Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists

Human immunodeficiency syndrome (HIV) infection leads to a progressive loss of T-cell-mediated immunity associated with T-cell apoptosis. We report here that CD4+ and CD8+ T cells from HIV-1-infected persons are sensitive to Fas (CD95/APO-1)-mediated death induced either by an agonistic anti-Fas antibody or by the physiologic soluble Fas ligand, although showing no sensitivity to tumor necrosis factor alpha-induced death. CD4+ and CD8+ T-cell apoptosis induced by Fas ligation was enhanced by inhibitors of protein synthesis and was prevented either by a soluble Fas receptor decoy or an antagonistic anti-Fas antibody. Fas- mediated apoptosis could also be prevented in a CD4+ or CD8+ T-cell- type manner (1) by several protease antagonists, suggesting the involvement of the interleukin-1beta (IL-1beta)-converting enzyme (ICE)- related cysteine protease in CD4+ T-cell death and of both a CPP32- related cysteine protease and a calpain protease in CD8+ T-cell death; and (2) by three cytokines, IL-2, IL-12, and IL-10, that exerted their effects through a mechanism that required de novo protein synthesis. Finally, T-cell receptor (TCR)-induced apoptosis of CD4+ T cells from HIV-infected persons involved a Fas-mediated death process, whereas TCR stimulation of CD8+ T cells led to a different Fas-independent death process. These findings suggest that Fas-mediated T-cell death is involved in acquired immunodeficiency syndrome (AIDS) pathogenesis and that modulation of Fas-mediated signaling may represent a target for new therapeutic strategies aimed at the prevention of CD4+ T-cell death in AIDS.     

Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing interferon-gamma and tumor necrosis factor- alpha secretion

We have recently shown that, in unfractioned peripheral blood mononuclear cells (PBMCs), the cross-linking of CD4 molecules (CD4XL) is sufficient to induce T-cell apoptosis. However, the underlying mechanism for the CD4XL-mediated T-cell apoptosis is largely unknown. Several recent studies have shown that Fas antigen (Ag), a cell-surface molecule, mediates apoptosis-triggering signals. We show here that cross-linking of CD4 molecules, induced either by anti-CD4 monoclonal antibody (MoAb) Leu3a or by human immunodeficiency virus-1 (HIV-1) envelope protein gp160, upregulates Fas Ag expression as well as Fas mRNA in normal lymphocytes. Addition of the tyrosine protein kinase inhibitor genistein or of the immunosuppressive agent cyclosporin A abrogated these effects. The upregulation of Fas Ag closely correlated with apoptotic cell death, as determined by flow cytometry. In addition, CD4XL resulted in the induction of interferon-gamma (IFN- gamma) and tumor necrosis factor-alpha (TNF-alpha) in the absence of interleukin-2 (IL-2) and IL-4 secretion in PBMCs. Both INF-gamma and TNF-alpha were found to contribute to Fas Ag upregulation and both anti- IFN-gamma and anti-TNF-alpha antibodies blocked CD4XL-induced Fas Ag upregulation and lymphocyte apoptosis. These findings strongly suggest that aberrant cytokine secretion induced by CD4XL and consequent upregulation of Fas Ag expression might play a critical role in triggering peripheral T-cell apoptosis and thereby contribute to HIV disease pathogenesis.     

Epstein-Barr virus induces Fas (CD95) in T cells and Fas ligand in B cells leading to T-cell apoptosis.

Epstein-Barr virus (EBV) acute infectious mononucleosis (AIM) is characterized by transient immunosuppression in vivo and increased T-cell apoptosis after ex vivo culture of AIM peripheral blood mononuclear cells. We undertook experiments to test whether EBV or purified virion envelope glycoprotein gp350 could contribute to Fas-mediated T-cell apoptosis. Our in vitro results indicate that EBV increased Fas expression in CD4(+) T cells and Fas ligand (FasL) expression in B cells and macrophages. Purified gp350 was also shown to significantly increase CD95 expression in CD4(+) T cells. When T-cell CD95 was cross-linked, EBV-stimulated T cells underwent apoptosis. The induction of T-cell CD95 by EBV followed by CD95 cross-linking with anti-CD95 monoclonal antibody resulted in a loss in the number of T cells responding to the T-cell mitogens, anti-CD3 antibody, and interleukin-2. These results indicate that, in addition to serving as a principal ligand for the attachment of virus to target cells, gp350 may also act as an immunomodulatory molecule that promotes T-cell apoptosis.     

