Plasma membrane signaling in HIV-1 infection

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4 + T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

NKT cells in HIV-1 infection

Natural killer T （NKT） cells are a unique T cell population that have important immunoregulatory functions and have been shown to be involved in host immunity against a range of microorganisms. It also emerges that they might play a role in HIV-1 infection, and therefore be selectively depleted during the early stages of infection. Recent studies are reviewed regarding the dynamics of NKT depletion during HIV-1 infection and their recovery under highly active antiretroviral treatment （HAART）. Possible mechanisms for these changes are proposed based on the recent developments in HIV pathogenesis. Further discussions are focused on HIV＇s disruption of NKT activation by downregulating CDld expression on antigen presentation cells （APC）. HIV-1 protein Nefis found to play the major role by interrupting the intracellular trafficking of nascent and recycling CDld molecules.     

A Study of Two Procedures of HIV-1 Isolation from Whole Blood Cultures

Culture techniques for isolation of HIV-1 from small amounts of whole blood (WB) treated with anticoagulant have been reported and gave results identical to those of culture of separated peripheral blood mononuclear cells. Some authors obtained much higher isolation rates when EDTA was used instead of heparin. We compared two previously described techniques for cultivation of HIV-1 from WB of adult HIV+ patients staged according to the CDC classification. In addition, we assessed the influence of the type of anticoagulant used for the collection of blood in viral replication in cell cultures from whole blood. Small volumes of WB treated with either heparin or EDTA were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMC) from healthy donors. We used two procedures for WB culture: procedure I, based on the culture of 250 μl of WB with 1 × 10⁶ PHA-PBMC from donors; and procedure II based on the culture of 500 μl of WB with 4× 10⁶ PHA-PBMC from donors. The cocultures were placed in 24-well plates and incubated for as long as 28 days in medium containing interleukin 2 (IL-2). Twice weekly half of the medium was replaced with fresh medium. In procedure II, one million fresh PHA-PBMC from donors was added on the 7th day of culture. The culture supernatant was assayed for the presence of HIV-1 p24 antigen in an enzyme immunoassay. The kinetics of HIV-1 replication in cultures of WB from 7 AIDS patients were similar using procedures I and II. In 8 HIV + patients the isolation rate was higher with heparin- than with EDTA-treated samples. The isolation rate was higher in AIDS patients (n = 8) than in others with both methods. In stage IV patients without AIDS (n = 8) we failed to isolate HIV-1 in 1 patient with procedure I, whereas we succeeded with procedure II. In stage II, HIV-I was isolated in 1 of 4 patients with both methods. HIV was isolated in cultures of WB from patients receiving zidovudine or related nucleoside analogues and in cultures of WB from untreated patients. HIV-1 could not be isolated from WB of patients with more than 400 CD4+ T lymphocytes in their peripheral blood (n=4); however, it was isolated from 14 of 16 patients with less than 400 CD4+ T lymphocytes. Our results suggest that procedure II is more sensitive than procedure I and that heparin is better than EDTA for collecting WB. We showed that the rate of HIV-1 isolation from WB increased in advanced-stage patients. Further studies are needed to define the clinical applications of WB culture.     

Decreased regulatory T‐cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4⁺/CD8⁺ ratio and CD4⁺CD25^hiFoxP3⁺ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4⁺/CD8⁺ ratio and Tregs in 82 patients and 50 controls. CD8⁺ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8⁺ T‐cell counts compared with stable GV patients (p = 0.001). The CD4⁺/CD8⁺ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4⁺/CD8⁺ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.     

