ABSTRACT. The ability of the human immunodeficiency virus type 1 (mHV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells ...
Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7998-8002, September 1991 Immunology
Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures E. LANGHOFF*, E. F. TERWILLIGER*, H. J. AND W. A. HASELTINE*
Bost, K. H. KALLAND*, M. C. POZNANSKY*, 0.
M. L. BACON*,
*Division of Human Retrovirology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; and tResearch Institute of Toxicology, University of Utrecht, Utrecht NL-3508TD, The Netherlands
Communicated by Baruj Benacerraf, May 13, 1991
ABSTRACT The ability of the human immunodeficiency virus type 1 (mHV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose 98% conservation), but BRU, BH10, and ELI do not contain the mutations that in HXB2 disable the vpr, vpu, and nef reading frames, respectively. DFCI-HT is therefore intact for all known HIV-1 gene functions. A viral stock of the monocyte tropic Ba-L strain of HIV-1 was obtained from the National Institutes of Health Repository. Primary viral patient isolates of HIV-1 prepared from peripheral blood mononuclear cell cultures were donated by V. A. Johnson. Titered viral stocks of the molecular clones of HIV-1 were obtained from Jurkat cell cultures transfected with 10 gg of proviral DNA by the DEAE-dextran procedure (17). Isolation and Culture of Primary Leukocyte Subsets. Purified populations of dendritic cells were obtained as described (3, 6, 18). The isolation protocol is summarized in Fig. 1. Unfractionated peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll separation. Monocytes either were depleted from the PBMC cultures by Fc adherence or were isolated by density separation on Percoll gradients and depleted of T cells by rosetting with neuraminidase-treated sheep erythrocytes (SRBCs) (3, 6, 18). The T cells (97% CD2 positive) were obtained from the macrophage-depleted (by adherence or Percoll) PBMCs by rosetting with SRBCs (6). The remaining leukocyte fraction was separated into B-celland dendritic cell-enriched populations by a refloatation in dense Percoll after 2 days of culture (6). The low-density cells contained most of the dendritic cells and were depleted of
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Abbreviations: HIV, human immunodeficiency virus; CAT, chloramphenicol acetyltransferase; PBMC, peripheral blood mononuclear cell. 7998
Immunology: Langhoff et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
7999
high density E rosette negative cells 48 culture and Percoll density gradient low density cells
Fc panning on lgG coatedplates depletion of monocytes
x
50-60% pure dendritic cells Panning against: _
CD45R:CDI lb CD22 CD5 I dendritic Cells
monocytes by Fc panning of IgG-coated dishes (3, 6) and of residual lymphocytes by panning with monoclonal antibodies to CD5 (2 ,ug/ml of Leul), CD45R [50% (vol/vol) 4G10 hybridoma medium], and CD11b [50o (vol/vol) OKM1 hybridoma medium]. In six consecutive experiments, dendritic cells isolated by this protocol and used for infection studies were 75% + 7% large (15-20 ,um) veiled cells (3, 18). All cell cultures were grown in medium of RPMI 1640 with 10% fetal calf serum, antibiotics, and 20%o phytohemagglutinin-conditioned medium made in our laboratory (3). The cell cultures were kept for 24-48 hr in culture medium before infection with 20 ng of p24 per well. After an overnight exposure to virus, the medium was totally replaced each day on the 2 following days. Thereafter, every third day the culture medium was totally replaced with fresh medium. These complete changes of growth medium should remove excess free virus that may remain from the initial inoculum. Immediately before the medium replacement, culture supernatants were harvested for viral p24 RIA assays (DuPont/ NEN). In Situ Hybridization and Immunoperoxidase Staining. Dendritic cells were isolated from peripheral blood and infected with the viral HXB-CAT and the ANRE-HXB-CAT clones, and the IIIB strain. Five days after infection cytospin preparations were made as described (19). The immunoperoxidase staining of acetone-fixed cytospin centrifuge preparations used standard procedures (20). A minimum of 200 cells was examined for each antibody. Immunoperoxidase staining was in some experiments done before in situ hybridization of the cytospin cell preparations.
