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S427 Mucosal Dendritic Cells and Immunodeficiency Viruses Melissa Pope Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York Dendritic cells [DCs] have been implicated in the pathogenesis of human immunodeficiency virus type 1 (HIV-1). When skin was used as a model for mucosae, the cutaneous DC–T cell milieu allowed the growth of HIV-1 and much of the newly produced virus could be detected in multinucleated DC-T cell syncytia. Such virus replication occurs irrespective of the genetic subtype, the syncytium- and non–syncytium-inducing capacities of the viruses, and whether they are classified as T cell– or macrophage-tropic. Similar DC-syncytia have been identified within the mucosal surfaces of the tonsillar tissue of HIV-1–infected persons. More recently, it was demonstrated that DC–T cell mixtures from the skin, mucosae, and blood of healthy macaques similarly support the replication of simian immunodeficiency virus. In both the human and monkey systems, active virus replication requires the presence of both DCs and T cells. Further studies using the macaque model are underway to elucidate the role of DCs in the transmission and spread of HIV infection. Replication of Human Immunodeficiency Virus Type 1 (HIV-1) Promoted by Cutaneous Dendritic Cells (DCs) DCs, potent antigen-presenting white blood cells, efficiently promote the replication of HIV-1 in vitro in collaboration with CD41 T cells [1–6]. DCs are found throughout the body, including in the blood, lymphoid tissues, and body surfaces (skin and mucosae) [7]. DCs interact efficiently with both T and B cells [8] and, therefore, play a major role in controlling immunity to incoming pathogens. As reviewed recently by Banchereau and Steinman [8], although DCs may be involved in the induction of immune responses to immunodeficiency virus infection, they are in fact exploited by viruses to provide a niche in which significant virus amplification can occur (see below). Therefore, understanding the balance between DCs “carrying” virus and inducing immune responses versus exacerbating infection is critical for the advancement of therapeutic and vaccine strategies. It has been proposed that DCs at the surface epithelia might be one of the first cells targeted by the virus and be critical in All human skin specimens used were authorized for use in research. Animal care operations were in compliance with the regulations detailed under the Animal Welfare Act and in the Guide for the Care and Use of Laboratory Animals. Financial support: NIH (AI-36082, AI-40045, AI-40874, AI-40877; “Mechanisms of AIDS Pathogenesis,” AI-38573; “Antigenic Variation of HIV-1 and Related Lentiviruses,” AI-35168); Dorothy Schiff Foundation; Mellam Family Fund; and Cooperative Agreement No. DAMD17-93-V3004, between the US Army Medical Research and Material Command and the Henry M. Jackson Foundation for the Advancement of Military Medicine. Reprints or correspondence: Dr. Melissa Pope, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave., New York, NY 10021 ([email protected]). The Journal of Infectious Diseases 1999; 179(Suppl 3):S427–30 q 1999 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/99/79S3-0010$02.00 the initiation and subsequent spread of infection to the draining lymphoid tissue [9]. Because the DC content of the mucosal epithelia is much like that of the skin (figure 1), we have used the more accessible skin-derived DCs as a model for the mucosal DCs to study the potential role of these cells in the transmission and spread of HIV-1 infection. In an organ culture system, DCs and memory T cells migrate from normal human skin [3, 10]. The DCs have a typical mature phenotype, expressing many costimulatory and adhesion molecules, high levels of class I and class II major histocompatibility complex proteins, moderate levels of CD83, CD25, and p55, and a perinuclear spot of CD68. Some of the DCs express the epidermal DC markers, CD1a and LAG-1, indicating that there is likely a mixture of dermal- and epidermal-derived DCs migrating from the skin. The memory T cells in these cultures consist of both CD41 and CD81 T cells, some of which are tightly bound to the DCs in DC–T cell conjugates. To investigate the contribution of each cell subset to HIV-1 replication, the single DCs, single T cells, and the DC–T cell conjugates can be separated by cell sorting [3, 10]. In our initial studies, we compared the replicative capacity of macrophage-tropic and T cell line–adapted, non–syncytiuminducing and syncytium-inducing viruses for their capacity to replicate in this DC–T cell environment. With all viruses tested, we found that virus growth is dependent on the presence of both the CD41 T cells and DCs and that most of the replication occurs in the DC–T cell conjugate fraction [2]. Virus-producing DC–T cell syncytia are the main source of new virus production irrespective of whether the virus had been previously characterized as syncytium-inducing or non–syncytium-inducing [2]. Although all viruses required T cells and DCs for virus