Previous studies indicated that MDDC contact with CD4+ T cells is required for efficient HIV-1 replication 8. Because MDDCs can transmit HIV-1 independently of DC-SIGN 5691011, these studies did not establish whether DMHT requires donor and target cell interactions. To evaluate whether cell contact is required for DMHT, Raji or Raji/DC-SIGN donor cells were preincubated with single-round infectious HIV-Luc/ADA, washed, and cocultured with Hut/CCR5 cells, a human T cell line. Alternatively, HIV-Luc/ADA-pulsed donor cells were separated from target cells by using transwell cell culture plates with permeable membranes. Compared with Raji donor cell controls, HIV-1 infection of Hut/CCR5 cells was enhanced significantly when in direct co-culture with Raji/DC-SIGN cells (Figure 1). In contrast, Raji/DC-SIGN cells did not transmit HIV-1 to Hut/CCR5 cells when the donor and target cells were separated in the coculture by a membrane (Figure 1). Placement of HIV-1-pulsed Raji/DC-SIGN donor cells on either the top or bottom of the transwell membrane opposite the target cells had no effect on the infection profiles (data not shown).

Raji and K562 cells expressing DC-SIGN were established via stable transfection of pcDNA3-DC-SIGN and several rounds of flow cytometry-based fluorescence-activated cell sorting (FACS) for cell populations expressing intermediate levels of DC-SIGN (Table 1 and Figure 2A). Parental Raji and K562 cells were uniformly negative for DC-SIGN expression.

We first examined whether the DC-SIGN transfectants would adsorb greater amounts of HIV-1 relative to the parental cell lines. Although HIV-1 can nonspecifically bind to a number of transformed cell types, high-level DC-SIGN expression increases HIV-1 adsorption by different transformed lines 1233334. HIV-1 binding to cell lines was detected by measuring the capture of pseudotyped HIV-Luc/ADA using an enzyme-linked immunosorbent assay (ELISA) for HIV-1 capsid (CA)-p24. Compared with the Raji and K562 parental cell controls, DC-SIGN transfectants bound an increased amount of HIV-1 (Figure 2B). Relative to the corresponding parental cells, results from six independent experiments indicate that HIV-1 binding was enhanced in DC-SIGN-expressing Raji or K562 cells by 1.5 ± 0.2 and 1.8 ± 0.2, respectively (mean ± SD, P > 0.05).

We next functionally assayed the DC-SIGN expressed in these cell lines. Binding to ICAM-3, a physiological ligand of DC-SIGN, was tested using a previously described flow cytometric assay 25. Low nonspecific binding to cells was observed in this assay. Less than 2% of ICAM-3-coated fluorescent beads bound to parental Raji and K562 cells (Figure 2C). In contrast, the adhesion to Raji/DC-SIGN and K562/DC-SIGN cells was 27% and 25%, respectively (Figure 2C). These data indicate that Raji and K562 transfectants express functionally authentic forms of DC-SIGN.

We next tested the efficiency of HIV-1 transmission by the two DC-SIGN-expressing cell lines. Virus donor cells were preincubated with HIV-Luc/ADA and washed to remove unbound virus, after which CD4+ T cells were added in coculture as infection targets. Raji/DC-SIGN cells stimulated HIV-1 transmission more than 100-fold relative to the Raji parental cells (Figure 2D). K562/DC-SIGN cells captured and transmitted HIV-1 less efficiently. These cells transmitted HIV-1 37-fold less efficiently than Raji/DC-SIGN cells and only 10-fold better than the K562 parental line. Using a smaller virus inoculum, we next tested whether the K562/DC-SIGN cells would enhance transmission of HIV-Luc/ADA that remained in co-culture with the CD4+ T cells. Compared with DC-SIGN-negative parental cells, Raji cells expressing moderate levels of DC-SIGN enhanced HIV-1 infection of T cells 10-fold (Figure 2E). In contrast, K562 cells expressing similar levels of DC-SIGN enhanced HIV-1 infection 3-fold under the same conditions. Similar results were observed when HIV-1 pseudotyped with simian immunodeficiency virus (SIV), X4-tropic HIV-1, or different R5-tropic HIV-1 Env proteins were used in transmission assays (data not shown). DMHT could be blocked by pre-incubation of donor cells either with monoclonal antibody against DC-SIGN or with mannan, a soluble ligand of C-type lectins (Figure 2D,2E).