Inhibition of CD4 cross-linking-induced lymphocytes apoptosis by vesnarinone as a novel immunomodulating agent: vesnarinone inhibits Fas expression and apoptosis by blocking cytokine secretion

Evidence is accumulating that T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals show accelerated cell death through apoptosis. We have recently demonstrated that the cross-linking of CD4 molecules (CD4XL) results in death of normal peripheral T cells through apoptosis and imbalanced cytokine secretion (ie, induction of tumor necrosis factor-alpha [TNF-alpha] and interferon-gamma [IFN-gamma] in the absence of interleukin-2 [IL-2] or IL-4 secretion). These upregulated cytokines (TNF-alpha/IFN-gamma) largely contributed to upregulation of the apoptosis-inducing cell surface molecule, Fas (APO- 1/CD95) and apoptosis induction. The present study investigated the effect of vesnarinone as a novel immunomodulating agent on CD4XL- induced T-cell apoptosis. The addition of vesnarinone to peripheral blood mononuclear cells (PBMC) significantly inhibited CD4XL-induced lymphocyte apoptosis. This apoptosis-inhibitory effect of vesnarinone was associated with the blocking of CD4XL-induced TNF-alpha IFN-gamma secretion and of Fas antigen upregulation. However, vesnarinone did not block effects of exogenously supplemented TNF-alpha/IFN-gamma on Fas induction. These data suggest that vesnarinone inhibits CD4XL-induced TNF-alpha/IFN-gamma secretion, thereby blocking subsequent Fas upregulation and apoptosis induction. Given the potent pathogenic role of imbalanced cytokine secretion observed in HIV-infection, an agent such as vesnarinone may be of therapeutic value in slowing disease progression.     

Activation-induced CD4+ T cell death in HIV-positive individuals correlates with Fas susceptibility, CD4+ T cell count, and HIV plasma viral copy number

The relevance of activation-induced cell death (AICD) of CD4+ T cells to AIDS pathogenesis is unknown. The present study investigates the relationship of AICD to a defined molecular mechanism regulating peripheral T cell homeostasis, Fas-mediated apoptosis, and clinical correlates of the pathogenesis of AIDS. Increased pokeweed mitogen (PWM)-induced AICD (22.8 versus 4.4%, p = 0.006) and Fas-mediated apoptosis (27.7 versus 12.0%, p = 0.002) of CD4+ T cells were observed in HIV+ versus HIV- individuals. Similarly, increased PWM-mediated AICD (16.2 versus 2.2%, p < 0.001) and Fas-mediated apoptosis (25.8 versus 7.6%, p = 0.005) were noted in CD8+ T cells from HIV+ versus HIV- individuals. PWM-induced AICD of CD4+ T cells was blocked (83% median specific inhibition) by Fas-blocking antibodies, whereas PWM-induced AICD of CD8+ T cells was Fas independent. Comparison between PWM- and anti-CD3-mediated AICD of CD4+ T cells indicated that PWM- and not CD3-induced AICD is Fas dependent. HIV+ individuals with an HIV RNA copy number of <30,000 copies/ml had lower PWM-induced AICD of CD4+ T cells than did those with an HIV RNA copy number of >30,000 copies/ml (5.7 versus 22.1%, p = 0.034), and PWM-induced AICD inversely correlated with CD4+ T cell count (R = -0.567, p = 0.043). Initiation of HAART decreased PWM-induced CD4+ T cell AICD from 24.4 to 9.4% posttreatment (p = 0.035). These results demonstrate that PWM-induced AICD of CD4+ T cells from HIV+ individuals is mediated by Fas/FasL, parallels the in vivo susceptibility of the CD4+ T cell to Fas-mediated apoptosis, and correlates with clinical markers of AIDS pathogenesis and response to HAART.     

Intrathyroidal CD4+ T lymphocytes express high levels of Fas and CD4+ CD8+ macrophages/dendritic cells express Fas ligand in autoimmune thyroid disease.