replication to ensue, the T cells did not need to be actively proliferating, and they did not require an exogenous stimulus [2, 3, 11]. Extending these studies, we have demonstrated that although S428 Pope JID 1999;179 (Suppl 3) Figure 1. Dendritic cell (DC) localization in skin and mucosal surfaces. Skin/Mucosa: Skin and skin-like epithelia of oral and genital tracts. DCs are located within stratified squamous epithelium of mucosae and epidermis of skin. DCs (and T cells) also can be found in underlying lamina propria and dermal regions. DCs and T cells migrate to lymph nodes via afferent lymph. Similar DC–T cell mixtures migrate from skin and mucosal tissues in organ culture. Tonsillar Mucosa: DCs but few, if any, T cells are present within lumenal surface stratified epithelium. Lymphoepithelium lining crypts contain DCs and numerous B and T cells positioned within loosely keratinized epithelium. DCs are also found just beneath epithelium in underlying T cell area of lymphoid tissue. Germinal center DCs (GC DC) and follicular DCs (FDC) are located in B cell areas. productive infection is not detectable when DCs are alone, small numbers of infected DCs (∼100 copies of provirus/5 3 10 4 DCs) could initiate a vigorous infection upon interaction with syngeneic CD41 T cells for up to 1.5 days after exposure of the DCs to the virus [3]. Furthermore, with all viruses tested, the capacity of the DCs to provide infectious virus was affected by treatment with zidovudine. Most recently, we have shown that this skin-derived DC–T cell milieu is supportive of infections with both E and B subtype viruses [6]. Although there is isolate-dependent variation with respect to the levels of virus growth, we did not observe any evidence of subtype-specific replication [6]. Similar observations that viruses from different genetic subtypes can replicate equally well in the cutaneous DC environment have also been reported by Dittmar et al. [12]. Our data and that presented by Dittmar et al. do not support the notion of subtype-restricted replication of HIV in the skin-derived DC milieu, which was described in an earlier study [13], suggesting that additional factors other than the genetic subtype of the virus are likely involved in determining transmission. There still appears to be some contradiction regarding the infectability of purified DCs [1, 5, 6, 12–16]. The contradiction may be explained, at least in part, by the method of cell isolation. Furthermore, more recent studies have revealed that the infectability of DCs may be related to the maturation state of the DC and the level of expression of relevant chemokine receptors [4, 15–18]. Nevertheless, DCs are clearly capable of promoting significant levels of virus replication, particularly in the presence of CD41 memory T cells. However, one must ask whether similar events occur in vivo, where such cellular locales may support the active growth of the virus. Virus-Producing Syncytia in the Tonsillar Mucosae of HIV-Infected Persons Numerous DCs are found at many body surfaces [7, 19, 20]. Notably, DCs are abundant within the mucosal epithelia covering the surfaces of the tonsillar tissue as well as the epithelium that lines the deep invaginations or tonsillar crypts [19, 20] (figure 1). In addition, there are many B and T lymphocytes within the epithelium lining the crypts; consequently, it is referred to as a “lymphoepithelium” [19, 20] (figure 1). Studies of the tonsils and adenoids from several HIV-1–infected persons identified many HIV-positive cells within these DC–T cell rich areas [19, 20]. Closer examination of the tissue sections revealed that many of these cells are multinucleated syncytia expressing high levels of intracellular HIV gag protein and that they are more prominent in the lymphoepithelia than in the lumenal surfaces of the tonsils and adenoids. Of interest, these virus-producing syncytia contain DCs (they stain for proteins known to be expressed by mature DCs). However, the inclusion of T cells into these syncytia has not been detected. These findings [19, 20] likely represent an in vivo correlate of the permissive DC–T cell environment we had described in our earlier in vitro studies [2, 3, 6] (i.e., an environment in which DCs and T cells can continuously interact with each other and provide a niche for virus replication). It is possible that virus circulating throughout the body (as cell-associated or cell-free JID 1999;179 (Suppl 3) HIV and SIV and Dendritic Cells virus) encounters such a permissive milieu and thereby exploits this site for further virus replication and spread. This could happen at similar cellular locales around the body and may be occurring at both acute and chronic stages of infection. Macaque DCs and the Replication of Simian Immunodeficiency Virus (SIV) To advance our understanding of the interactions between DCs and immunodeficiency viruses and to determine what role DCs have in virologic and immunologic aspects of disease, we have used the SIV-macaque system. This is a reliable system in which to study immunodeficiency diseases [21]. In our