Pathogen-specific induction of CD154 is impaired in CD4+ T cells from human immunodeficiency virus-infected patients

The pathogenesis of immunodeficiency associated with human immunodeficiency virus (HIV) infection remains incompletely understood. CD154, a molecule that is expressed primarily on activated CD4(+) T cells, is pivotal for regulation of cell-mediated and humoral immunity and is crucial for control of many opportunistic infections. We investigated whether CD4(+) T cells from HIV-infected patients exhibit defective induction of CD154 in response to opportunistic pathogens. Incubation of purified human CD4(+) T cells with monocytes plus antigenic preparations of either Candida albicans, cytomegalovirus, or Toxoplasma gondii resulted in induction of CD154. Expression of CD154 in response to these pathogens was impaired in CD4(+) T cells from HIV-infected patients. This defect correlated with decreased production of interleukin (IL)-12 and interferon (IFN)-gamma in response to T. gondii. Recombinant CD154 partially restored secretion of IL-12 and IFN-gamma in response to T. gondii in cells from HIV-infected patients. Together, defective induction of CD154 is likely to contribute to impaired cell-mediated immunity against opportunistic pathogens in HIV-infected patients.     

Dexamethasone inhibits CD4 T cell deletion mediated by macrophages from human immunodeficiency virus-infected persons.

Prednisolone slows the loss of CD4 T cells in individuals with human immunodeficiency virus (HIV) disease and inhibits antigen-induced apoptosis of recently HIV-infected CD4 cells in vitro. This study investigated whether dexamethasone inhibits the ability of macrophages to delete CD4 T cells via anti-CD4 antibody or immune-complexed HIV envelope protein gp120. Peripheral blood mononuclear cells from HIV-negative persons were incubated with CD4-reactive ch412 monoclonal antibody or with gp120/IgG immune complexes and resident macrophages, with and without dexamethasone. Dexamethasone inhibited CD4 cell deletion in a dose-dependent manner. The deletion of normal CD4 cells by macrophages from HIV-infected patients also was inhibited by dexamethasone. Furthermore, up-regulation of CD95 expression on T cells exposed to anti-CD4 and gp120/IgG, which predisposes T cells to CD95-mediated apoptosis, is inhibited by dexamethasone in a dose-dependent fashion. Dexamethasone inhibits the macrophage-mediated deletion of CD4 lymphocytes in HIV-infected persons.     

Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase

Human neutrophil elastase (HNE) and proteinase 3 (PRO3) are myeloid tissue-restricted serine proteases, aberrantly expressed by myeloid leukemia cells. PRO3 and HNE share the PR1 peptide sequence that induces HLA-A*0201-restricted cytotoxic T cells (CTLs) with antileukemia reactivity. We studied the entire HNE protein for its ability to induce CTLs. In an 18-hour culture, HNE-loaded monocytes stimulated significant intracellular interferon gamma (IFN-gamma) production by CD4+ and CD8+ T cells in 12 of 20 and 8 of 20 healthy individuals, respectively. Lymphocytes from 2 HNE responders were pulsed weekly for 4 weeks to generate HNE-specific CTLs. One of 2 HLA-A*0201-negative individuals inhibited the colony formation of HLA-identical chronic myelogenous leukemia progenitor cells (73% inhibition at 50:1 effector-target [E/T] ratio), indicating that peptides other than PR1 can induce leukemia-reactive CTLs. Repetitive stimulations with HNE in 2 of 5 HLA-A*0201+ individuals increased PR1 tetramer-positive CD8+ T-cell frequencies from 0.1% to 0.29% and 0.02% to 0.55%, respectively. These CTLs recognized PR1 peptide or killed HNE-loaded targets. These results indicate that exogenously processed HNE is a source of PR1 peptide as well as other peptide sequences capable of inducing leukemia-specific CD8+ and CD4+ T cells. HNE could, therefore, be used in an HLA-unrestricted manner to induce leukemia-reactive CTLs for adoptive immunotherapy.     