In autoimmune thyroid disease (AITD), the proportion of CD4 lymphocytes is lower in the thyroid than in the peripheral blood. We examined both Fas and Fas ligand (FasL) expression in lymphocyte subsets and nonlymphoid mononuclear cells including monocytes, macrophages, and dendritic cells (M/DCs) in both peripheral blood and thyroid specimens from 11 patients with Graves' disease and 1 with Hashimoto's disease by three-color flow cytometry. Proportions and intensities of Fas expression were increased in CD4 single-positive (SP) (CD4(+) CD8(-)), CD8 SP (CD8(+) CD4(-) ), and CD4(+) CD8(+) double-positive (DP) lymphocytes in AITD thyroids compared to those in blood, and were much higher in CD4(+) (CD4 SP and DP) lymphocytes than in CD8 SP lymphocytes in the thyroid. In the blood, most M/DCs expressed only CD4, but approximately 60% of M/DCs expressed both CD4 and CD8 in AITD thyroid. The proportion of DP M/DCs expressing FasL was higher in thyroid than in blood; proportion and intensity of FasL expression were much higher in DP M/DCs than in CD4 SP and CD8 SP M/DCs in the thyroid. These data indicate that increased Fas expression in intrathyroidal CD4(+) T lymphocytes may be the cause of CD4 lymphocyte reduction in AITD thyroid, and that intrathyroid DP M/DCs with high FasL expression may be related to the reduction in AITD.     

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.     

A liver tolerates a portal antigen by generating CD11c+ cells,which select Fas ligand+ Th2 cells via apoptosis

Administration of an antigen (Ag) per oral route leads to apoptosis of Ag-specific CD4(+) T cells and to development of Th2 cells expressing Fas ligand (FasL) in the liver. We determined whether presentation of an ingested Ag in the liver alone was enough to select these FasL(+)Th2 cells and explored how this selection was achieved in the liver. Ovalbumin (OVA) administered orally was colocalized with class II(+) cells in the periportal and parenchymal area of the liver. On coculture with naive OVA-specific CD4(+) T cells, hepatic CD11c(+) cells from mice fed OVA generated Ag-specific Th2 cells. This was achieved by apoptosis of CD4(+) T cells, decrease of interleukin 12 (IL-12) secretion, and increase of IL-18 secretion by the CD11c(+) cells. Addition of IL-12 to this coculture prevented apoptosis of the CD4(+) T cells, which was associated with up-modulation of IL-2 receptor beta chain expression. Administration of IL-12 to mice fed OVA prevented apoptosis of OVA-specific CD4(+) T cells in the liver. Moreover, adoptive transfer of hepatic CD11c(+) cells from mice fed OVA together with OVA-specific CD4(+) T cells led to development of Th2 cells as well as apoptosis of the transferred CD4(+) T cells in the lymph nodes of the recipient mice on immunization with OVA. In conclusion, presentation of an ingested Ag by hepatic CD11c(+) cells selects Th2 cells resistant to apoptosis in the liver, which is mediated in part by down-regulation of IL-12 secretion by the former cells.     

gp120-mediated induction of the MAPK cascade is dependent on the activation state of CD4(+) lymphocytes

The capacity of the HIV-1 envelope glycoprotein gp120 to induce intracellular signals is thought to contribute to HIV-1 pathogenesis. Here, we report that gp120 binding resulted in activation of the mitogen-activated protein kinase (MAPK) in CD4(+) lymphocytes prestimulated through their T-cell receptor (TCR). However, gp120 did not activate this pathway in either freshly isolated quiescent T cells or nonproliferating CD4(+) lymphocytes prestimulated with the interleukin-7 (IL-7) cytokine. This response was not solely dependent on proliferation per se because proliferating IL-7-prestimulated umbilical cord (UC)-derived T lymphocytes did not exhibit significant MAPK activation upon gp120 binding. Nevertheless, like peripheral blood lymphocytes, MAPK recruitment was induced by gp120 in UC T cells following TCR prestimulation. The lack of a gp120-mediated signaling response was not due to decreased gp120 receptor levels; CD4 expression was modified neither by IL-7 nor by TCR engagement, and high levels of functional CXCR4 were present on IL-7-treated lymphocytes. In addition to CD4 and CXCR4, recent evidence suggests that glycosphingolipids in raft microdomains serve as cofactors for HIV-1 fusion. The ganglioside GM1, a marker of rafts, was augmented in TCR-stimulated but not IL-7-stimulated T lymphocytes, and disruption of rafts inhibited gp120-induced signaling. Thus, stimulation of a mitogenic pathway by gp120 appears to require receptor binding in the context of membrane microdomains. These studies reveal a mechanism via which gp120 may differentially modulate the fate of activated and quiescent T cells in vivo.     

