Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

HIV-1 Induces DCIR Expression in CD4+ T Cells

The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4⁺ T cells found in the synovial tissue from rheumatoid arthritis (RA) patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4⁺ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4⁺ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons) and cells acutely infected in vitro (seen in both virus-infected and uninfected cells). Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4⁺ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals) and -independent intrinsic apoptotic pathways (involving the death effector AIF). Finally, we demonstrate that the higher surface expression of DCIR in CD4⁺ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4⁺ T cells, a process that might promote virus dissemination throughout the infected organism.     

Strong Ability of Nef-Specific CD4+ Cytotoxic T Cells To Suppress Human Immunodeficiency Virus Type 1 (HIV-1) Replication in HIV-1-Infected CD4+ T Cells and Macrophages

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4⁺ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4⁺ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4⁺ T cells. The CD4⁺ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4⁺ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4⁺ T cells from an HLA-DRB1*0803⁺ donor. In addition, these Nef-specific cytotoxic CD4⁺ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4⁺ T cells in vitro. Nef187-203-specific cytotoxic CD4⁺ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803⁺ donors, respectively. These results suggest that HIV-1-specific CD4⁺ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.Human immunodeficiency virus (HIV)-specific CD8⁺ cytotoxic T cells (CTLs) play a central role in the control of HIV type 1 (HIV-1) during acute and chronic phases of an HIV-1 infection (5, 29, 34). However, HIV-1 escapes from the immune surveillance of CD8⁺ CTLs by mechanisms such as mutations of immunodominant CTL epitopes and downregulation of major histocompatibility complex class I (MHC-I) molecules on the infected cells (9, 11, 12, 49). Therefore, most HIV-1-infected patients without highly active antiretroviral therapy (HAART) develop AIDS eventually.HIV-1-specific CD4⁺ T cells also play an important role in host immune responses against HIV-1 infections. An inverse association of CD4⁺ T-cell responses with viral load in chronically HIV-1-infected patients was documented in a series of earlier studies (8, 36, 39, 41, 48), although the causal relationship between them still remains unclear (23). Classically, CD4⁺ T cells help the expansion of CD8⁺ CTLs by producing growth factors such as interleukin-2 (IL-2) or by their CD40 ligand interaction with antigen-processing cells and CD8⁺ CTLs. In addition, CD4⁺ T cells provide activation of macrophages, which can professionally maintain CD8⁺ T-cell memory (17). On the other hand, the direct ability of virus-specific cytotoxic CD4⁺ T cells (CD4⁺ CTLs) to kill target cells has been widely observed in human virus infections such as those by human cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus, Dengue virus, and HIV-1 (2, 4, 10, 19, 30, 31, 38, 50). Furthermore, one study showed that mouse CD4⁺ T cells specific for lymphocytic choriomeningitis virus have cytotoxic activity in vivo (25). These results, taken together, indicate that a subset of effector CD4⁺ T cells develops cytolytic activity in response to virus infections.HIV-1-specific CD4⁺ CTLs were found to be prevalent in HIV-1 infections, as Gag-specific cytotoxic CD4⁺ T cells were detected directly ex vivo among peripheral blood mononuclear cells (PBMCs) from an HIV-1-infected long-term nonprogressor (31). Other studies showed that up to 50% of the CD4⁺ T cells in some HIV-1-infected donors can exhibit a clear cytolytic potential, in contrast to the fact that healthy individuals display few of these cells (3, 4). These studies indicate the real existence of CD4⁺ CTLs in HIV-1 infections.The roles of CD4⁺ CTLs in the control of an HIV-1 infection have not been widely explored. It is known that Gag-specific CD4⁺ CTLs can suppress HIV-1 replication in a human T-cell leukemia virus type 1-immortalized CD4⁺ T-cell line (31). However, the functions of CD4⁺ T cells specific for other HIV-1 antigens remain unclear. On the other hand, the abilities of CD4⁺ CTLs to suppress HIV-1 replication in infected macrophages and CD4⁺ T cells may be different, as in the case of CD8⁺ CTLs for HIV-1-infected macrophages (17). In this study, we identified Nef-specific CD4⁺ T cells and investigated their ability to kill HIV-1 R5 virus-infected macrophages and HIV-1 X4 virus-infected CD4⁺ T cells and to suppress HIV-1 replication in the infected macrophages and CD4⁺ T cells. The results obtained in the present study show for the first time the ability of HIV-1-specific CD4⁺ CTLs to suppress HIV-1 replication in natural host cells, i.e., macrophages and CD4⁺ T cells.     