Analysis of intracellular cytokines in CD4+ and CD8+ lung and blood T cells in sarcoidosis

In pulmonary sarcoidosis, activated T cells accumulate in the lungs. We hypothesized that the balance between the T-helper type 1 (Th1) cytokines (interferon [IFN]-gamma and interleukin [IL]-2) and Th2 cytokines such as IL-4, IL-5, and IL-10 might explain differences in clinical outcome in pulmonary sarcoidosis, such as why patients of human leukocyte antigen (HLA) type DR17 have a much better prognosis than those of other HLA types. Peripheral blood lymphocytes (PBL) and lymphocytes obtained by bronchoalveolar lavage (BAL) from HLA-typed sarcoidosis patients, as well as PBL from healthy controls, were stimulated in vitro, fixed, and permeabilized with saponin. Thereafter, cells were stained with fluorescence- labeled antibodies specific for intracellular cytokines (IL-2, IL-4, IFN-gamma, and tumor necrosis factor (TNF)-alpha and cell surface markers CD4 and CD8, and were subjected to flow-cytometric analysis. In bronchoalveolar lavage fluid (BALF), there were significantly greater frequencies of T cells positive for IFN-gamma and TNF-alpha than there were among PBL, and significantly fewer cells positive for IL-4, in both the CD4+ and CD8+ subsets. HLA-DR17-positive patients showed a tendency toward a less pronounced Th1 response that may be related to their good prognosis. Sarcoidosis patients had higher frequencies of cells positive for IFN-gamma, IL-4, and IL-2 in their blood than did healthy controls, a finding that may reflect the systemic nature of sarcoidosis. A clear Th1 cytokine profile of CD4+ as well as of CD8+ T cells was demonstrated in BALF from sarcoidosis patients. This was most pronounced for CD8+ cells, which may therefore make an important contribution to the inflammatory process in the lungs in pulmonary sarcoidosis.     

Simian immunodeficiency virus infection of CD4+CD8+ T cells in a macaque with an unusually high peripheral CD4+CD8+ T lymphocyte count

We assessed the possible role in vivo CD4(+) CD8(+) T cells as a viral reservoir for simian immunodeficiency virus (SIV), in a macaque with 50% CD4(+) CD8(+) T cells in peripheral blood. During primary infection (day 14) of this rhesus macaque with the pathogenic SIVmac251 strain, proviruses were detected at similar frequencies in CD4(+) CD8(+) T cells (1/10) and CD4(+) T cells (1/10) and at a lower frequency in CD8(+) T cells (1/800). On day 235, no viral DNA was detected in CD8(+) cells, despite the persistent high viral load, indicating that CD8(+) cells do not constitute a reservoir during the chronic phase of SIV infection. Infection induced early lymphopenia of CD4(+), CD4(+) CD8(+), and CD8(+) cells; only the CD8(+) cell population returned to initial levels and expanded further. We found that CD4(+) CD8(+) T cells expressed the costimulatory CD28 molecule less and were more prone to die in vitro after phytohemagglutinin/interleukin 2 stimulation than were CD4(+) T cells. Taken together, massive death of CD4(+) CD8(+) T cells during acute stages of SIV infection may explain why CD8(+) T cells did not represent a major reservoir for SIV at the onset of infection.     

CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells.

We have investigated the ability of human immunodeficiency virus (HIV)-infected cells to kill uninfected CD4+ lymphocytes. Infected peripheral blood mononuclear cells were cocultured with autologous 51Cr-labeled uninfected cells. Rapid death of the normal CD4-expressing target population was observed following a brief incubation. Death of blood CD4+ lymphocytes occurred before syncytium formation could be detected or productive viral infection established in the normal target cells. Cytolysis could not be induced by free virus, was dependent on gp120-CD4 binding, and occurred in resting, as well as activated, lymphocytes. CD8+ cells were not involved in this phenomenon, since HIV-infected CEMT4 cells (CD4+, CD8- cells) mediated the cytolysis of uninfected targets. Reciprocal isotope-labeling experiments demonstrated that infected CEMT4 cells did not die in parallel with their targets. The uninfected target cells manifested DNA fragmentation, followed by the release of the 51Cr label. Thus, in HIV patients, infected lymphocytes may cause the depletion of the much larger population of uninfected CD4+ cells without actually infecting them, by triggering an apoptotic death.     

In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals

Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3- mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N- acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.     

Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects

We have genetically engineered CD4(+) and CD8(+) T cells with human immunodeficiency virus (HIV) specificity by inserting a gene, CD4zeta, containing the extracellular domain of human CD4 (which binds HIV env) linked to the zeta (zeta) chain of the T-cell receptor (which mediates T-cell activation). Twenty-four HIV-positive subjects received a single infusion of 2 to 3 x 10(10) autologous CD4zeta-modified CD4(+) and CD8(+) T cells administered with (n = 11) or without (n = 13) interleukin-2 (IL-2). Subjects had CD4 counts greater than 50/microL and viral loads of at least 1000 copies/mL at entry. T cells were costimulated ex vivo through CD3 and CD28 and expanded for approximately 2 weeks. CD4zeta was detected in 1% to 3% of blood mononuclear cells at 8 weeks and 0.1% at 1 year after infusion, and survival was not enhanced by IL-2. Trafficking of gene-modified T cells to bulk rectal tissue and/or isolated lamina propria lymphocytes was documented in a subset of 5 of 5 patients at 14 days and 2 of 3 at 1 year. A greater than 0.5 log mean decrease in rectal tissue-associated HIV RNA was observed for at least 14 days, suggesting compartmental antiviral activity of CD4zeta T cells. CD4(+) counts increased by 73/microL at 8 weeks in the group receiving IL-2. There was no significant mean change in plasma HIV RNA or blood proviral DNA in either treatment arm. This sustained, high-level persistence of gene-modified T cells demonstrates the feasibility of ex vivo T-cell gene therapy in HIV-infected adults and suggests the importance of providing HIV-specific T-helper function.     

CD4+ T-cell induction of Fas-mediated apoptosis in Burkitt's lymphoma B cells

Cytotoxic function of CD4+ Th1 cells is mediated by Fas (CD95, APO-1) and its ligand (Fas ligand). Recent studies using nontransformed B cells and the Ramos Burkitt's lymphoma (BL) B-cell line cells show that CD40 ligation at the B-cell surface by activated, CD40 ligand (CD40L)- bearing, CD4+ T cells upregulates Fas expression on B cells and primes B cells for Fas-mediated death signals. In this work, we examine whether this CD4+ T-cell-dependent molecular pathway for Fas upregulation and B-cell apoptosis reflects a peculiarity of the Ramos B- cell line or is applicable to other Burkitt's tumors as well. In 5 of the 6 Epstein-Barr virus-negative BL cell lines examined, the cells constitutively express undetectable or low levels of Fas and are resistant to Fas-mediated signals induced by monoclonal anti-Fas antibody. All 6 of the BL cell line B cells upregulate Fas in response to CD40 ligation, and in 4 of the cases they become sensitive to Fas- mediated death signals. In one BL cell line, the cells are constitutively sensitive to Fas-mediated cytolysis and are unaffected by CD40 signals. Next, we applied these immunologic manipulations to cells from a refractory clinical sample and observed that the tumor cells could be induced to express Fas and undergo apoptosis in our system. These results establish CD4+ T cells and the Fas-Fas ligand system as important immune regulators of Burkitt's lymphoma B cells and indicate that the susceptibility of tumor cells to Fas-mediated death signals can be modulated by specific activation events at the cell surface.     

A factor from CD8 cells of human immunodeficiency virus-infected patients suppresses HLA self-restricted T helper cell responses.

Defective in vitro T helper cell (Th) function can occur in asymptomatic human immunodeficiency virus (HIV)-seropositive (HIV+) individuals. A characteristic, early finding is the loss of an in vitro response to recall antigens, such as influenza A virus (FLU), despite an intact Th response to alloantigen (ALLO). To determine whether suppressor cells and/or inhibitory factors could contribute to this HIV-associated Th immunodeficiency, coculture studies were performed using peripheral blood leukocytes (PBLs) from monozygotic twins, one of whom was HIV-infected (HIV+) and one of whom was uninfected (HIV-seronegative, HIV-). In vitro Th function was measured as interleukin 2 production or proliferation to FLU and ALLO. Two pairs of twins were repetitively studied. A single HIV+ individual with multiple samples of cryopreserved PBLs over 6 years (including a HIV- specimen) was also studied. PBLs from the HIV+, but not from the HIV-, individuals demonstrated defective in vitro Th function in response to FLU but not to ALLO. PBLs from HIV+ individuals could induce a similar defect in the Th function of syngeneic or autologous HIV- PBLs. This suppression was generated by CD4-depleted, but not by CD8-depleted, PBLs. A suppressive factor from CD8+ cells of HIV+ donors was generated by 24-hr unstimulated cultures of HIV+ PBLs. This factor inhibited FLU but not ALLO responses of autologous, syngeneic, or allogeneic HIV- PBLs. This suppressive effect could not be explained by HIV infection or replication during the culture period. These results demonstrate that selective abrogation of Th function to recall antigens in HIV+ individuals is associated with an inhibitory factor produced by CD8+ T cells.     