CD4+ T lymphocytes mediate in vivo clearance of plasmid DNA vaccine antigen expression and potentiate CD8+ T-cell immune responses

There is evidence that the limited immunogenicity of plasmid DNA vaccines is the result, at least in part, of the rapid clearance of vaccine antigen expression by antigen-specific immune responses. However, the cell types responsible for the clearance of plasmid DNA vaccine antigens are not known. Here we demonstrate that macrophages, NK cells, and CD8(+) T cells did not significantly contribute to the DNA antigen clearance but CD4(+) T cells played the crucial role in attenuating plasmid DNA vaccine antigen expression. Adoptive transfer experiments demonstrate that CD4(+) T cells facilitated DNA vaccine antigen clearance in a Fas/FasL-dependent manner. Furthermore, we show that depletion of CD4(+) T cells prevented the clearance of vaccine antigen and the appearance of a CD8(+) T-cell immune response. Inoculation of major histocompatibility complex class II KO mice with the plasmid DNA led to persistent antigen expression and abolition of a CD8(+) T-cell immune response. Importantly, the prolongation of antigen expression by disrupting the CD4(+) T-cell Fas/FasL myocytes signaling led to a 3- to 5-fold increase of antigen-specific CD8(+) T-cell responses. These data demonstrate a dominant role of CD4(+) T cell-mediated cytotoxicity in plasmid DNA vaccine antigen clearance.     

Oligoclonal expansions of CD4+ and CD8+ T-cells in the target organ of patients with biliary atresia

BACKGROUND & AIMS: Biliary atresia is an inflammatory, fibrosclerosing neonatal cholangiopathy, characterized by a periductal infiltrate composed of CD4(+) and CD8(+) T cells. The pathogenesis of this disease has been proposed to involve a virus-induced, subsequent autoreactive T cell-mediated bile duct injury. Antigen-specific T-cell immunity involves clonal expansion of T cells expressing similar T-cell receptor (TCR) variable regions of the beta-chain (Vbeta). We hypothesized that the T cells in biliary atresia tissue expressed related TCRs, suggesting that the expansion was in direct response to antigenic stimulation. METHODS: The TCR Vbeta repertoire of T cells from the liver, extrahepatic bile duct remnants, and peripheral blood of biliary atresia and other cholestatic disease controls were characterized by fluorescent-activated cell sorter analysis, and TCR junctional region nucleotide sequencing was performed on expanded TCR Vbeta regions to confirm oligoclonality. RESULTS: FACS analysis revealed Vbeta subset expansions of CD4(+) and CD8(+) T cells from the liver or bile duct remnant in all patients with biliary atresia and only 1 control. The CD4(+) TCR expansions were limited to Vbeta3, -5, -9, and -12 T-cell subsets and the CD8(+) TCR Vbeta expansions were predominantly Vbeta20. Each Vbeta subset expansion was composed of oligoclonal populations of T cells. CONCLUSIONS: Biliary atresia is associated with oligoclonal expansions of CD4(+) and CD8(+) T cells within liver and extrahepatic bile duct remnant tissues, indicating the presence of activated T cells reacting to specific antigenic stimulation. Future studies entail identifying the specific antigen(s) responsible for T-cell activation and bile duct injury.     

The dual functions of fas ligand in the regulation of peripheral CD8+ and CD4+ T cells

Although Fas ligand (FasL) is well characterized for its capacity to deliver a death signal through its receptor Fas, recent work demonstrates that FasL also can receive signals facilitating antigen (Ag)-specific proliferation of CD8(+) T cells. The fact that the gld mutation differentially influences the proliferative capacity of CD8(+) and CD4(+) T cells presented the intriguing possibility that a single molecule may play opposing roles in these two subpopulations. The present study focuses on how these positive and negative regulatory roles are balanced. We show that naive CD4(+) T cells are responsive to FasL-mediated costimulation on encounter with Ag when Fas-mediated death is prevented. Thus, the machinery responsible for transducing the FasL positive reverse signal operates in both CD4(+) and CD8(+) T cells. Instead, differential control of FasL expression distinguishes the role of FasL in these two T cell subpopulations. FasL costimulation occurs immediately on T cell receptor ligation and correlates with the up-regulation of FasL expression on CD8(+) and naive CD4(+) T cells, both of which are sensitive to the FasL costimulatory signal. Conversely, FasL-initiated death occurs late in an immune response when high levels of FasL expression are maintained on CD4(+) T cells that are sensitive to Fas-mediated death, but not on CD8(+) T cells that are relatively insensitive to this signal. This careful orchestration of FasL expression during times of susceptibility to costimulation and conversely, to death, endows FasL with the capacity to both positively and negatively regulate the peripheral T cell compartment.     

