Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1

We investigated whether CD4+ T cells that do not produce HIV-1 are killed in HIV-infected human lymphoid tissue. Tissue blocks were inoculated with high amount of doxycycline-dependent HIV-rtTA. Doxycycline triggered productive infection and loss of CD4+ T cells in these tissues, whereas without doxycycline, neither productive infection nor CD4+ T cell depletion was detected in spite of the massive presence of virions in the tissue and of viral DNA in the cells. Thus, HIV-1 alone is sufficient to deplete productively infected CD4+ T cells but is not sufficient to cause the death of uninfected or latently infected CD4+ T cells.     

HIV-1 actively replicates in naive CD4(+) T cells residing within human lymphoid tissues.

Although HIV-1 gene expression is detected in naive, resting T cells in vivo, such cells are resistant to productive infection in vitro. However, we found that the endogenous microenvironment of human lymphoid tissues supports de novo infection and depletion of this population. Cell cycle analysis and DNA labeling experiments established that these cells were definitively quiescent and thus infected de novo. Quantitation of the "burst size" within naive cells further demonstrated that these cells were productively infected and contributed to the local viral burden. These findings demonstrate that lymphoid tissues support active HIV-1 replication in resting, naive T cells. Moreover, these cells are not solely reservoirs of latent virus but are permissive hosts for viral replication that likely targets them for elimination.     

Mutagen-mediated enhancement of HIV-1 replication in persistently infected cells

Lethal mutagenesis, a new antiviral strategy to extinguish virus through elevated mutation rates, was explored in H61-D cells an HIV-1 persistently infected lymphoid cell line. Three mutagenic agents: 5-hydroxy-2^′-deoxycytidine (5-OHdC), 5-fluorouracil (5-FU) and 2,2^′-difluoro-2^′-deoxycytidine (gemcitabine) were used. After 54 passages, treatments with 5-FU and gemcitabine reduced virus infectivity, p24 and RT activity. Treatment with the pyrimidine analog 5-OHdC resulted in increases of p24 production, RT activity and infectivity. Rise in viral replication by 5-OHdC during HIV-1 persistence is in contrast with its inhibitory effect in acute infections. Viral replication enhancement by 5-OHdC was associated with an increase in intracellular HIV-1 RNA mutations. Mechanisms of HIV-1 replication enhancement by 5-OHdC are unknown but some potential factors are discussed. Increase of HIV-1 replication by 5-OHdC cautions against the use, without previous analyses, of mutagenic nucleoside analogs for AIDS treatment.     

Enumeration of latently infected CD4+ T cells from HIV-1-infected patients using an HIV-1 antigen ELISPOT assay.

BACKGROUND: Latently infected resting CD4(+) T cells carrying replication-competent HIV-1 are present in naive, chronically infected individuals as well as in those who are receiving highly active antiretroviral therapy (HAART). These cells serve as a potential source of reactivation of viral replication and remain a major obstacle for the eradication of HIV-1. OBJECTIVES: The enzyme-linked immunospot (ELISPOT) assay was adapted to the detection and the enumeration of HIV-1 antigen-secreting cells at the single cell level. We applied this test to count latently HIV-1-infected CD4(+) T cells. STUDY DESIGN: Latently infected CD4(+) T cells were assessed in an in vitro model of HIV-1-infected resting CD4(+) T cells as well as in eighteen HAART-treated and in four HIV-1-infected untreated patients. Enriched CD4(+) T cells were cultured with or without antibodies against CD3 and CD28 T cell receptors and with irradiated peripheral blood mononuclear cells from HIV-1 seronegative individuals. At the term of the cell culture, CD4(+) T lymphocytes were tested using the HIV-1 antigen ELISPOT assay. RESULTS: In the experimental HIV-1 infection model, 5579+/-4190 CD4(+) T cells secreting HIV-1 antigen were enumerated after polyclonal activation. In contrast, only 15+/-6 HIV-1 immunospots were obtained from unstimulated T cells. In all patients tested, induced HIV-1 antigen-secreting cells were measured at a frequency of 55+/-108/10(6) CD4(+) T cells. CONCLUSION: As each immunospot represents one HIV-1 antigen-secreting cell, the HIV-1 ELISPOT assay is a powerful to enumerate circulating CD4(+) T lymphocytes latently infected with HIV-1.     

Efficient induction of HIV-1 replication in latently infected cells through contact with CD4+ T cells: involvement of NF-kappaB activation

Reservoir cells latently infected with HIV-1 pose one of the major obstacles that hamper ultimate eradication of HIV-1 from infected patients. In this report, we showed that direct contact with MOLT-4 T cells induced HIV-1 replication in J(22)-HL-60 latently infected cells without any additional stimulus. Neutralization experiments revealed that pro-inflammatory cytokines, whose production was increased following cell-cell contact, were unlikely to be primarily involved in the induced HIV-1 replication. Cell-cell contact, but not soluble components in the culture supernatant, caused a rapid phosphorylation and degradation of IkappaBalpha, which led to elevated NF-kappaB DNA binding activity in J(22)-HL-60 cells. Furthermore, forced expression of a super-repressor form of IkappaBalpha or pretreatment with ritonavir efficiently blocked the activation of NF-kappaB and HIV-1 replication in J(22)-HL-60 cells co-cultured with MOLT-4 T cells. Moreover, either resting or PHA stimulated primary CD4(+) T cells induced HIV-1 replication in J(22)-HL-60 cells in a similar way with that of MOLT-4 cells. These results indicated that direct contact with CD4(+) T cells induced HIV-1 replication in latently infected cells and provide insight into the molecular mechanism of virus release from myeloid progenitor cells latently infected with HIV-1.     

Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells

To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.     

Regulation of primary HIV-1 isolate replication in dendritic cells

The potential role of dendritic cells (DC) in the immunopathology of human immunodeficiency virus 1 (HIV-1) disease remains controversial. This study examines replication of a panel of HIV-1 strains (both laboratory adapted and primary) within DC, in the context of the well-established monocyte-DC and monocyte-macrophage transition. Viral replication was assessed by p24 ELISA assay. All strains of HIV-1 tested replicated in DC. Only CCR5-tropic virus replicated in macrophages. Lipopolysaccharide (LPS) induced DC maturation (as reflected in altered cell phenotype) and at the same time diminished the ability of DC to support HIV-1 replication. In contrast the presence of activated T cells, which had been fixed to prevent them acting as a site for viral replication, enhanced the ability of the DC to support viral replication, as has been reported previously for macrophages. Thus cells that are DC by phenotype, but are not activated, act as the optimum reservoir for HIV-1 replication. If this form of DC is present in peripheral tissues, this will be permissive for amplification of the in vivo viral load at sites where there are few responder cells available, and hence contribute to the persistent immunopathology.     

A recombinant vesicular stomatitis virus encoding HIV-1 receptors and human OX40 ligand efficiently eliminates HIV-1-infected CD4-positive T cells expressing OX40

OX40 protein is highly expressed on activated CD4-positive T cells that are susceptible for human immunodeficiency virus type 1 (HIV-1) infection. To target and kill HIV-1-infected OX40(+) T cells, we used a recombinant vesicular stomatitis virus (rVSV) lacking its envelope glycoprotein (ΔG) and instead expressing HIV-1 receptors CD4/CXCR4 and OX40 ligand (OX40L). Expression of OX40L as well as HIV-1 receptors on the VSV particles led to specific infection of OX40(+) T cells, including primary cells, either acutely or chronically infected with X4 HIV-1. Consequently, the rVSV rapidly eliminated these infected cells and caused a marked reduction of HIV-1 viral load in culture. Inclusion of the OX40L gene in the VSV recombinant led to significantly better infection and HIV-1 elimination compared with an rVSVΔG expressing only HIV-1 receptors. A novel rVSVΔG encoding both HIV-1 receptors and OX40L has a potentially greater therapeutic value than an rVSVΔG expressing only HIV-1 receptors.     

Microanatomic relationships between CD8+ cells and HIV-1-producing cells in human lymphoid tissue in vivo

OBJECTIVE: Host immune responses are unable to fully suppress HIV-1 replication in lymphoid tissues. Microanatomic relationships between HIV-1-producing cells and CD8+ cells in lymphoid tissues were analyzed to determine whether there was evidence for an immune privileged site or impaired recognition of virus-producing cells. METHODS: CD8+ cell phenotypes were determined on disaggregated inguinal lymph node cells by flow cytometry for seven untreated HIV-1-infected subjects. Microanatomic relationships between HIV-1-producing cells and CD8+ cells were analyzed in lymph node sections from 15 HIV-1-infected individuals using in situ hybridization and immunohistochemical staining. RESULTS: Most (median, 96%) lymph node CD8+ cells coexpressed CD3. Frequencies of virus-producing cells detected by in situ hybridization correlated with plasma HIV-1 RNA concentration (Spearman rho = 0.70; p =.02; n = 11). The percentage of lymph node cells adjacent to virus-producing cells that were CD8+ (median, 29%) was not statistically different from the percentage of CD8+ cells in lymphoid tissue overall (median, 34%; p =.09). CONCLUSIONS: Multiple explanations could account for the observation that CD8+ cells do not preferentially accumulate around virus-producing cells including the possibility that HIV-1-specific CD8+ cells cannot recognize virus-producing cells. Further studies are necessary to determine whether HIV-1-specific CD8+ T cells aggregate around virus-producing cells in lymphoid tissue.     

