Interleukin-15 is a potent survival factor in the prevention of spontaneous but not CD95-induced apoptosis in CD4 and CD8 T lymphocytes of HIV-infected individuals. Correlation with its ability to increase BCL-2 expression

IL-15 shares many biological properties with IL-2, a cytokine whose administration to HIV-infected individuals has been effective in enhancing depleted CD4 T lymphocyte numbers. The present study examined whether exogenous IL-15 could protect lymphocytes of HIV-infected individuals from spontaneous apoptosis, associated with growth factor deprivation, and CD95-induced apoptosis, which is believed to play a major role in T lymphocyte loss and HIV pathogenesis. Although IL-15, like IL-2, failed to inhibit CD95-induced lymphocyte apoptosis in vitro, IL-15 was found to act as a potent survival factor in the prevention of spontaneous apoptosis. The greater enhancement of lymphocyte survival, promoted by IL-15 as compared with IL-2 when used at an equivalent concentration, was associated with higher up-regulation of bcl-2 expression. In addition, IL-15 was more potent than IL-2 in stimulating lymphocyte proliferation. Despite the strong ability of IL-15 to promote both lymphocyte survival and proliferation, the increases in representation and total numbers of viable cells induced by IL-15 were not higher than those induced by IL-2. This appears to be associated with the greater ability of IL-15 to activate lymphocytes and increase their apoptosis-susceptibility. Therefore, lymphocyte loss occurring by growth factor deprivation in HIV infection may be potentially prevented by IL-15, although its benefits for survival need to be closely assessed against its ability to augment lymphocyte activation.     

Mechanisms of HIV envelope-induced T lymphocyte apoptosis

Infection by the human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4 T lymphocytes, which leads to dysfunction of the immune system. Although a variety of mechanisms may contribute to the gradual T cell decline that occurs in HIV-infected patients, abnormal apoptosis of infected or bystander T lymphocytes is an important event leading to immunodeficiency. The HIV envelope glycoprotein plays a crucial role in HIV associated apoptosis through both death receptor-mediated and mitochondria-dependent pathways. This review summarizes current knowledge of Env-mediated T lymphocyte apoptosis.     

Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.     

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase-independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathione levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS-, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL-2 production. Overall, our data demonstrate that HS- reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL-2, therefore de-activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti-inflammatory effects of sulfur compounds.     

Mechanisms of HIV envelope-induced T lymphocyte apoptosis

Infection by the human immunodeficiency virus (HIV) is characterized by a progressive depletion of CD4 T lymphocytes, which leads to dysfunction of the immune system. Although a variety of mechanisms may contribute to the gradual T cell decline that occurs in HIV-infected patients, abnormal apoptosis of infected or bystander T lymphocytes is an important event leading to immunodeficiency. The HIV envelope glycoprotein plays a crucial role in HIV associated apoptosis through both death receptor-mediated and ...     

Differential expression of interleukin-2 and gamma interferon in human immunodeficiency virus disease

Subnormal T-cell production of interleukin-2 (IL-2) in human immunodeficiency virus (HIV) disease has been described; however, it is not clear whether failure to synthesize IL-2 represents a selective or global defect in T-cell cytokine production. We evaluated the intracellular production of gamma interferon (IFN-gamma) and IL-2 in CD4(+) cells that were stimulated with staphylococcal enterotoxin B or cytomegalovirus antigen. Strikingly, IFN-gamma and IL-2 are differentially regulated in T cells of HIV-infected patients such that the numbers of CD69(+) cells or IFN-gamma-positive cells that make IL-2 are proportionally decreased in CD4(+) T cells from HIV-infected patients. These findings demonstrate a selective defect in IL-2 production and suggest that enumeration of IFN-gamma-producing cells in response to T-cell receptor stimulation, while providing some estimate of antigen-reactive cell frequency, may not reflect or predict "normal" T-cell function in HIV-infected patients.     

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.     

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.Copies of articles are available through ISI Document Delivery Services c/o The Genuine Article, 3501 Market Street, Philadelphia, PA 19104.     