Random T-cell receptor recruitment in human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells from genetically identical twins infected with the same HIV-1 strain

Human immunodeficiency virus type 1 (HIV-1) cytotoxic T-lymphocyte escape mutations represent both a major reason for loss of HIV immune control and a considerable challenge for HIV-1 vaccine design. Previous data suggest that initial HIV-1-specific CD8⁺ T-cell responses are determined largely by viral and host genetics, but the mechanisms influencing the subsequent viral evolution are unclear. Here, we show a random recruitment of T-cell receptor (TCR) alpha and beta clonotypes of the initial HIV-1-specific CD8⁺ T cells during primary infection in two genetically identical twins infected simultaneously with the same virus, suggesting that stochastic TCR recruitment of HIV-1-specific CD8⁺ T cells contributes to the diverse and unpredictable HIV-1 sequence evolution.     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.     

Human Erythrocytes Selectively Bind and Enrich Infectious HIV-1 Virions

Although CD4(+) cells represent the major target for HIV infection in blood, claims of complement-independent binding of HIV-1 to erythrocytes and the possible role of Duffy blood group antigen, have generated controversy. To examine the question of binding to erythrocytes, HIV-1 was incubated in vitro with erythrocytes from 30 healthy leukapheresis donors, and binding was determined by p24 analysis and adsorption of HIV-1 with reduction of infectivity for CD4(+) target cells. All of the cells, regardless of blood group type, bound HIV-1 p24. A typical preparation of erythrocytes bound <2.4% of the added p24, but erythrocytes selectively removed essentially all of the viral infectivity as determined by decreased infection of CD4(+) target cells; however, cell-associated HIV-1 was approximately 100-fold more efficient, via trans infection, than unadsorbed virus for infection of CD4(+) cells. All of the bound HIV-1 p24 was released by treatment of the cells with EDTA, and binding was optimized by adding Ca²⁺ and Mg²⁺ during the washing of erythrocytes containing bound HIV-1. Although the small number of contaminating leukocytes in the erythrocyte preparation also bound HIV-1 p24, there was no significant binding to CD4, and it thus appears that the binding occurred on leukocytes at non-CD4 sites. Furthermore, binding occurred to erythrocyte ghosts from which contaminating leukocytes had been previously removed. The results demonstrate that erythrocytes incubated in vitro with HIV-1 differentially adsorb all of the infectious HIV-1 virions (as opposed to non-infectious or degraded virions) in the absence of complement and independent of blood group, and binding is dependent on divalent cations. By analogy with HIV-1 bound to DC-SIGN on dendritic cells, erythrocyte-bound HIV-1 might comprise an important surface reservoir for trans infection of permissive cells.     

APOPTOSIS-INDUCED BY TRAIL AND TNF-α IN HUMAN MULTIPLE MYELOMA CELLS IS NOT BLOCKED BY BCL-2

TRAIL, the ligand for the newly discovered DR-4 and DR-5 receptor is a member of the tumour necrosis factor (TNF) family of death signal tranduction proteins with a mechanism of cell death, similar to the Fas and Fas ligand (Fas-L) system. Here, we provide first time evidence that TRAIL and TNF-α are potent inducers of apoptosis in multiple myeloma (MM) cell lines and freshly isolated myeloma cells. TRAIL effectively induced extensive apoptosis in 8226 and ARP-1 MM cells in a time- and dose-dependent manner reaching 80% within 48 h of treatment with a dose of 160 ng/ml. Bcl-2 transfected 8226 and ARP-1 cells were equally sensitive to apoptosis by TRAIL. Apoptosis with TNFα, reached >60% within 48 h of treatment with a dose of 160 ng/ml. In addition to MM cell lines, freshly isolated, flow-sorted myeloma cells from 8 different MM patients expressing variable levels of bcl-2 were equally sensitive to both TRAIL and TNF-α. We have previously shown that anti-Fas-induced apoptosis is not blocked by endogenous or ectopic bcl-2 in MM cell lines. Here we extend our observation with Fas to include TNF-α and TRAIL to the apoptotic signals that are not be blocked by bcl-2, in MM cells.     