From pathogen to medicine: HIV-1-derived lentiviral vectors as vehicles for dendritic cell based cancer immunotherapy

Over the years, the unique capacity of dendritic cells (DC) for efficient activation of naive T cells has led to their extensive use in cancer immunotherapy protocols. In order to be able to fulfil their role as antigen-presenting cells, the antigen of interest needs to be efficiently introduced and subsequently correctly processed and presented by the DC. For this purpose, a variety of both viral and non-viral antigen-delivery systems have been evaluated. Amongst those, HIV-1-derived lentiviral vectors have been used successfully to transduce DC.This review considers the use of HIV-1-derived lentiviral vectors to transduce human and murine DC for cancer immunotherapy. Lentivirally transduced DC have been shown to present antigenic peptides, prime transgene-specific T cells in vitro and elicit a protective cytotoxic T-lymphocyte (CTL) response in animal models. Different parameters determining the efficacy of transduction are considered. The influence of lentiviral transduction on the DC phenotype and function is described and the induction of immune responses by lentivirally transduced DC in vitro and in vivo is discussed in detail. In addition, direct in vivo administration of lentiviral vectors aiming at the induction of antigen-specific immunity is reviewed. This strategy might overcome the need for ex vivo generation and antigen loading of DC. Finally, future perspectives towards the use of lentiviral vectors in cancer immunotherapy are presented.     

Tumor Necrosis Factor Receptor Expression in HIV-1-Infected CD4+ T Cells

We investigated whether HIV-1 can regulate tumor necrosis factor receptor (TNFR) expression in SupT-1, a CD4 + T-cell line. The cells were infected with HIV-1 containing 1,000 cpm RT activity, as early as day 3 after infection and all along the culture the supernatant level of core protein p24 was >250 pg/ml, and on days 6 and 9 after infection, p24 was found in 10 % of the cells as determined by indirect immunofluorescence assay. The cells were growing without loss of viability. The study of TNFR expression was based on a microassay for measurement of binding of ¹²⁵I-TNFα to cells, in which free and cell-bound ligand separation was performed by centrifugation through oil. Scatchard analysis of TNFα binding on days 6 and 9 after infection revealed a 90 % increase in the expression of high-affinity membrane receptors in HIV + SupT-1 culture compared with uninfected cells (mean +/-S.D. = 501 +/-148.5 vs. 263 +/-77.8 receptors/cell, n = 9, P< 0.001) with no change in dissociation constants (mean +/? S.D. = 4.36 +/?1.06 vs. 4.00 +/?1.12 × 10^?10 m ).     

Human neutrophil alpha-defensin 4 inhibits HIV-1 infection in vitro

Human neutrophil alpha-defensin 4 (HNP4) is more effective than HNP1-3 in protecting human peripheral blood mononuclear cells from infection by both X4 and R5 HIV-1 strains. HNP4 binds to both CD4 and gp120 approximately two orders of magnitude weaker than does HNP1, and is less effectively sequestered by glycosylated serum proteins than HNP1. These results suggest that the HIV-1 inhibition by HNP4 stems at least partially from a unique and lectin-independent property of HNP4 with CD4 and/or gp120. Our finding identifies an anti-HIV-1 property of HNP4 and may have implications in the development of new antiviral agents for AIDS therapy.     

The neuropathogenesis of HIV-1 infection

HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mononuclear phagocytes (MP) (macrophages and microglia) are reservoirs for persistent viral infection. When activated, MP contribute to neuronal damage. Such activated and virus-infected macrophages secrete cellular and viral factors, triggering neural destructive immune responses. Our Center's laboratories have begun to decipher the molecular and biochemical pathways for MP-mediated neuronal damage in HAD. This review will discuss the salient clinical and pathological features of HAD and highlight the recent advances made, by our scientists and elsewhere, in unraveling disease mechanisms, including the role of chemokines and their receptors in the neuropathogenesis of HIV-1 encephalitis.     

