone (Dex). In this work we characterize this model morphologically and biochemically. Three unreported morphological changes observed by us--namely: I) fringing of nucleus with advancement of fringes up to the plasma membrane; II) segmentation and peripheral migration of condensed chromatin through a rotation movement; III) "flowering of the cell" consisting in the radial separation of the lymphocyte into centrally united "petals" with the tendency to form apically multiple apoptotic bodies--completed the classical morphology of the apoptosis phenomenon. The apoptotic death was confirmed by the oligonucleosomal (multiples of 200 bp) and mononucleosomal fragmentation of DNA isolated from lymphocytes.     

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4+ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4+ T cell attrition in vivo.     

Differential regulation of translation and eIF4E phosphorylation during human thymocyte maturation

Activation of peripheral blood T cells by cross-linking of CD3 results in a rapid and substantial rise in translation rates and proliferation, which coincides with an increase in the cap-binding protein, eIF4E activity. In contrast, immature CD4+ CD8+ double-positive (DP) thymocytes undergo apoptosis in response to anti-CD3 mAb. We have investigated translation initiation in the response of immature thymocytes to activating signals. Activation by anti-CD3 + anti-CD4 of immature CD4+ CD8+ DP thymocytes results in a rapid decrease in protein synthesis. In contrast, similar treatment of CD4+ or CD8+ single-positive (SP) thymocytes results in an increase in protein synthesis. The rate of protein synthesis is linked to the phosphorylation status of eIF4E. Following anti-CD3 + anti-CD4 stimulation, eIF4E phosphorylation strongly decreases in immature DP thymocytes, whereas it increases in mature SP thymocytes. The expression of 4E-BP2, a specific repressor of eIF4E function, is high in DP cells but decreases during maturation, raising the possibility of a role for 4E-BP2 in repressing eIF4E phosphorylation. These data provide evidence for differential regulation of the translational machinery during T cell development.     

Productive infection of neonatal CD8+ T lymphocytes by HIV-1

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.     

Requirement of CD28-CD86 co-stimulation in the interaction between antigen-primed T helper type 2 and B cells

The interaction between CD28 and its ligands, CD80 and CD86, is crucial for an optimal activation of antigen-specific T cells. However, the requirement of CD80 or CD86 co-stimulation in Th2 cell differentiation and activation is controversial. Freshly isolated murine CD4+ and CD8+ T cells were incubated with P815 transfectants expressing a similar level of either CD80 or CD86 in the presence of anti-CD3 mAb. Both CD80 and CD86 co-stimulated the proliferation of CD4+ and CD8+ T cells at comparable time-kinetics and magnitude, but CD86 alone was able to co-stimulate IL-4 and especially IL-10 production in CD4+ T cells. In typical Th2-dependent immune responses elicited by Nippostrongylus brasillensis infection, the anti-CD86 mAb treatment but not the anti-CD80 mAb treatment efficiently inhibited antigen-specific IgE and IgG1 production, which was accompanied with the reduced IL-4 production. Our results suggest that CD86 co-stimulation plays a dominant role not only in the primary activation of Th2 cells but also in the secondary interaction between antigen-primed Th2 cells and B cells.     

Potent and broad antitumor effects of DX-8951f, a water-soluble camptothecin derivative, against various human tumors xenografted in nude mice

Previous studies have shown that in vitro culture of human CD4+ T cells with antibodies to CD3 and CD28 immobilized on beads induced an antiviral effect to HIV-1 infection. Herein, we have used CD4+ T cells from nonhuman primates to address issues critical for use of such cells for therapy and immune reconstitution of humans and nonhuman primates infected with HIV and simian immunovirus (SIV). These studies include definition of the kinetics of the antiviral effect, the relative stability of the acquired phenotype, and whether such activated and expanded CD4+ T cells retain their immune function. Results of our studies show that antiviral effect is induced rapidly following activation with anti-CD3/CD28-coated beads. Additionally, the antiviral effect is not stable in these cells and requires continuous culture with anti-CD3/CD28 beads. Removal of CD4+ T cells from anti-CD3/CD28 stimulation renders these cells susceptible to infection, demonstrating that the resistant phenotype is not stable in these cultures. However, anti-CD3/CD28 expanded CD4+ T cells do retain immune function. Thus, although these findings imply a note of caution for therapeutic strategies aimed at providing patients with virus-resistant CD4+ T cells, the present study suggests that transfusion of such cells with retained immune function may have immune restoration capability.     