Identification of a potential HIV-induced source of bystander-mediated apoptosis in T cells: Upregulation of TRAIL in primary human macrophages by HIV-1 tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.     

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef⁺ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.     

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses,Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4⁺ and CD8⁺ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4⁺ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4⁺ T cell depletion which renders the newly activated HIV-specific CD4⁺ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.     

Ligand-independent exhaustion of killer immunoglobulin-like receptor-positive CD8+ T cells in human immunodeficiency virus type 1 infection

Virus-specific CD8⁺ T cells play a central role in the control of viral infections, including human immunodeficiency virus type 1 (HIV-1) infection. However, despite the presence of strong and broad HIV-specific CD8⁺ T-cell responses in chronic HIV-1 infection, these cells progressively lose critical effector functions and fail to clear the infection. Mounting evidence suggests that the upregulation of several inhibitory regulatory receptors on the surface of CD8⁺ T cells during HIV-1 infection may contribute directly to the impairment of T-cell function. Here, we investigated the role of killer immunoglobulin receptors (KIR), which are expressed on NK cells and on CD8⁺ T cells, in regulating CD8⁺ T-cell function in HIV-1 infection. KIR expression was progressively upregulated on CD8⁺ T cells during HIV-1 infection and correlated with the level of viral replication. Expression of KIR was associated with a profound inhibition of cytokine secretion, degranulation, proliferation, and activation by CD8⁺ T cells following stimulation with T-cell receptor (TCR)-dependent stimuli. In contrast, KIR⁺ CD8⁺ T cells responded potently to TCR-independent stimulation, demonstrating that these cells are functionally competent. KIR-associated suppression of CD8⁺ T-cell function was independent of ligand engagement, suggesting that these regulatory receptors may constitutively repress TCR activation. This ligand-independent repression of TCR activation of KIR⁺ CD8⁺ T cells may represent a significant barrier to therapeutic interventions aimed at improving the quality of the HIV-specific CD8⁺ T-cell response in infected individuals.     

ULTRAVIOLET B IRRADIATION MODULATES SUSCEPTIBILITY TO TUMOUR NECROSIS FACTOR-α-INDUCED APOPTOSIS VIA INDUCTION OF DEATH RECEPTORS IN MURINE FIBROBLASTS

Ultraviolet B (UVB) irradiation causes cell death by apoptosis in murine fibroblast cells. Tumor necrosis factor-α (TNF-α) is also a well known inducer of apoptosis, although the physiological significance of this activity is poorly understood. We investigated the effects of pretreatment with UVB (312 nm) on TNF-α-induced apoptosis in murine fibroblast cells. UVB enhanced susceptibility to cell death by TNF-α in a dose-dependent manner. UVB but not TNF-α induced the expression of TNF receptor type-1 (TNFR-1) and type-2 (TNFR-2) in a dose-dependent manner. Expression of Fas (CD95) and Fas-ligand (Fas-L), and significant DNA fragmentation were observed in the cells that died. These results suggest that UVB irradiation modulates susceptibility to TNF-α-induced apoptosis through the induction of TNFRs, Fas, and Fas-L in murine fibroblasts.     

Inability of Plasmacytoid Dendritic Cells To Directly Lyse HIV-Infected Autologous CD4+ T Cells despite Induction of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