Influence of fibronectin on HIV-1 infection and capability of binding to platelets

We have previously demonstrated that fibronectin (FN) can bind HIV-1 envelope proteins, in particular gp120. The aim of the present study was to determine some biological effects of this phenomenon. Pretreating HIV-1 with human FN increased the infectivity of HIV-1, when a low concentration of the virus was used. In contrast, an RGD-containing pentapeptide (Gly-Arg-Gly-Asp-Ser), which is a fundamental binding site of FN, reduced the infectivity of a suspension of HIV-1 at high concentrations of the virus. It is likely that FN bridges the cell surface and the virions, while the RGD-containing pentapeptide may saturate the HIV-1 binding sites for cell surface receptors. Moreover, gp120 was bound to the FN present on the surface of platelets. The specifity of this binding was confirmed by the inhibition obtained by pretreating platelets with anti-FN antibodies. The consequence of the surface modifications of the platelets could explain the thrombocytopenia that frequently occurs in patients infected with HIV and suggests also the possibility that platelets could be a vehicle for the virus in the circulation.     

Mathematical analysis of delay differential equation models of HIV-1 infection

Models of HIV-1 infection that include intracellular delays are more accurate representations of the biology and change the estimated values of kinetic parameters when compared to models without delays. We develop and analyze a set of models that include intracellular delays, combination antiretroviral therapy, and the dynamics of both infected and uninfected T cells. We show that when the drug efficacy is less than perfect the estimated value of the loss rate of productively infected T cells, delta, is increased when data is fit with delay models compared to the values estimated with a non-delay model. We provide a mathematical justification for this increased value of delta. We also provide some general results on the stability of non-linear delay differential equation infection models.     

HIV-1-specific functional immune measurements as markers of disease progression

The impairment of lymphocytes to proliferate to HIV antigen is a relatively early functional defect of cell-mediated immunity found in HIV-infected individuals. The finding of strong proliferative responses in nonprogressive HIV disease as well as its inverse association with viral load and clinical manifestation of AIDS supports the further use of this marker as a surrogate of disease progression. The observation that HIV-specific lymphocyte proliferation is associated with the production of CD8-derived HIV suppressive factors such as the -chemokines further supports this conclusion. These functional immune measurements provide an additional marker to monitor disease progression in HIV-infected individuals, along with the current standards of CD4 counts and viral load.     

Extrachromosomal human immunodeficiency virus type-1 DNA can initiate a spreading infection of HL-60 cells

In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to less than 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was less than 0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.     

Intrarectal inoculation of macaques by the simian immunodeficiency virus,SIVmne E11S: CD4 + depletion and AIDS

Macaca nemestrina and Macaca fascicularis were inoculated with various doses of a single-cell clone of SIV_mne-infected HuT 78 cells (E11S) by both the intravenous and intrarectal routes. Animals inoculated intravenously at each dose seroconverted and virus was isolated from peripheral blood mononuclear cells, but only the high-dose intrarectally exposed macaques became viremic and seroconverted. However, some seronegative, virus isolation negative intrarectally inoculated macaques showed evidence of infection and disease.     

Systemic and mucosal immunity in rhesus macaques immunized with HIV-1 peptide and gp120 conjugated to Brucella abortus

HIV vaccine testing in primates is an important method for determining the possibility of vaccine benefit in humans. Goals of HIV-1 vaccination include establishing neutralizing antibodies and a strong CD8(+) T-cell response. We tested a novel vaccine conjugate for its ability to elicit relevant immune responses to HIV proteins and peptides in rhesus macaques. A neutralizing epitope, V3 loop peptide from HIV-1 envelope, was coupled to heat-inactivated Brucella abortus (V3-HKBA). Rhesus macaques were immunized with this conjugate in the anterior thigh. After two immunizations V3-specific antibodies were found in the sera and at mucosal sites. Neutralizing activity of these antibodies was demonstrated by syncytia inhibition assays. Cellular immune recall responses were demonstrated by antigen-specific induction of interferon-gamma and Regulation on Activation Noraml T Cell Expressed and Secreted (RANTES) secretion in vitro. These results confirm and extend preliminary studies in mice that suggest HKBA is an effective carrier that promotes neutralizing antibody secretion at relevant mucosal sites, as well as cellular immune responses that are correlated with viral protection.     