Allergen immunotherapy decreases specific allergen-induced expression of Fas and FasL on CD4+ and CD8+ cells.

Apoptosis programmed cell death without induction of an inflammatory response. It is mediated by Fas--a cell surface protein which is expressed on activated lymphocytes. Interaction with its counterpart--the Fas ligand induces the apoptosis of Fas bearing cells. The mechanism underlying successful immunotherapy has not been identified. The aim of this study was to investigate whether specific immunotherapy (SIT) might affect Fas and FasL expression after stimulation with specific allergen. The study was conducted on 8 allergic subjects and 8 healthy volunteers as controls. The allergic patients were treated with conventional SIT (Pollinex). Blood samples were collected before the first day and 7 days after the last injection. Isolated CD4+ and CD8+ cells were incubated in various concentrations of specific allergen (1, 10, 100 ng/ml) or in medium alone. Indirect immunofluorescence test with rabbit IgG against human Fas and FasL was performed. The percentage of positive cells was determined under fluorescence microscope. The expression of Fas and FasL before SIT was significantly increased on CD4+ and CD8+ cells under the influence of specific allergen (10, 100 ng/ml). After SIT, significant decrease in the expression of both molecules was observed, although elimination of allergen-reactive cells was not complete and their number was still higher than in the controls. CONCLUSION: The exposure of CD4+ and CD8+ cells on allergen may induce the Fas-FasL apoptotic pathway. Significant decrease in number of allergen-reactive CD4+, CD8+ cells after SIT suggests participation of the phenomenon in deletion of clones, which may be a part of the allergen tolerance mechanism achieved naturally or during SIT.     

Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals

This study investigates apoptosis as a mechanism for CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1) infection. Although several recent studies have shown that T cells of HIV-infected individuals show enhanced susceptibility to cell death by apoptosis, the mechanisms responsible for apoptosis are largely unknown. By using a flow cytometric technique and by morphology, we have quantitated the percentage of cells undergoing apoptosis in peripheral blood mononuclear cells (PBMCs) from HIV-seronegative donors and from HIV- infected asymptomatic patients. The PBMCs were cultured without any stimulus or with staphylococcus enterotoxin B, anti-T-cell receptor (TCR) alpha beta monoclonal antibody WT-31, or phytohemagglutinin for periods up to 6 days. In addition, we sought to determine whether cross- linking of CD4 followed by various modes of TCR stimulation in vitro could induce apoptosis in normal PBMCs. Here we show that (1) patient PMBCs undergo marked spontaneous apoptosis; (2) stimulation of T cells of patients as well as normal donors results in increased apoptosis; and (3) cross-linking of CD4 molecules is sufficient to induce apoptosis in CD4+ T cells if cross-linking is performed in unfractioned PBMCs, but not if CD4 molecules are cross-linked in purified T-cell preparations. These observations strongly suggest that accelerated cell death through apoptosis plays an important role in the pathogenesis of HIV-1 infection. At the same time, our observations implicate cross- linking of CD4 in vivo as a major contributor to this mechanism of accelerated cell death in HIV infection.     

Increased intensities of fas expression on peripheral T-cell subsets in severe autoimmune thyroid disease.