The function of plasmacytoid dendritic cells (PDC) in chronic human immunodeficiency virus type 1 (HIV-1) infection remains controversial with regard to its potential for sustained alpha interferon (IFN-α) production and induction of PDC-dependent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity of HIV-infected cells. We address these areas by a study of chronically HIV-1-infected subjects followed through antiretroviral therapy (ART) interruption and by testing PDC cytolytic function against autologous HIV-infected CD4⁺ T cells. Rebound in viremia induced by therapy interruption showed a positive association between TRAIL and viral load or T-cell activation, but comparable levels of plasma IFN-α/β were found in viremic ART-treated and control subjects. While PDC from HIV-infected subjects expressed less interferon regulator factor 7 (IRF-7) and produced significantly less IFN-α upon Toll-like receptor 7/9 (TLR7/9) engagement than controls, membrane TRAIL expression in PDC from HIV⁺ subjects was increased. Moreover, no significant increase in death receptor 5 (DR5) expression was seen in CD4⁺ T cells from viremic HIV⁺ subjects compared to controls or following in vitro infection/exposure to infectious and noninfectious virus or exogenous IFN-α, respectively. Although activated PDC killed the DR5-expressing HIV-infected Sup-T1 cell line, PDC did not lyse primary autologous HIV⁺ CD4⁺ T cells yet could provide accessory help for NK cells in killing HIV-infected autologous CD4⁺ T cells. Taken together, our data show a lack of sustained high levels of soluble IFN-α in chronic HIV-1 infection in vivo and document a lack of direct PDC cytolytic activity against autologous infected or uninfected CD4⁺ T cells.Human immunodeficiency virus (HIV) infection is associated with chronic immune activation, progressive immune suppression, and deletion of memory adaptive responses, resulting in increased susceptibility to opportunistic infections (23, 51, 52). Loss of CD4⁺ T cells is the hallmark of HIV infection, with multiple mechanisms proposed as contributing to this loss (activation-induced cell death, direct cytopathic effect, immune cells, and death receptor-mediated apoptosis induction) (reviewed in references 33 and 34). One of the most puzzling observations in AIDS pathogenesis has been the progressive depletion of bystander T cells, especially in lymphoid tissues (25, 33, 34, 55). While antiretroviral therapy (ART) initiated in the early stages of HIV infection, when CD4⁺ T-cell counts are high (>500 cells/μl), may prevent the destruction of lymph node (LN) tissue and the massive depletion of CD4⁺ T lymphocytes by decreasing the rate of virally induced apoptosis (20), a persistent, albeit decreased, level of apoptosis of peripheral blood CD4⁺ and CD8⁺ T cells is seen in ART-treated HIV⁺ subjects despite long-term viral suppression (36).A member of the tumor necrosis factor (TNF) family, TNF-related apoptosis-inducing ligand (TRAIL), has been shown to be involved in HIV-1-associated T-cell apoptosis (33, 34). TRAIL (soluble or membrane bound) induces apoptosis upon binding to death receptor 4 (DR4; also named TRAIL-R1) or DR5 (also named TRAIL-R2, TRICK2, or Killer/DR5).On the basis of the in vitro observation that alpha interferon (IFN-α) and interferon regulator factor 7 (IRF-7) are increased in plasmacytoid dendritic cells (PDC) exposed to HIV-1 (40), the hypothesis that PDC activation by HIV-1 is responsible for an increased level of IFN-α throughout chronic disease has been proposed. It has also been proposed that the activation of the PDC compartment by HIV-1 participates in the initial immune activation following acute infection and contributes to CD4⁺ T-cell depletion by inducing, through IFN-α, the production of TRAIL, which mediates apoptosis of DR5-expressing CD4⁺ T cells following HIV-1 infection (37, 38, 40). However, several lines of evidence question the direct involvement of PDC in the loss of T cells during HIV infection, as PDC numbers are depleted during chronic HIV infection and PDC remaining in circulation are functionally impaired (10). Recent data show that circulating PDC in HIV-infected subjects, although unable to secrete IFN-α after Toll-like receptor (TLR)-mediated activation, constitutively express an increased level of IFN-α mRNA, indicating that during HIV infection PDC are activated yet impaired (71). Rodriguez et al. demonstrated the prevention of spontaneous apoptosis of CD4⁺ and CD8⁺ T cells by IFN-α (63), a major product of PDC following HIV-1 stimulation (3, 28). In addition, Audige et al. (2) showed that blockade of IFN-α and IFN-α receptor during in vitro HIV infection of CD4⁺ T cells isolated from human tonsils did not prevent apoptosis or TRAIL production, suggesting a lack of a central link between IFN-α production and apoptosis of tonsillar CD4⁺ T cells in HIV-1 infection. These data are also consistent with the observation that, in the human peripheral blood lymphocyte-transplanted SCID mouse (hu-PBL-SCID) model, IFN-α efficiently increases the survival of CD4⁺ T cells (49). Thus, controversy remains on the role of IFN-α as an indirect or direct inducer of apoptosis of CD4⁺ T cells through PDC/TRAIL induction, whereas the possibility that IFN-α acts as an antiviral agent by controlling HIV-1 replication and thus reducing virally mediated T-cell loss appears to be supported by several studies (reviewed in references 26, 47, and 58). In this regard, endogenous IFN-α produced by PDC has been shown to play an important role in controlling HIV infection in the human thymus (35), upregulating host antiviral factors such as APOBEC (1, 32, 44, 70) and stimulating NK cell-mediated cytotoxic activity against autologous HIV-infected targets (72).In this report, we investigated the in vivo correlates of viremia in chronically infected subjects by studying the relationship between therapy interruption-associated viremia and plasma IFN-α and TRAIL levels. We also tested in vitro the functional outcome of HIV-1-activated PDC in terms of their ability to mediate lysis of primary autologous CD4 T cells (infected or not with HIV-1), compared to indirect PDC-mediated lysis effects on the NK-dependent antiviral cytotoxic response.     