Upregulation of HIV-1 replication in chronically infected cells by ingenol derivatives

Summary.  We have previously reported that ingenol derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in acutely infected cells. In this study, however, we have found that some ingenol derivatives strongly enhance the replication of HIV-1 in chronically infected cells at nanomolar concentrations. One of the derivatives could activate nuclear factor κB(NF-κB), a potent inducer of HIV-1 replication, through the activation of protein kinase C (PKC). Whereas another derivative, which affected neither PKC nor NF-κB, significantly enhanced HIV-1 replication, suggesting that a PKC-independent mechanism may also exist in ingenol derivative-induced HIV-1 upregulation. Received February 23, 1998 Accepted May 21, 1998     

The role of upstream U3 sequences in HIV-1 replication and CD4+ T cell depletion in human lymphoid tissue ex vivo

The LTRs of all primate lentiviruses contain long U3 regions overlapping the nef gene. To assess the relevance of the modulatory U3 region for HIV-1 replication, we inactivated the T-rich region, the Polypurine tract and attachment (att) sequences in nef by silent mutations and inserted intact cis-regulatory elements just upstream of the core enhancer. These modifications severely truncated the U3 region and eliminated the nef overlap. The resulting HIV-1 mutants expressed functional Nef, replicated efficiently and caused CD4+ T cell depletion in ex vivo-infected lymphoid tissue suggesting that the modulatory U3 region might not be essential for efficient HIV-1 gene expression and AIDS pathogenesis.     

Extracts of hamster cells abortively infected with human adenovirus type 12 are competent to support initiation of viral DNA replication

Baby hamster kidney (BHK-21) cells do not allow replication of human adenovirus type 12 (Ad12) DNA during abortive infection by this virus. However, we have determined that crude extracts of BHK-21 cells abortively infected with Ad12 support in vitro the initiation reaction of Ad12 DNA replication. Synthesis of the Ad12 pTP-dCMP initiation complex by BHK extracts is two- to five-fold less than when crude infected human (KB) cell extracts are used in the reaction. Combining infected KB cytoplasmic and uninfected BHK nuclear extracts in the reaction indicates that the decreased efficiency is probably due to a lesser ability of hamster nuclear extracts to support the initiation reaction, rather than to decreased synthesis of Ad12 pTP and DNA polymerase during abortive infection, or to the presence of an inhibitor in BHK cells.     

Restricted replication of primary HIV-1 isolates using both CCR5 and CXCR4 in Th2 but not in Th1 CD4(+) T cells

We have previously reported that CCR5-dependent human immunodeficiency virus type-1 (HIV-1; R5), but not CXCR4-restricted (X4) virus, efficiently replicates in T helper cell type 1 (Th1), Th2, or Th0 polyclonal T cells obtained from human umbilical cord blood (CB lines). The X4 virus restriction was env-dependent but did not occur at the level of viral entry. Here, we describe that in contrast to these monotropic HIVs, primary HIV-1 isolates capable of using CCR5 or CXCR4 indifferently for entry (i.e., R5X4 viruses) efficiently replicated in Th2 but not in Th1 CB lines. Although Th1 cells secreted significantly higher amounts of the three CCR5-binding chemokines in comparison with Th2 cells, this restriction was not explained by a defective infection of Th1 cells. Interferon-gamma (IFN-gamma) down-regulated CCR5 in Th1 cells and inhibited, whereas interleukin-4 (IL-4) up-regulated CXCR4 and enhanced the spreading of R5 and R5X4 viruses in polarized CB lines. However, both cytokines did not rescue the replication of X4 and dualtropic viruses in both types of CB lines or in Th1 cells, respectively, whereas addition of anti-IL-4- or anti-IFN-gamma-neutralizing antibodies did not activate virus expression. These findings together suggest the existence of post-entry restriction pathways influenced by gp120 Env/chemokine coreceptor interaction that may significantly contribute to the superior capacity of R5 and R5X4 HIV-1 strains to spread in vivo in comparison to X4 monotropic viruses.     

Variable fate of virus-specific CD4(+) T cells during primary HIV-1 infection.

Impairment of CD4(+) T lymphocyte responses to human immunodeficiency virus (HIV)-derived antigens is the classic immunological defect observed during the chronic phase of HIV-1 infection. Early intervention with potent antiretroviral therapy (ART) can preserve HIV-specific CD4(+) T lymphocyte reactivity, providing indirect evidence that such responses are mounted during primary infection and subsequently lost in the majority of infected individuals. Here, we demonstrate early and dramatic expansions of functional HIV-specific CD4(+) T lymphocyte frequencies directly ex vivo. These responses are initially of broad specificity, and can disappear rapidly during the natural course of primary infection. This process of loss is variable, such that the rapidity and extent of functional compromise differs between individuals. Institution of ART during these early phases of HIV-1 infection preserves patterns of functional reactivity within the HIV-specific CD4(+) T lymphocyte population. However, there was no evidence for the restoration of deleted responses. These findings indicate that, in some individuals at least, ART must be administered within a narrow window of opportunity during primary HIV-1 infection to effect substantial immune preservation.     

CD3和CD20阳性细胞在人胎腭扁桃体的发育方法

目的 探讨T、B细胞在人胎儿腭扁桃体内的发育情况.方法 收集9～20周因故终止妊娠胎儿腭扁桃体22例,采用免疫组织化学方法,以细胞表面分化群(CD)抗体(CD3和CD20)染色分别显示T细胞和B细胞,用BioMiaspro图像分析软件对免疫反应阳性细胞进行计数,有关数据做统计学分析.结果 妊娠9周时,胎儿腭扁桃体原基内...