Reduction of fecal contamination of street-vended beverages in Guatemala by a simple system for water purification and storage, handwashing, and beverage storage

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

Increased CD4-positive monocytes in HIV-infected haemophilic patients

The monocyte-macrophage system is known to play a central role in HIV infection, and expression of CD4 on the surface of monocytes/macrophages is important, since this molecule is a key factor for the entrance of HIV into susceptible cells. In this paper we evaluated the expression of CD4 in monocytes of haemophilic patients (He) who had been infected with HIV (HIV + He) through transfusion of contaminated plasma concentrates. Thirty seropositive patients (HIV + He), 10 seronegative He patients (HIV-He) and 20 voluntary normal blood donors were studied. Phenotypic evaluation of monocytes was performed by flow cytometry of peripheral blood stained with anti-CD45, -CD3, -CD4 and -CD14 monoclonal antibodies. The percentage of CD4 monocytes was increased in all HIV+ patients groups, but it was highest in those belonging to Groups III and IV A of the CDC classification. Furthermore, the median of fluorescence intensity of CD4+ monocytes from individual patients was shifted to the right, indicating expression of increased numbers of CD4 molecules on the cell membrane of monocytes. This could in turn favour HIV infection and viral persistence, facilitating in vivo dissemination of the virus.     

Production of HIV-1 by human B cells infected in vitro: characterization of an EBV genome-negative B cell line chronically synthetizing a low level of HIV-1 after infection

Human immunodeficiency virus type 1 (HIV-1) has tropism for helper T lymphocytes and cells of the monocyte/ macrophage lineages. HIV-1 can also infect other cell types, including B cells. We show here that 10% of fresh circulating B cells from HIV-1-seronegative donors (i) express the CD4 receptor and CCR5 and CXCR4, two recently described coreceptors for HIV-1 and (ii) are permissive to HIV-1 with de novo proviral DNA integration following ex vivo infection by either SI (syncytium-inducing) or NSI (non-syncytium-inducing) isolates. To get further information on the interaction between HIV and B cells, the susceptibility of several EBV-positive or -negative B cell lines to infection by SI and NSI isolates was checked. Following infection of an EBV- CD4+ CXCR4+ CCR5- B cell line (DG75) by an SI HIV-1 isolate, we obtained a cell line which chronically produced low-level infectious HIV-1 for 2 years (HIV-DG75). Immunocytochemical data, combined with in situ PCR data, established that HIV-DG75 cells consist of at least three populations uninfected cells, infected virus-producing cells, and infected but nonproducing cells. Moreover, HIV-DG75 cells which express p24 antigen do not go into apoptosis, contrary to T lymphocytes. We infer from these results that B cells could constitute a reservoir of infectious virus in infected patients.     

Activation-induced NK cell death triggered by CD2 stimulation

Both T cells and natural killer (NK) cells express CD2, the target of an alternative activation pathway that induces the proliferation of both cell types. The mitogenic response to CD2 ligation requires the co-expression of CD3:TCR in T cells and FcgammaRIII in NK cells, suggesting that these receptors are involved in transducing the response initiated by CD2. The ability of FcgammaRIII to trigger the activation-induced death of IL-2-primed NK cells led us to investigate the potential for CD2 to trigger activation-induced NK cell death. Our results reveal that the same anti-CD2 monoclonal antibodies (mAb) that activate freshly isolated NK cells induce apoptosis in IL-2-primed NK cells. CD2-induced apoptosis results in chromatin condensation, DNA fragmentation and cleavage of caspase-3. Activation-induced NK cell death triggered by CD2 ligation is extremely rapid (DNA fragmentation is first observed at 90 min) and it is not inhibited by neutralizing antibodies reactive with TNF-alpha or Fas ligand. Whereas mAb reactive with distinct CD2 epitopes (i.e. T11.1, T11.2, and T11.3) are required for activation-induced T cell death, mAb reactive with a single CD2 epitope are sufficient for activation-induced NK cell death. The ability of CD2, CD16, and CD94 to induce apoptosis in IL-2-primed lymphocytes suggests that cytokine priming changes the response to a signaling cascade that is common to each of these activation receptors.     