Tetrahydrobiopterin enhances apoptotic PC12 cell death following withdrawal of trophic support

(6R)-Tetrahydro-l-biopterin (BH(4)) is the rate-limiting cofactor in the production of catecholamine and indoleamine neurotransmitters and is also essential for the synthesis of nitric oxide by nitric-oxide synthase. We have previously reported that BH(4) administration induces PC12 cell proliferation and that nerve growth factor- or epidermal growth factor-induced PC12 cell proliferation requires the elevation of intracellular BH(4) levels. We show here that BH(4) accelerates apoptosis in undifferentiated PC12 cells deprived of serum and in differentiated neuron-like PC12 cells after nerve growth factor withdrawal. Increased production of catecholamines or nitric oxide cannot account for the enhancement of apoptosis by BH(4). Furthermore, increased calcium influx by exogenous BH(4) administration is not involved in the BH(4) proapoptotic effect. Our data also argue against the possibility that increased oxidative stress, due to BH(4) autoxidation, is responsible for the observed BH(4) effects. Instead, they are consistent with the hypothesis that BH(4) induces apoptosis by increasing cell cycle progression. Elevation of intracellular BH(4) during serum withdrawal increased c-Myc (and especially Myc S) expression earlier than serum withdrawal alone. Furthermore, N-acetylcysteine and the cyclin-dependent kinase inhibitor olomoucine ameliorated the BH(4) proapoptotic effect. These data suggest that BH(4) affects c-Myc expression and cell cycle-dependent events, possibly accounting for its effects on promoting cell cycle progression or apoptosis.     

In vivo inhibition of NF-kappa B in T-lineage cells leads to a dramatic decrease in cell proliferation and cytokine production and to increased cell apoptosis in response to mitogenic stimuli, but not to abnormal thymopoiesis.

To understand the role of NF-kappa B complexes in T cell development and activation, we have generated transgenic mice in which RelA and c-Rel complexes were selectively inhibited in the T-lineage cells by specific expression of a trans-dominant form of I kappa B alpha. Transgene expression did not affect the thymic development, but led to lowered numbers of splenic T cells and to a dramatic decrease in the ex vivo proliferative response of splenic T lymphocytes. Analysis of IL-2 and IL-2R alpha expression demonstrated that the perturbation of the proliferation response was not attributable to an abnormal expression of these genes. In contrast, expression of IL-4, IL-10, and IFN-gamma was strongly inhibited in the transgenic T cells. The proliferative deficiency of the transgenic T cells was associated with an increased apoptosis. These results point out the involvement of NF-kappa B/Rel family proteins in growth signaling pathways by either regulating proteins involved in the IL-2 signaling or by functionally interfering with the cell cycle progression.     

Perturbations of cell cycle control in T cells contribute to the different outcomes of simian immunodeficiency virus infection in rhesus macaques and sooty mangabeys

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

CD4 epitope masking by gp120/anti-gp120 antibody complexes. A potential mechanism for CD4+ cell function down-regulation in AIDS patients.

The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency.     

Immunologic effects of interleukin 2 in primary immunodeficiency diseases

Five children with primary deficiencies of T cell function were studied to assess the effects of highly purified exogenous Interleukin 2 (IL 2) on their in vitro T cell responses. The lymphocytes from one child with Nezelof's T cell deficiency demonstrated absence of endogenous IL 2 production and improved proliferative responses to mitogen or alloantigen in the presence of exogenous IL 2. Moreover, during in vitro mixed lymphocyte culture in the presence of exogenous IL 2, his lymphocytes were able to develop into cytotoxic effector cells. A second child with Nezelof's syndrome demonstrated a different type of defect. The lymphocytes from this child had less impairment of endogenous IL 2 production. Although IL 2 increased the proliferation of his cells in response to PHA, similar augmentation was not seen after stimulation with OKT3 or alloantigen. In cell-mediated cytotoxicity assays, after mixed lymphocyte culture, natural killer-like activity was strongly boosted in the cultures that contained IL 2, but T cell-mediated cytotoxicity was not. The lymphocytes from three patients with severe combined immunodeficiency did not show improved proliferative responses in the presence of IL 2. Thus, only one of the five patients demonstrated the combination of defective endogenous IL 2 production, but preservation of the ability to respond appropriately to exogenous IL 2. This child may therefore have suffered from a T cell defect pathophysiologically similar to that seen in nude or aged mice.     

