Melissa Pope

Conjugates of dendritic cells and memory T lymphocytes from skin facilitate productive infection with HIV-1

Experimentally, a productive infection with HIV-1 requires that virus be administered to T cells that are activated by mitogens. We describe a productive milieu for HIV-1 within the confines of normal skin that does not require standard stimuli. The milieu consists of dendritic cells and T cells that emigrate from skin and produce distinctive stable, nonproliferating conjugates. These conjugates, upon exposure to each of seven different HIV-1 isolates, begin to release high levels of virus progeny within 4 days. Numerous infected syncytia, comprised of both dendritic and T cells, rapidly develop. We propose that conjugates of dendritic cells and T cells, as found in the external linings of organs involved in sexual transmission of HIV-1, represent an important site for the productive phase of HIV-1 infection. Because the affected T cells carry the memory phenotype, this site additionally provides a mechanism for the chronic depletion of CD4+ memory cells in HIV-1 disease.

Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120

A topical microbicide reduces the probability of virus transmission when applied to the vagina or rectum of a person at risk of sexually acquiring HIV-1 infection. An effective microbicide could significantly reduce the global spread of HIV-1, particularly if women were able to use it covertly to protect themselves. A microbicide could target the incoming virus and either permanently inactivate it or reduce its infectivity, or it could block receptors on susceptible cells near the sites of transmission. We describe here how vaginal administration of the broadly neutralizing human monoclonal antibody b12 can protect macaques from simian-human immunodeficiency virus (SHIV) infection through the vagina. Only 3 of 12 animals receiving 5 mg b12 vaginally in either saline or a gel and then challenged vaginally (up to 2 h later) with SHIV-162P4 became infected. In contrast, infection occurred in 12 of 13 animals given various control agents under similar conditions. Lower amounts of b12 were less effective, suggesting that protection was dose dependent. These observations support the concept that viral entry inhibitors can help prevent the sexual transmission of HIV-1 to humans.

Transmission, acute HIV-1 infection and the quest for strategies to prevent infection.

By the acute stage of HIV-1 infection, the immune system already faces daunting challenges. Research on mucosal barriers and the events immediately after heterosexual transmission that precede this acute stage could facilitate the development of effective microbicides and vaccines.

Exploiting dendritic cells to improve vaccine efficacy

The challenges to vaccine biology are dramatized by the current situation with an AIDS vaccine (see Letvin, this Perspective series, ref. 1). For years, data have been available on the HIV-1 genome and its proteins, as well as numerous antigens recognized by the immune system. Still, this information has not been readily translated into candidate vaccines that induce the broad and long-lasting T cell–mediated immunity thought to be necessary to protect people from acquiring AIDS (2–5). Vaccines are also lacking for many other serious infections in which T cell–mediated immunity should be protective. These include pathogens whose genomic sequences and antigenic proteins are well characterized: tuberculosis, malaria, and the herpes simplex, papilloma, Epstein-Barr, and hepatitis C viruses. In essence, the identification of foreign antigens is necessary but not sufficient for producing vaccines that are effective in the T cell sphere. Better vaccine delivery and vaccine adjuvants, or enhancers of immunity, are required (6, 7). We propose that dendritic cell (DC) physiology should be considered and exploited in meeting each of the challenges in vaccine biology that lie ahead (see Table ​Table1).1). DCs act as nature’s adjuvants for regulating antigen-specific immunity. As antigen-presenting cells, DCs capture antigens, process them into peptides, and present them on products of the MHC to T cells. DCs are both efficient and specialized in antigen presentation, and they control the magnitude, quality, and memory of the ensuing immune response. DCs have been used successfully as cellular adjuvants in mice to elicit protective T cell–mediated immunity against pathogens and tumors (8, 9). These cells are now being used to prime and expand T cells specific for human cancers (refs. 10–12; see also Yu and Restifo, this Perspective series, ref. 13). The responding T cells include helper cells, especially Th1 CD4+ cells, which produce IFN-γ; and killer cells, especially CD8+ cytolytic T lymphocytes (CTLs), which exocytose granules rich in perforin and granzyme. New information indicates that DCs control responses by other classes of lymphocytes (B, NK, and NKT cells) and elicit T cell memory, a critical goal of vaccination. Table 1 Challenges in vaccine biology requiring improved control of antigen presentation Developing the capacity to harness DCs for vaccination seems particularly urgent in confronting infectious agents that, like HIV-1, pose unusual demands with respect to safety; the time-honored approach of microbial attenuation is now being set aside as vaccine biologists turn to defined antigens, poorly replicating vectors, and DNA. Although these vaccines introduce foreign microbial products, they often generate weak immunity, especially T cell–mediated immunity. Consequently, greater emphasis on underlying immunologic processes is needed, notably the strong adjuvant roles of DCs. Interestingly, as we discuss below, even the classical vaccine approach of microbial attenuation, used successfully for smallpox and measles, may have unknowingly exploited the adjuvant roles of DCs.