Induction of lymphomonocyte activation by HIV-1 glycoprotein gp120. Possible role in AIDS pathogenesis

Dysfunction of cytokine secretion pattern has been suggested to play a central role in the immunopathogenesis of HIV infection. In fact a shift of T helper cell functions from a Th1-type to TH0- or TH2-type has been observed in HIV-1 infected subjects undergoing disease progression. The inhalance of cytokine network is accompanied by persistent activation of the immune system, impaired ability to mount a proper activation response (anergy), and priming to apoptosis. Extensive investigation during the last decade has been conducted on the influence of HIV-1 gp120 or of its precursor gp160 on several lymphocyte and monocyte functions. Gp120 is able to rise intracellular calcium concentration and to induce the formation of inositol triphosphate, can block mitogen- or antigen-driven T cell activation, can induce altered cytokine production by activated PBMC subpopulations, determines impaired cytotoxicity and chemotactic response to antigens, interferes with the activity of antigen presenting cells, enhances or induces apoptosis, stimulates polyclonal B cell activation and induces or up-modulates a number of cytokines, including IL-6. TNF, IL-1-alpha and -beta, IL-10 and IL-8. Furthermore, both IFN-alpha and -gamma, as well as several markers of IFN activity, such as beta 2-microglobulin and neopterin, are induced in gp120-stimulated PBMC. However, neither IL-4 (Th2-type) nor IL-2 (Th1-type), nor DNA synthesis are activated by gp120. On the other hand gp120-stimulated PBMC express increased IL-2 receptors, and can be induced by exogenous IL-2 to proliferate, suggesting that they are in a state of at least partial activation. According to this hypothesis, other activation markers, both early (such as CD69), and late (such as CD45RO and CD71), are induced by gp120, but this even partial activation does not lead to the ability of PBMC to support productive infection by HIV-1, unless in the presence of exogenous IL-2. The HIV-induced cytokines can influence HIV infection either directly, by up- or down-modulating virus replication, or indirectly, by modulating the expression of cellular molecules. In fact, during the budding process, the HIV envelope captures a number of cell membrane proteins, including cytokine receptors such as IL-2R, adhesion molecules such as LFA-1, ICAM-1, -2, HLA Class I and II, as well as cell lineage markers. Gp120-induced cytokines, particularly IFN-gamma, upmodulate the cellular expression of intercellular adhesion molecules, such as ICAM-1. We have shown that the IFN-gamma-driven increase of the expression of ICAM-1 by cells chronically infected with HIV-1 can be transmitted to the virus progeny, resulting in phenotypic alteration of the virus, and leading to the expansion of its host cell spectrum to CD4-negative cells expressing the appropriate ligands, i.e. LFA-1. Intercellular adhesion molecules are also involved in the cell-mediated transmission of HIV infection, and the increased ICAM-1 expression induced by IFN-gamma determines a stimulation of the transmission of HIV from abortively infected endothelial cells to permissive CD4 lymphocytes. On the whole, these data indicate that HIV, or its soluble products such as gp120, can modify several PBMC functions, by inducing a number of cytokines and a partial state of immune activation. It is possible that the gp120-driven changes of PBMC functions are not only an epiphenomenon of HIV infection, but rather, it is likely that they can participate in the immunopathological events responsible for disease progression.     

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.