CD4+ T cell kinetics and activation in human immunodeficiency virus-infected patients who remain viremic despite long-term treatment with protease inhibitor-based therapy.

T cell dynamics were studied in human immunodeficiency virus-infected patients who continued using antiretroviral therapy despite detectable plasma viremia (RNA copies >2500 /mL). CD4(+) cell fractional replacement rates, measured by the deuterated glucose technique, were lower in treated patients with detectable viremia than in untreated patients and were similar to those in patients with undetectable viremia. Cell cycle and activation markers exhibited similar trends. For any level of viremia, CD4(+) cell fractional replacement rates were lower in patients with drug-resistant virus than in patients with wild-type virus, which suggests that the resistant variant was less virulent. Interruption of treatment in patients with drug-resistant viremia resulted in increased CD4(+) cell activation, increased CD4(+) cell turnover, and decreased CD4(+) cell counts. These data indicate that partial virus suppression reduces CD4(+) cell turnover and activation, thereby resulting in sustained CD4(+) cell gains, and that measurements of T cell dynamics may provide an in vivo marker of viral virulence.     

Microarray analysis reveals similarity between CD8+CD28- T cells from young and elderly persons, but not of CD8+CD28+ T cells

We isolated highly purified CD8⁺CD28⁺ and CD8⁺CD28⁻ T cell populations from healthy young and elderly persons for gene expression profiling using Affymetrix oligonucleotide microarrays. We demonstrate that the gene expression profile of CD8⁺CD28⁻ T cells is very similar in young and elderly persons. In contrast, CD8⁺CD28⁺ in elderly differ from CD8⁺CD28⁺ in young persons. Hierarchical clustering revealed that CD8⁺CD28⁺ in elderly are located between CD8⁺CD28⁺ in young and CD8⁺CD28⁻ (young and old) T cells regarding their differentiation state. Our study demonstrates a dichotomy of gene expression levels between CD8⁺CD28⁺ T cells in young and elderly persons but a similarity between CD8⁺CD28⁻ T cells in young and elderly persons. As CD8⁺CD28⁺ T cells from elderly and young persons are distinct due to a different composition of the population, these results suggest that the gene expression profile does not depend on chronological age but depends on the differentiation state of the individual cell types.     

人类免疫缺陷病毒感染者CD4+T淋巴细胞计数与结核分枝杆菌共感染的相关性

目的 探讨HIV合并结核分枝杆菌感染患者外周血CD4+T淋巴细胞的变化及其对酶联免疫斑点试验(ELISPOT)的影响.方法 应用结核酶联免疫斑点试验(T-SPOT.TB)对云南省和上海地区193例明确诊断的HIV感染者的血标本进行结核分枝杆菌特异性T淋巴细胞检测,同时应用流式细胞仪检测外周血CD4+T淋巴细胞计数,采用t检验进行统计学分析.结果 HIV感染者中潜伏性结核发生率达30.6％.HIV合并活动性结核患者的外周血CD4+T淋巴细胞计数平均值为190×106/L,显著低于HIV合并潜伏性结核组的484×106/L(t=6.665,P＜0.01).根据CD4+T淋巴细胞计数＞500×106/L、200×106～500×106/L、＜200×106/L进行分层分析发现,活动性结核组和潜伏性结核组构成比依次为1∶16.2、1∶1.3、5.6∶1.79例T-SPOT.TB阳性的病例中,共有20例HIV合并活动性结核患者,其中14例患者CD4+T淋巴细胞计数平均值＜200×106/L,5例为200×106/L～500×106/L,1例＞500×106/L.59例潜伏性结核患者中,52例CD4+T淋巴细胞计数＞200×106/L.结论 我国HIV感染人群中有较高的潜伏性结核发生率,HIV合并活动性结核患者的总体细胞免疫应答功能严重受损,随着CD4ˉT淋巴细胞计数的下降,HIV患者合并的潜伏性结核更易进展为活动性结核.当CD4+T淋巴细胞＜200×106/L时,对T-SPOT.TB检测结果可能有影响.     