Our findings reinforced the notion that DMHT is cell type dependent, implying that cell-specific factors account for differences in HIV-1 transmission by different donor cells. We thus screened for differential K562 surface expression of immune synapse and cell adhesion molecules common to immature MDDCs and Raji cells (Table 2). Because cell contact is required for DMHT, we reasoned that the lack of such factors on K562/DC-SIGN cells could impair the efficient transmission of HIV-1. Surveyed ligands included the MHC class I and II complexes involved in antigen presentation; intercellular adhesion molecules and their cognate ligands, such as ICAM-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50), LFA-1 (CD11a/CD18), LFA-3; and molecules involved in T cell activation, such as B7-1 (CD80) and B7-2 (CD86). We also assayed for the presence of HIV-1 receptors on the different donor cell types. Staining and flow cytometric analysis of surface expression of these molecules is summarized in Table 2. Notably, immature MDDCs and Raji/DC-SIGN cells, which transmit HIV-1 efficiently, expressed similar levels of MHC class II (HLA-DR, -DP and -DQ), and LFA-1. In contrast, neither LFA-1 nor MHC class II molecules were expressed by K562 cells. As expected, Raji and K562 cells did not express CD4 or CCR5, ligands that could compete for interaction with the HIV-1_ADA Env.

The MHC class II transactivator (CIITA) is a master regulator of the class II locus as well as related proteins that influence MHC class II sorting 35. Expression of CIITA is sufficient to reconstitute the MHC class II presentation pathway in different cell types. In addition, dominant-negative (DN) versions of CIITA have been developed that are capable of suppressing expression of CIITA-regulated genes in APCs 35. We took advantage of these tools to manipulate expression of MHC class II molecules in Raji/DC-SIGN and K562/DC-SIGN cell populations.

After stable transfection of K562 and K562/DC-SIGN cells with wild-type (WT) CIITA, MHC class II-positive cells were detected and enriched via FACS (Figure 3A). In contrast, stable transfection of DN-CIITA in Raji and Raji/DC-SIGN cells yielded cells with significantly impaired expression of MHC class II molecules that were further enriched by FACS (Figure 3B). Antibody staining for DC-SIGN and HLA-DR expression confirmed that greater than 95% of K562/DC-SIGN/WT-CIITA cells were double-positive for both molecules, and 93% of the DC-SIGN-positive Raji/DC-SIGN/DN-CIITA cells were largely negative for expression of MHC class II.

To assess the role of LFA-1 in DMHT, we generated K562/LFA-1, K562/DC-SIGN/LFA-1, and K562/DC-SIGN/WT-CIITA/LFA-1 cell lines. Comparison of the surface expression of DC-SIGN, HLA-DR, and LFA-1 on these cells is shown in Figure 3C. DC-SIGN expression in the K562-derived cells was similar or even higher than that in Raji/DC-SIGN cells. In addition, three-color staining and FACS analysis of sorted K562/DC-SIGN/WT-CIITA/LFA-1 cells also confirmed that more than 96% of cells were triple-positive for DC-SIGN, HLA-DR, and LFA-1 (data not shown).

The MHC class II- and LFA-1-manipulated donor cells were next tested for their ability to transmit DC-SIGN-captured HIV-1. Neither induction nor repression of MHC class II expression in K562/DC-SIGN/WT-CIITA cells or Raji/DC-SIGN/DN-CIITA cells, respectively, increased or reduced the efficiency of HIV-1 transmission significantly (Figure 3D). In fact, HIV-1 transmission mediated by Raji/DC-SIGN/DN-CIITA cells was somewhat higher than that mediated by Raji/DC-SIGN cells, potentially due to increased DC-SIGN expression levels (Figure 3C). In addition, expression of LFA-1 in the different DC-SIGN-expressing K562 transfectants did not enhance DMHT (Figure 3D). In contrast, Raji/DC-SIGN cells with equal or lower levels of DC-SIGN relative to K562/DC-SIGN lines transmitted HIV-1 efficiently (Figure 3D).