Fas (CD95)-Fas ligand (FasL; CD178)-induced apoptosis is necessary for the maintenance of self-tolerance. To clarify whether or not any abnormalities in the Fas-FasL system exist in patients with autoimmune thyroid disease (AITD), we examined the expression of Fas and FasL on peripheral T lymphocytes by three-color flow cytometry in 113 patients with AITD and 49 healthy controls. The intensities of Fas expression in both CD4(+) and CD8(+) T cells decreased in thyrotoxic patients with Graves' disease (GD), but increased in both patients with severe Hashimoto's disease (HD) undergoing treatment and seriously intractable patients with GD continuously positive for thyrotropin (TSH) receptor antibody despite treatment with antithyroid drugs for more than 5 years. The proportion of Fas expression was increased in CD4(+) T cells from patients with untreated GD, and in CD8(+) T cells from patients with severe HD. The proportion of CD8(+) T cells decreased in patients with severe HD. FasL were not expressed on T cells in controls and patients with AITD. These results indicate that (1). the intensities of Fas expression on peripheral T cells increase in severe autoimmune thyroid diseases and (2). both the intensity and the proportion of Fas expression may be important for the induction of apoptosis.     

Human immunodeficiency virus type 1 protease inhibitor modulates activation of peripheral blood CD4(+) T cells and decreases their susceptibility to apoptosis in vitro and in vivo.

CD4(+) T cells from patients with human immunodeficiency virus (HIV) infection undergo apoptosis at an increased rate, which leads to their depletion during disease progression. Both the Fas-Receptor (Fas-R) and interleukin-1beta (IL-1beta)-converting enzyme (ICE; caspase 1) appear to play a role in the mechanism of apoptosis of CD4(+) lymphocytes. Although Fas-R is upregulated on both CD4(+) and CD8(+) cells in HIV-infected patients, results from our laboratory and others indicate that, in patients with advanced disease, CD4(+) cells preferentially express ICE. Protease inhibitors have successfully halted the progression of HIV disease and increased CD4(+) T counts. In this study, we examined the effect of protease inhibitors on Fas-R (CD95), ICE (caspase 1) expression, apoptosis, and cell death in CD4(+) T cells of (1) HIV-infected patients who were receiving protease inhibitors, and (2) normal and patient CD4(+) T cells cultured with a protease inhibitor in vitro. Fifteen patients with advanced HIV disease on treatment showed dramatically decreased CD4(+) T-cell ICE expression, diminished apoptosis, and increased numbers of CD4(+) cells within 6 weeks of institution of protease inhibitor therapy, and before down-modulation of Fas-R (CD95) expression was evident. To determine the role of HIV infection, we studied the effect of ritonavir, a protease inhibitor, on normal and patient cells in vitro. Stimulated and unstimulated normal CD4(+) T cells, cultured with protease inhibitor, demonstrated markedly decreased apoptosis and ICE expression (P =. 01). While Fas-R expression was not significantly altered during short-term culture by such treatment, Fas-Ligand (Fas-L) membrane expression of phytohemagglutinin (PHA)-stimulated blood lymphocytes was decreased by protease inhibitor. In the presence of ritonavir, CD4(+) T cells from HIV-infected patients showed similar changes in ICE intracellular levels without alteration of Fas expression. In conclusion, protease inhibitors appear to decrease CD4(+) T-cell ICE expression and apoptosis before they affect Fas-R expression in HIV-infected patients. This action was independent of HIV infection, as similar effects were seen in CD4(+) T cells from normal controls. Some of the benefit of protease inhibitors may be related to modification of programmed cell death, which increases CD4(+) T-cell number. Whether this is due to directly to the changes effected in the caspase system remains to be determined.     

Activation of the CD95 (APO-1/Fas) system in T cells from human immunodeficiency virus type-1-infected children

Increased apoptosis of CD4+ T cells is considered to be involved in CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1)- infected individuals progressing toward acquired immunodeficiency syndrome (AIDS). We have recently shown that CD95 (APO-1/Fas) expression is strongly increased in T cells of HIV-1-infected children. In this report we provide further evidence for a deregulated CD95 system in AIDS. CD95 expression in HIV-1+ children is not restricted to previously activated CD45RO+ T cells but is also increased on freshly isolated naive CD45RA+ T cells. In addition, specific CD95-mediated apoptosis is enhanced in both CD4+ and CD8+ T cells. Furthermore, levels of CD95 ligand mRNA are profoundly increased. Specific T-cell receptor/CD3-triggered apoptosis in HIV-1+ children is more enhanced in CD8+ than in CD4+ T cells. Accelerated activation induced cell death of T cells could partially be inhibited by blocking anti-CD95 antibody fragments. These data suggest an involvement of the CD95 receptor/ligand system in T-cell depletion and apoptosis in AIDS and may open new avenues of rational intervention strategies.     