RESULTS Fractionation of Peripheral Blood Lymphocytes. A specific surface marker for human dendritic cells has not yet been described. Consequently, purification ofthese cells must rely on subtraction of leukocytes, which have distinctive surface markers, as well as on the physical properties of the dendritic cells themselves, as shown in Fig. 1 and previously described (6, 18). Two similar methods were used for the purification. In some preparations, monocytes and macrophages were removed from the lymphocyte preparations by adherence to Fc-coated plates. In a second procedure, monocytes were
I
FIG. 1. Isolation scheme for preparation of purified leukocyte subsets of adherent and nonadherent monocytes, T cells, and dendritic cells.
separated by Percoll gradient centrifugation (Fig. 1). Purified adherent or nonadherent monocyte and macrophage populations result from these different purification techniques. The isolation results are summarized in Table 1 and Fig. 2. The dendritic cell population is almost entirely free of cells with T-cell-, B-cell-, and monocyte/macrophage-specific antigens. All cell types react with antibodies that recognize a common determinant on the major histocompatibility complex class II protein, but dendritic cells react much more strongly than do T cells or monocyte/macrophages with this antibody (Table 1 and Fig. 2). The morphology of the cells in the populations differs dramatically. The cells differ in average diameter, as determined by scanning and transmission electron microscopy (Fig. 2). The shape of the cells also differs. The dendritic cells are large, irregular cells that have extensive cytoplasmic veils. The nuclei of the dendritic cells are large and irregular. HIV-1 Infection of Blood Dendritic Cells in Vitro. The cells were infected with a low multiplicity of virus. The amount of virus used, about 20 ng of p24 capsid antigen, was determined to be the minimum necessary to ensure infection of all cultures. A low concentration of virus was used to amplify differences among cell types. The virus used for the initial studies was HXB-CAT, in which the nefgene is replaced with the CAT gene. Infection was monitored by measurement of extracellular p24 antigen as well as CAT enzyme activity in cell extracts (17). Table 2 demonstrates that the HXB-CAT virus replicates well in the purified dendritic cell populations. The number of cells in each experiment is not the same; 20-40 times more monocytes and T cells were used than dendritic cells. Lower numbers of dendritic cells were used as these cells comprised 96 200 ng/ml, was found in the supernatant of SupT1 cells.
DISCUSSION Use of highly purified dendritic cell populations permits determination of the ability of HIV-1 to replicate in this unusual cell type. The experiments presented here demonstrate that HIV-1 can productively infect the nondividing primary dendritic cells in culture. When normalized for cell number, the dendritic cell population produces more virus than do other primary cell types. Indeed, the level of virus replication in dendritic cell populations approaches that observed in the replicating cultures of a CD4' T tumor cell line. The possibility exists that other contaminating cell types might contribute to the results. The B cells represent the largest fraction of cell contaminants with specific leukocyte markers, and a contribution from this leukocyte subset cannot be ruled out. It should be noted that B cells isolated from peripheral blood do not themselves support HIV-1 replication (26). Purified dendritic cells, in contrast to T cells and monocytes, are permissive for replication of all tested virus isolates. T-cell tropic and monocyte/macrophage tropic virus strains replicate in the dendritic cells, which also supported virus growth after -t-irradiation of the cultures. The HXBCAT virus, defective for viral genes vpr, vpu, and nef, also replicates in dendritic cell cultures as does the DFCI-HT virus, which expresses all three proteins from all of these genes. Dendritic cells also support the replication of two primary patient isolates of HIV-1. The basis for preferential prolific replication in the primary dendritic cells is not known. These cells and the related Langerhans cells (27) express low but detectable levels of the CD4 receptor (28-30). It is remarkable that prolific virus replication is observed in this nonreplicating terminally differentiated cell population even after irradiation. This observation raises a question of great interest regarding the requirement of cell replication for virus production (31, 32). However, HIV-1 virus is produced from cultures of monocytes or macrophages that are not dividing (21). The present studies provide a starting point for systematic investigations of this phenomenon. Experiments with transgenic mice suggest that the HIV-1 long terminal repeat functions preferentially in skin Langerhans cells (33). Langerhans cells are present at or near mucosal surfaces and may be among the first cells to become infected upon mucosal HIV-1 exposure (16, 34). It is possible that transcription factors present in dendritic cells favor active transcription initiation from the HIV-1 long terminal repeat. The observation that purified dendritic cells support continued replication of HIV-1 replication without notable cytopathic effects suggests that these cells may play an important role in viral pathogenesis. In the lymph nodes the dendritic cells are in close proximity to T cells (1, 2), where infected dendritic cells may serve as a constant source of infection for CD4+ T cells. It is reported that HIV-1 interferes with the normal antigen-presenting function of this cell type (14, 35, 36). However, recent studies indicate that HIV-1 infection does not affect their ability to support long-term growth of T cells (19).
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