initial studies using the SIV-macaque system, we endeavored to isolate and characterize DCs from the body surfaces of healthy macaques. Using the organ culture method developed for human skin [10], we can isolate DCs and T cells from the nasopharyngeal and genital mucosae and from the skin [22]. The DCs in these preparations exhibit the typical morphologic and phenotypic characteristics of mature human DCs (table 1), expressing high levels of major histocompatibility complex proteins and many costimulatory and adhesion molecules. The percentages of DCs in the suspensions isolated from the genital mucosae are comparable to those seen in skin-derived suspensions (30%–50%). In contrast, !2% of the leukocytes isolated from the tonsillar tissues are DCs, the majority being T cells and B cells. In all suspensions, the T cells consist of both CD41 and CD81 subsets that can be free or bound to some of the DCs. As we have described in the human system [2, 3, 6, 11], the macaque skin–derived DC–T cell mixtures supported SIV replication [22]. Of interest, virus replication also occurred in the DC–T cell suspensions isolated from the tonsillar and vaginal mucosae but not in the cervical DC–T cell mixtures. Both SIVmac251 and SIVmac239 replicated equally well in these cultures, with much of the new viral protein production occurring in multinucleated syncytia. Using the skin-derived cells that were separated by cell-sorting techniques into the single DCs, single T cells, and DC–T cell conjugates, we recently demonstrated that SIV replication is dependent on the presence of both the DCs and T cells [23], just as we reported for the HIV-human skin system [2, 3, 6]. To more readily facilitate studies using macaque DCs, we adapted a method for the generation of significant numbers of DCs from monocyte-derived blood precursors [24]. After blood monocytes are cultured in the presence of the cytokines granulocyte-macrophage colony-stimulating factor and interleukin4, a population of immature DCs can be obtained and further differentiated into a mature DC population by additional culture in a monocyte-conditioned medium (table 1). Initial studies on the infectability of these cells have revealed that, much like the cutaneous DCs, the mature blood-derived Table 1. Source Skin Mucosae Blood Blood S429 Isolation and characterization of macaque dendritic cells. Isolation method a Phenotype Organ culture Organ culture GM-CSF, IL-4, MCM Mature HLA-DR1111 CD86111 CD2511 CD831 CD681/spot p5511 GM-CSF, IL-4 Immature HLA-DR111 CD8611 CD252 CD832 CD68111 p552 NOTE. GM-CSF 5 granulocyte-macrophage colony-stimulating factor; IL 5 interleukin; MCM 5 monocyte-conditioned medium. a HLA-DR, CD86, CD25, and CD83 expression on cell surface by fluorescence-activated cell sorter analysis (i.e., immature DCs contain intracellular CD83). CD68 and p55 staining by immunoperoxidase labeling of acetone-fixed cytospins. DCs do not support significant levels of virus replication in the absence of T cells [23]. In fact, syngeneic T cells from various sites around the body can collaborate with the blood-derived DCs to support active virus replication. Similar observations have been made when skin-derived DCs are cultured with syngeneic T cells from skin, blood, lymph node, or spleen. Fluorescence-activated cell sorter analysis of the DC–T cell cocultures also revealed that both skin- and blood-derived DCs can form DC–T cell conjugates with each the T cell populations examined [23]. Therefore, the capacity of the mature DC–T cell milieu to allow virus replication is not unique to the DCs and T cells isolated from the body surfaces. However, since the T cells in the skin and mucosae are memory T cells, the possibility that it is the memory T cell subset that is contributing to the support of virus replication in the T cell suspensions isolated from blood, lymph node, and spleen is under investigation. Concluding Remarks It has become clear from human and monkey studies that DCs promote the vigorous replication of HIV [2, 3, 6] and SIV [22, 23], respectively. In the immune system, DCs are pivotal in the initiation of antigen-specific T cell immune responses [7]. It is quite possible that DCs play two roles in the pathogenesis of immunodeficiency virus infection. One role could be during the onset and spread of infection in acute and (perhaps) chronic phases of disease. DCs have been implicated, particularly following mucosal exposure to immunodeficiency viruses, as one of the first cells targeted by the virus, after which they traffic the virus to the draining nodes where it can be amplified [9]. S430 A second role would be to induce virus-specific immune responses in the draining lymphoid tissues, which might control viral replication and subsequent spread. Continued studies investigating the biology of the interaction of immunodeficiency viruses with DCs are being performed in the monkey system. Such studies are critical to advance our understanding of the involvement of DC and other cells in the pathogenesis of infection. 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