Fas/Fas ligand expression and characteristics of primed CD45RO+ T cells in the inflamed mucosa of ulcerative colitis

BACKGROUND: Chronic immune activation in the colon is characteristic of ulcerative colitis (UC). Fas/Fas ligand (FasL) system is a mechanism responsible for activation-induced cell death (AICD), which maintains homeostasis within the immune system. Thus, Fas/FasL expression on activated colonic T cells of UC patients, as well as the susceptibility of such T cells to AICD was investigated in order to determine the role of activated colonic T cells in the long lasting inflammation in UC. METHODS: Fas, FasL, and CD45RO expression on peripheral blood and colonic T cells of UC patients were assayed by flow cytometry. Apoptosis of colonic T cells induced by anti Fas antibody was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. RESULTS: The majority of colonic T cells expressed both CD45RO and Fas in the colonic mucosa, a situation that was quite different from that in the peripheral blood. The number of CD45RO+CD8+ and Fas+CD8+ T cells was significantly lower in UC patients than the controls, unlike the number of Fas+CD4+ T cells. In contrast, the number of both CD45RO+CD4+ and CD45RO+CD8+ T cells in UC mucosa expressing FasL was significantly higher than in the controls. While Fas mediated apoptosis of CD45RO+CD8+ T cells was higher in UC patients than the controls, the number of apoptotic CD45RO+CD4+ T cells from UC mucosa was not. CONCLUSIONS: In UC patients, CD45RO+CD4+ T cells are less sensitive to apoptotic signals mediated by Fas. These phenomena may contribute to the pathogenesis of UC.     

Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL

Myeloid-derived suppressor cells (MDSCs) inhibit adaptive and innate immunity and accumulate in the blood of persons with cancer, chronic inflammation, trauma, infection, and stress. Some of the factors inducing their accumulation are known; however, mechanisms regulating their turnover have not been identified. Mass spectrometry showed prominent expression of apoptosis pathway proteins, suggesting that MDSC turnover may be regulated by Fas-FasL-mediated apoptosis. This hypothesis was confirmed by showing that blood MDSCs induced by 3 mouse tumors were Fas(+) and apoptosed in response to Fas agonist in vitro and to activated FasL(+) T cells in vivo. FasL-deficient mice contained significantly more blood MDSCs than FasL(+/+) mice, and after removal of primary tumors MDSCs regressed in STAT6(-/-) and CD1(-/-) mice but not in STAT6(-/-)FasL(-/-) or CD1(-/-)FasL(-/-) mice. Fas(+) macrophages and dendritic cells did not apoptose in response to activated T cells, indicating that Fas-FasL regulation of myeloid cells was restricted to MDSCs. These results identify a new mechanism regulating MDSC levels in vivo and show a retaliatory relationship between T cells and MDSCs in that MDSCs suppress T-cell activation; however, once activated, T cells mediate MDSC apoptosis.     

The magnitude and breadth of hepatitis C virus-specific CD8+ T cells depend on absolute CD4+ T-cell count in individuals coinfected with HIV-1

CD8(+) T-cell responses are an essential antiviral host defense in persistent viral infections, and their sustained effectiveness is thought to be critically dependent on CD4(+) T-helper cells. To determine the relationship between HIV-1-induced CD4(+) T-cell depletion and hepatitis C virus (HCV)-specific CD8(+) T-cell responses during viral persistence, we studied 103 persons positive for HCV, 74 coinfected with HIV-1. CD8(+) T-cell responses to the entire HCV polyprotein were determined by using an interferon-gamma enzyme-linked immunospot (ELISpot) assay. Although HIV-1 infection by itself was not associated with a diminished HCV-specific response, HIV-1-associated CD4(+) depletion was associated with significantly lower HCV-specific CD8(+) T cells (R = 0.48, P < .0001). In contrast, declining CD4(+) counts over the same range were not associated with diminished Epstein-Barr virus (EBV)- (R = 0.19, P = .31) or HIV-1-specific (R = -0.13, P = .60) CD8(+) T-cell responses in persons infected with all viruses. These data indicate that frequencies of circulating HCV-specific CD8(+) T-cell responses are sensitive to absolute CD4(+) T-cell counts and provide a possible explanation for the accelerated HCV disease course in persons coinfected with HIV-1 and HCV.