To determine whether K562 cells express negative factors that impair DMHT or lack positive factors necessary for DMHT, we sought to make fusions with K562/DC-SIGN and Raji cells to test in HIV-1 transmission assays. We first attempted to make cell-cell fusions through a hybridoma protocol that used polyethylene glycol (PEG)-3000. However, this method resulted in a low proportion of cell fusions that had a transient cell culture life. Because DMHT can occur in less than a few hours, we were curious what fraction of DMHT-permissive cells could be detected under conditions simulating a cell fusion experiment. We observed that under conditions where Raji/DC-SIGN cells were 10% or lower in a mixed population with K562 cells, DMHT was inefficient and comparable to the level of DMHT by a uniform K562/DC-SIGN population (data not shown). We next assayed whether HIV-1 transmission could be detected when Raji/DC-SIGN or Raji cells were mixed in equal proportion with K562/DC-SIGN or K562 lines. Strikingly, we found that HIV-1 transmission by Raji/DC-SIGN cells was strongly inhibited in the presence of K562 cells (Figure 4A). This effect was observed irrespective of whether the K562 cells were added before or after HIV-Luc/ADA adsorption to Raji/DC-SIGN cells (data not shown). In contrast, cocultured Raji cells did not significantly affect HIV-1 transmission by Raji/DC-SIGN or K562/DC-SIGN cells (Figure 4A). Compared with the Raji and Raji/DC-SIGN mixture, HIV-1 transmission was 14-fold reduced by the K562 and Raji/DC-SIGN mixture.

To examine whether the K562 cells were exerting a negative effect on the HIV-1 susceptibility of Hut/CCR5 targets cells, Hut/CCR5 cells alone or mixed with an equal amount of Raji or K562 cells were infected with HIV-Luc/ADA. Co-culture with K562 cells did not result in any detectable inhibition of HIV-1 infection of Hut/CCR5 cells (Figure 4B).

To determine whether the trans inhibition of HIV-1 transmission was dependent on direct interactions between K562 and Raji/DC-SIGN cells, we assayed HIV-1 transmission efficiency when the K562 cells were separated from Raji/DC-SIGN cells by a transwell membrane. As expected, K562 cells were able to significantly diminish Raji/DC-SIGN-mediated HIV-1 transmission when these cells were cultured in the same compartment with Hut/CCR5 target cells (Figure 4C, "mixed" donor cells). However, when K562 cells were placed on the top of the permeable membrane to separate them from cocultured Raji/DC-SIGN and Hut/CCR5 cells on the bottom, no significant inhibition of HIV-1 transmission was observed (Figure 4C, "separated" donor cells).

PMX-DC-SIGN and pBABE-DC-SIGN expression constructs containing human DC-SIGN cDNA have been previously described 5. Human DC-SIGN cDNA obtained from pBABE-DC-SIGN was subcloned into the polylinker of the pcDNA3 expression construct (Invitrogen) between the BamHI and EcoRV sites to derive pcDNA3-DC-SIGN. Constructs encoding the WT- and DN-CIITA truncation mutant (300–1130) 3538 were gifts from Jenny Ting (University of North Carolina, Chapel Hill). Constructs pCDL1 39 and pCDB1 40 encoding the αL and β2 subunits of LFA-1, respectively, were kindly provided by Timothy Springer (Harvard Medical School, Boston). The pCMV-hph construct encoding hygromycin selection resistance was a gift from Michael Emerman (Fred Hutchinson Cancer Research Center, Seattle).

Hut/CCR5 and GHOST/X4/R5 cell lines have previously been described 5. Raji B cells used in this study have been previously described as B-THP-1 cells 21. The human erythroleukemic K562 cell line was purchased from the American Type Culture Collection (ATCC number CCL-243). A panel of stable cell lines generated and used in this study is summarized in Table 1. Stable cell lines derived from parental Raji and K562 cells were subjected to FACS to obtain cells with the desired enhanced or reduced expression levels of specific target molecules. Constructs used for expression of target genes and conditions for selective drugs are also listed (Table 1).