Early disturbance of the circadian rhythm of T and B lymphocytes in human immunodeficiency virus infection

Circadian rhythms in circulating B and T (CD3, CD4, CD8) lymphocyte subsets and in plasma cortisol were studied in 13 HIV-infected men and 14 healthy male controls. The circadian maximum (acrophase) of plasma cortisol was similar in both groups, approximately 8.00 A.M., however, a statistically significant increase was found in the 24 hour-mean value (mesor) of infected patients as compared to healthy controls. Circadian rhythms were statistically validated in all lymphocyte subsets of healthy controls, whereas, large alterations were found in patients with acquired immunodeficiency syndrome (AIDS), already in asymptomatic infected individuals. The alterations concern the mesor and the amplitude for B and CD4 lymphocytes and all cycle parameters for CD3 and CD8 lymphocytes.     

Characteristics of the specific cell-mediated immune response in human immunodeficiency virus infection.

The human immunodeficiency virus (HIV)-specific lymphocyte proliferation response was determined for 40 persons at different stages of HIV infection. The specific response to purified HIV virion antigens from strain HTLV-IIIB was poor, occurred in only 9 of the 40 subjects, was not improved with the addition of interleukin-2, and was more frequent in symptom-free individuals (46%) than in patients with lymphadenopathy syndrome (10%). Reactivity to subcomponent p24 was better than that to whole HIV; reactivity was present in five of six infected persons and increased with the addition of exogenous interleukin-2. Reactivities to subcomponents (g)p41 and gp120 were also measured. This is the first evidence of a specific cell-mediated immune response to HIV antigen in HIV-infected persons. Monkeys immunized with purified HIV or with purified p24 displayed cellular immunoreactivity both to whole HIV and to subcomponents. In contrast to the poor reactivity to HIV antigen, the lymphocytes of the patients had good specific cell proliferation responses to cytomegalovirus and herpes simplex virus challenge and a normal response to the addition of phytohemagglutinin. The results suggest a functional defect in peripheral lymphocytes of some HIV-infected individuals on the basis of their response to whole HIV antigen and a better response to gag protein.     

Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes.

Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.     

Primary human T lymphocytes engineered with a codon-optimized IL-15 gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous cytokine

IL-15 is a common gamma-chain cytokine that has been shown to be more active than IL-2 in several murine cancer immunotherapy models. Although T lymphocytes do not produce IL-15, murine lymphocytes carrying an IL-15 transgene demonstrated superior antitumor activity in the immunotherapy of B16 melanoma. Thus, we sought to investigate the biological impact of constitutive IL-15 expression by human lymphocytes. In this report we describe the generation of a retroviral vector encoding a codon-optimized IL-15 gene. Alternate codon usage significantly enhanced the translational efficiency of this tightly regulated gene in retroviral vector-transduced cells. Activated human CD4+ and CD8+ human lymphocytes expressed IL-15Ralpha and produced high levels of cytokine upon retroviral transduction with the IL-15 vector. IL-15-transduced lymphocytes remained viable for up to 180 days in the absence of exogenous cytokine. IL-15 vector-transduced T cells showed continued proliferation after cytokine withdrawal and resistance to apoptosis while retaining specific Ag recognition. In the setting of adoptive cell transfer, IL-15-transduced lymphocytes may prolong lymphocyte survival in vivo and could potentially enhance antitumor activity.     

Cutting edge: class I presentation of host peptides following HIV infection

Class I MHC molecules bind intracellular peptides for presentation to cytotoxic T lymphocytes. Identification of peptides presented by class I molecules during infection is therefore a priority for detecting and targeting intracellular pathogens. To understand which host-encoded peptides distinguish HIV-infected cells, we have developed a mass spectrometric approach to characterize HLA-B*0702 peptides unique to or up-regulated on infected T cells. In this study, we identify 15 host proteins that are differentially presented on infected human T cells. Peptides with increased expression on HIV-infected cells were derived from multiple categories of cellular proteins including RNA binding proteins and cell cycle regulatory proteins. Therefore, comprehensive analysis of the B*0702 peptide repertoire demonstrates that marked differences in host protein presentation occur after HIV infection.