Replication of HIV-1 in dendritic cell-derived syncytia at the mucosal surface of the adenoid.

Human immunodeficiency virus-type 1 (HIV-1) replicates actively in infected individuals, yet cells with intracellular depots of viral protein are observed only infrequently. Many cells expressing the HIV-1 Gag protein were detected at the surface of the nasopharyngeal tonsil or adenoid. This infected mucosal surface contained T cells and dendritic cells, two cell types that together support HIV-1 replication in culture. The infected cells were multinucleated syncytia and expressed the S100 and p55 dendritic cell markers. Eleven of the 13 specimens analyzed were from donors who did not have symptoms of acquired immunodeficiency syndrome (AIDS). The interaction of dendritic cells and T cells in mucosa may support HIV-1 replication, even in subclinical stages of infection.

Blockade of Attachment and Fusion Receptors Inhibits HIV-1 Infection of Human Cervical Tissue

Identification of cellular factors involved in HIV-1 entry and transmission at mucosal surfaces is critical for understanding viral pathogenesis and development of effective prevention strategies. Here we describe the evaluation of HIV-1 entry inhibitors for their ability to prevent infection of, and dissemination from, human cervical tissue ex vivo. Blockade of CD4 alone or CCR5 and CXCR4 together inhibited localized mucosal infection. However, simultaneous blockade of CD4 and mannose-binding C-type lectin receptors including dendritic cell–specific intercellular adhesion molecule–grabbing integrin was required to inhibit HIV-1 uptake and dissemination by migratory cells. In contrast, direct targeting of HIV-1 by neutralizing mAb b12 and CD4-IgG2 (PRO-542) blocked both localized infection and viral dissemination pathways. Flow cytometric analysis and immunostaining of migratory cells revealed two major populations, CD3+HLA-DR− and CD3−HLA-DR+ cells, with a significant proportion of the latter also expressing dendritic cell–specific intercellular adhesion molecule–grabbing integrin. Bead depletion studies demonstrated that such HLA-DR+ cells accounted for as much as 90% of HIV-1 dissemination. Additional studies using immature monocyte-derived dendritic cells demonstrated that although mannose-binding C-type lectin receptors and CD4 are the principal receptors for gp120, other mechanisms may account for virus capture. Our identification of the predominant receptors involved in HIV-1 infection and dissemination within human cervical tissue highlight important targets for microbicide development.

The Interaction of Immunodeficiency Viruses with Dendritic Cells

Dendritic cells (DCs) can influence HIV-1 and SIV pathogenesis and protective mechanisms at several levels. First, HIV-1 productively infects select populations of DCs in culture, particularly immature DCs derived from blood monocytes and skin (Langerhans cells). However, there exist only a few instances in which HIV-1- or SIV-infected DCs have been identified in vivo in tissue sections. Second, different types of DCs reliably sequester and transmit infectious HIV-1 and SIV in culture, setting up a productive infection in T cells interacting with the DCs. This stimulation of infection in T cells may explain the observation that CD4+ T lymphocytes are the principal cell type observed to be infected with HIV-1 in lymphoid tissues in vivo. DCs express a C-type lectin, DC-SIGN/CD209, that functions to bind HIV-1 (and other infectious agents) and transmit virus to T cells. When transfected into the THP-1 cell line, the cytosolic domain of DC-SIGN is needed for HIV-1 sequestration and transmission. However, DCs lacking DC-SIGN (Langerhans cells) or expressing very low levels of DC-SIGN (rhesus macaque monocyte-derived DCs) may use additional molecules to bind and transmit immunodeficiency viruses to T cells. Third, DCs are efficient antigen-presenting cells for HIV-1 and SIV antigens. Infection with several recombinant viral vectors as well as attenuated virus is followed by antigen presentation to CD4+ and CD8+ T cells. An intriguing pathway that is well developed in DCs is the exogenous pathway for nonreplicating viral antigens to be presented on class I MHC products. This should allow DCs to stimulate CD8+ T cells after uptake of antibody-coated HIV-1 and dying infected T cells. It has been proposed that DCs, in addition to expanding effector helper and killer T cells, induce tolerance through T cell deletion and suppressor T cell formation, but this must be evaluated directly. Fourth, DCs are likely to be valuable in improving vaccine design. Increasing DC uptake of a vaccine, as well as increasing their numbers and maturation, should enhance efficacy. However, DCs can also capture antigens from other cells that are initially transduced with a DNA vaccine or a recombinant viral vector. The interaction of HIV-1 and SIV with DCs is therefore intricate but pertinent to understanding how these viruses disrupt immune function and elicit immune responses.