A retrospective evaluation of the role of T cells in the development of malaria vaccine

Due to the fact that the life cycle of malaria parasites is complex, undergoing both an extracellular and intracellular phases in its host, the human immune system has to mobilize both the humoral and cellular arms of immune responses to fight against this parasitic infection. Whereas humoral immunity is directed toward the extracellular stages which include sporozoites and merozoites, cell-mediated immunity (CMI), in which T cells play a major role, targets hepatic stages - liver stages - of the parasites. In this review, the role of T cells in protective immunity against liver stages of the malaria infection is being re-evaluated. Furthermore, this review intends to address how to translate the findings regarding the role of T cells obtained in experimental systems to actual development of malaria vaccine for humans.     

打靶恒河猴CD4~+ T细胞的TRIM5α基因影响其感染HIV的能力

目的:探讨打靶恒河猴CD4~+ T细胞的TRIM5α基因对其感染HIV-1能力的影响。方法:从恒河猴的外周血中通过磁珠分选获得CD4~+ T细胞,并采用流式检测阳性率。构建打靶TRIM5α基因的TALEN质粒,通过电转导入CD4~+ T细胞,流式分选出转染TALEN质粒的细胞,提取基因组T7E1酶切检测打靶效率。HIV-1病毒感染打靶TRIM5α的CD4~+ T细胞,并通过ELASA检测病毒感染的情况。结果:成功地从恒河猴的外周血中分选出了CD4~+ T细胞,流式检测阳性率为99.5%。打靶TRIM5α基因的TALEN质粒转染CD4~+ T细胞的转染效率约为24.8%,并可成功打靶TRIM5α,打靶效率约为40%。ELASA检测结果表明打靶TRIM5α的恒河猴CD4~+ T细胞对HIV病毒的感染能力增强。结论打靶恒河猴CD4~+ T细胞的TRIM5α基因可使其易感HIV病毒,为进一步建立恒河猴HIV-1感染动物模型奠定基础。     

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.     

Expression of PD‐1/LAG‐3 and cytokine production by CD4+ T cells during infection with Plasmodium parasites

CD4⁺ T cells play critical roles in protection against the blood stage of malarial infection; however, their uncontrolled activation can be harmful to the host. In this study, in which rodent models of Plasmodium parasites were used, the expression of inhibitory receptors on activated CD4⁺ T cells and their cytokine production was compared with their expression in a bacterial and another protozoan infection. CD4⁺ T cells from mice infected with P. yoelii 17XL, P yoelii 17XNL, P. chabaudi, P. vinckei and P. berghei expressed the inhibitory receptors, PD‐1 and LAG‐3, as early as 6 days after infection, whereas those from either Listeria monocytogenes‐ or Leishmania major‐infected mice did not. In response to T‐cell receptor stimulation, CD4⁺ T cells from mice infected with all the pathogens under study produced high concentrations of IFN‐γ. IL‐2 production was reduced in mice infected with Plasmodium species, but not in those infected with Listeria or Leishmania. In vitro blockade of the interaction between PD‐1 and its ligands resulted in increased IFN‐γ production in response to Plasmodium antigens, implying that PD‐1 expressed on activated CD4⁺ T cells actively inhibits T cell immune responses. Studies using Myd88^?/?, Trif^?/? and Irf3^?/? mice showed that induction of these CD4⁺ T cells and their ability to produce cytokines is largely independent of TLR signaling. These studies suggest that expression of the inhibitory receptors PD‐1 and LAG‐3 on CD4⁺ T cells and their reduced IL‐2 production are common characteristic features of Plasmodium infection.

     

T helper 1 effector memory CD4+ T cells protect the skin from poxvirus infection

1. Download : Download high-res image (224KB)
2. Download : Download full-size image