Antigen-Presenting Cells Represent Targets for R5 HIV-1 Infection in the First Trimester Pregnancy Uterine Mucosa

Background

During the first trimester of pregnancy, HIV-1 mother-to-child transmission is relatively rare despite the permissivity of placental cells to cell-to-cell HIV-1 infection. The placenta interacts directly with maternal uterine cells (decidual cells) but the physiological role of the decidua in the control of HIV-1 transmission and whether decidua could be a source of infected cells is unknown.

Methodology/Principal Findings

To answer to this question, decidual mononuclear cells were exposed to HIV-1 in vitro. Decidual cells were shown to be more susceptible to infection by an R5 HIV-1, as compared to an X4 HIV-1. Infected cells were identified by flow cytometry analysis. The results showed that CD14⁺ cells were the main targets of HIV-1 infection in the decidua. These infected CD14⁺ cells expressed DC-SIGN, CD11b, CD11c, the Fc gamma receptor CD16, CD32 and CD64, classical MHC class-I and class-II and maturation and activation molecules CD83, CD80 and CD86. The permissivity of decidual tissue was also evaluated by histoculture. Decidual tissue was not infected by X4 HIV-1 but was permissive to R5 HIV-1. Different profiles of infection were observed depending on tissue localization.

Conclusions/Significance

The presence of HIV-1 target cells in the decidua in vitro and the low rate of in utero mother-to-child transmission during the first trimester of pregnancy suggest that a natural control occurs in vivo limiting cell-to-cell infection of the placenta and consequently infection of the fetus.     

Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8⁺ T-cells and the use of an in vitro model of naïve CD8⁺ T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8⁺ T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8⁺ T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8⁺ and CD4⁺ T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.     

Immune Suppression by Neutrophils in HIV-1 Infection: Role of PD-L1/PD-1 Pathway

HIV-1 infection is associated with a progressive loss of T cell functional capacity and reduced responsiveness to antigenic stimuli. The mechanisms underlying T cell dysfunction in HIV-1/AIDS are not completely understood. Multiple studies have shown that binding of program death ligand 1 (PD-L1) on the surface of monocytes and dendritic cells to PD-1 on T cells negatively regulates T cell function. Here we show that neutrophils in the blood of HIV-1-infected individuals express high levels of PD-L1. PD-L1 is induced by HIV-1 virions, TLR-7/8 ligand, bacterial lipopolysaccharide (LPS), and IFNα. Neutrophil PD-L1 levels correlate with the expression of PD-1 and CD57 on CD4⁺ and CD8⁺ T cells, elevated levels of neutrophil degranulation markers in plasma, and increased frequency of low density neutrophils (LDNs) expressing the phenotype of granulocytic myeloid-derived suppressor cells (G-MDSCs). Neutrophils purified from the blood of HIV-1-infected patients suppress T cell function via several mechanisms including PD-L1/PD-1 interaction and production of reactive oxygen species (ROS). Collectively, the accumulated data suggest that chronic HIV-1 infection results in an induction of immunosuppressive activity of neutrophils characterized by high expression of PD-L1 and an inhibitory effect on T cell function.     

Naïve and Memory CD4 T Cells Differ in Their Susceptibilities to Human Immunodeficiency Virus Type 1 Infection following CD28 Costimulation: Implications for Transmission and Pathogenesis

In vitro evidence suggests that memory CD4⁺ cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA⁺ CD4⁺ (naïve) and CD45RO⁺ CD4⁺ (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4⁺ cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4⁺ subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the β-chemokines RANTES, MIP-1α, and MIP-1β upon stimulation. Neutralization of these β-chemokines rendered memory CD4⁺ cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and β-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4⁺ T cells serve as the targets for early rounds of HIV-1 replication.