Infectious and Whole Inactivated Simian Immunodeficiency Viruses Interact Similarly with Primate Dendritic Cells (DCs): Differential Intracellular Fate of Virions in Mature and Immature DCs

ABSTRACT As potential targets for human immunodeficiency virus type 1 and simian immunodeficiency virus (HIV-1 and SIV), dendritic cells (DCs) likely play a significant role in the onset and spread of infection as well as in the induction of antiviral immunity. Using the SIV-macaque system to study the very early events in DC-virus interactions, we compared chemically inactivated SIV having conformationally and functionally intact envelope glycoproteins (2,2′-dithiodipyridine [AT-2] SIV) to infectious and heat-treated SIV. Both human and macaque DCs interact similarly with SIV without detectable effects on DC viability, phenotype, or endocytic function. As assessed by measuring cell-associated viral RNA, considerable amounts of virus are captured by the DCs and this is reduced when the virus is heat treated or derived from a strain that expresses low levels of envelope glycoprotein. Immunostaining for SIV proteins and electron microscopy indicated that few intact virus particles are retained at the periphery of the endocytically active, immature DCs. This contrasts with a perinuclear localization of numerous virions in large vesicular compartments deeper within mature DCs (in which macropinocytosis is down-regulated). Both immature and mature DCs are capable of clathrin-coated pit-mediated uptake of SIV, supporting the notion that the receptor-mediated uptake of virus can occur readily in mature DCs. While large numbers of whole viruses were preferentially found in mature DCs, both immature and mature DCs contained similar amounts of viral RNA, suggesting that different uptake/virus entry mechanisms are active in immature and mature DCs. These findings have significant implications for cell-to-cell transmission of HIV-1 and SIV and support the use of AT-2 SIV, an authentic but noninfectious form of virus, as a useful tool for studies of processing and presentation of AT-2 SIV antigens by DCs.

Dendritic cells and the replication of HIV-1.

Dendritic cells (DCs) are a distinct lineage of white cells that arise from CD34+ progenitors in the bone marrow. DCs exhibit many specializations that lead to efficient antigen capture and presentation to T cells, both CD4+ helpers and CD8+ killers. In several human tissues, DCs express the CD4 receptor for HIV‐1. Some early reports described the explosive infection of blood‐derived DCs by HIV‐1 and a severe compromise of their presenting function. In contrast, other studies described active HIV‐1 replication when DCs were interacting with CD4+ T cells. This productive infection could begin with a low viral burden in DCs but required that the DCs retain their normal binding and stimulatory function for T cells. In this review we first summarize those features of the DC system that seem pertinent to HIV‐1 infection. Then we consider the current literature on the interaction of HIV‐1 with DCs, from several different tissues, in HIV‐1‐infected patients or following challenge with HIV‐1 in vitro. The literature leads to the hypothesis that HIV‐1 infection is a battleground in which DCs could be leading both of the armies, the aggressor that promotes HIV‐1 replication from relatively small numbers of infected cells and the defender that mediates T cell‐dependent resistance.

Canarypox Virus-Induced Maturation of Dendritic Cells Is Mediated by Apoptotic Cell Death and Tumor Necrosis Factor Alpha Secretion

ABSTRACT Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.