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.     

Interferon-alpha differentially rescues CD4 and CD8 T cells from apoptosis in HIV infection

OBJECTIVE: To examine the effects of interferon-alpha (IFN-alpha) on T cell survival and activation in HIV infection. DESIGN: The effects of IFN-alpha on spontaneous apoptosis and CD38 expression among T cell subsets were determined in vitro and studied in relation to CD4 cell counts, plasma HIV RNA levels and the age of the subjects. METHODS: Peripheral blood mononuclear cells from 48 HIV-infected persons and 17 healthy donors were incubated in vitro overnight with or without the addition of IFN-alpha. Percentages of apoptotic cells (positive for annexin V) and CD38 cells were determined among T cell subsets. RESULTS: IFN-alpha inhibited spontaneous apoptosis of CD4 and CD8 T lymphocytes. This protective activity was impaired in CD4 T cells from HIV-infected persons. The reduced protection of IFN-alpha among CD4 cells from HIV-infected persons was not related to the percentages of activated (CD38 or CD45RO+CD38+) cells. Surprisingly, IFN-alpha induced CD38 expression among CD8 T cells from HIV-infected persons, and the magnitude of this effect was directly related to circulating CD4 T cell count. The CD8 T cell subset that expressed CD38 in response to IFN-alpha was defined as CD28 negative, CD62 ligand (CD62L) intermediate/negative. CONCLUSIONS: Heightened expression of IFN-alpha in HIV infection may contribute to the phenotypic activation state that characterizes chronic infection while a diminished responsiveness of CD4 T cells to the protective effect of this cytokine may contribute to differential survival of CD4 and CD8 T cells in HIV disease.     

CD25+CD4+ T cells contribute to the control of memory CD8+ T cells

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. In animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.     

Discordant increases in CD4+ T cells in human immunodeficiency virus-infected patients experiencing virologic treatment failure: role of changes in thymic output and T cell death

Some patients infected with human immunodeficiency virus (HIV) who are experiencing antiretroviral treatment failure have persistent improvement in CD4+ T cell counts despite high plasma viremia. To explore the mechanisms responsible for this phenomenon, 2 parameters influencing the dynamics of CD4+ T cells were evaluated: death of mature CD4+ T cells and replenishment of the CD4+ T cell pool by the thymus. The improvement in CD4+ T cells observed in patients with treatment failure was not correlated with spontaneous, Fas ligand-induced, or activation-induced T cell death. In contrast, a significant correlation between the improvement in CD4+ T cell counts and thymic output, as assessed by measurement of T cell receptor excision circles, was observed. These observations suggest that increased thymic output contributes to the dissociation between CD4+ T cell counts and viremia in patients failing antiretroviral therapy and support a model in which drug-resistant HIV strains may have reduced replication rates and pathogenicity in the thymus.     

Interleukin 7 reduces the levels of spontaneous apoptosis in CD4+ and CD8+ T cells from HIV-1-infected individuals

Apoptosis has been suggested as one of the major mechanisms of CD4+ T cell depletion during the course of HIV type 1 (HIV-1) infection. Here, we show that interleukin 7 (IL-7), a nonredundant cytokine that plays essential roles in the generation and homeostasis of the T cell compartment of the immune system, exerts strong antiapoptotic effects ex vivo on both CD4+ and CD8+ T cells derived from HIV-1-infected subjects. The level of IL-7-mediated reduction of apoptosis was inversely correlated with the number of circulating CD4+ T cells, indicating a higher sensitivity to IL-7 effects in patients with more advanced disease. The antiapoptotic effect of IL-7 was uncoupled from the induction of cellular proliferation or endogenous HIV-1 replication. These results provide a further rationale for consideration of IL-7 as an agent of immune reconstitution in HIV-1 infection.