Immature DCs were generated from CD14+ monocyte precursors treated with a cocktail of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) as described 541. One week after differentiation, these cells uniformly expressed high levels of HLA-DR, HLA-I, CD11b, CD11c, DC-SIGN, and ICAM-1; moderate levels of LFA-1 and CD86; and low levels of CD14.

Raji, K562-derived cell lines, and Hut/CCR5 cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories) and selective drugs as indicated in Table 1. HEK293T and GHOST/X4/R5 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (Atlanta Biologicals). DCs were maintained in OptiMEM medium (Invitrogen) supplemented with 20% FBS (HyClone Laboratories) and specific cytokines as previously described 5.

Fluorescein isothiocyanate (FITC), phycoerythrin (PE), and tri-color or Cy-chrome-conjugated mouse anti-human MAbs against the following molecules were used: DC-SIGN (clone 120526) and CXCR4 (clone 44717) (R&D Systems); LFA-1 (clone MEM-25), HLA-I (clone TÜ 149), HLA-DR (clone TÜ 36), ICAM-1 (clone MEM-111), ICAM-3 (clone TP1/25.1), CD4 (clone S3.5), and goat anti-mouse immunoglobulin (IgG, Caltag Laboratories); CD80 (clone L307.4), CD86 (clone 2331), HLA-DQ (clone TÜ 169), HLA-DR (clone TÜ 36), LFA-3 (clone 1C3), CCR5 (2D7/CCR5), and Blood Group Fy6 (Duffy, clone NaM185-2C3) (PharMingen); ICAM-2 (clone CBR-IC2/2, Biosource) and HLA-DP (clone B7/21, Leinco Technologies). MAbs to HLA-1 identify MHC class I expression, and MAbs to HLA-DP, -DQ, and -DR identify MHC class II expression.

Single-round infectious, pseudotyped HIV-1 stocks (HIV-Luc/ADA) were generated by calcium phosphate cotransfections of HEK293T cells with the proviral vector plasmid NL-Luc-E^-R^- (HIV-Luc) containing a firefly luciferase reporter gene 42 and an expression plasmid for the R5-tropic HIV-1_ADA envelope glycoprotein. Viral stocks were evaluated by limiting dilution on GHOST/X4/R5 cells.

To assess the expression of DC-SIGN or other surface molecules, cells (2 × 10⁵) were stained with an MAb directed against the specific antigen and compared with isotype-matched IgG controls. For direct staining, cells were incubated at 4°C in phosphate-buffered saline (PBS) containing 2% FBS (FACS buffer) and 2 μg of FITC- or PE-conjugated MAbs per milliliter in a total volume of 100 μl. For indirect staining, after a 30-min incubation with the first specific MAb at 4°C, the cells were washed with FACS buffer and resuspended in 100 μl of FACS buffer containing 2 μg of FITC- or PE-conjugated antibody against mouse IgG per milliliter. DC-SIGN specific MAbs 5 were obtained from R&D Systems. Cells were incubated for 30 min at 4°C, washed with FACS buffer, and analyzed using a FACSCalibur flow cytometer (Becton Dickinson).

DC-SIGN-expressing Raji and K562 transfectants as well as DC-SIGN-negative parental cells (2 × 10⁵) were incubated with pseudotyped HIV-Luc/ADA containing 20 ng of CA-p24 for 2 h at 37°C, and washed extensively with PBS to remove unbound virus. Cells were subsequently lysed with 0.5% Triton X-100 to release adsorbed virus, which was quantified using HIV-1 p24 ELISA kits (Coulter Beckman).

Soluble, recombinant ICAM-3 was obtained from R&D Systems. Carboxylate-modified TransFluorSpheres (1.0 μm, 488 nm excitation/645 nm emission, Molecular Probes) were coated with ICAM-3 as described previously 43. Adhesion of ICAM-3 to DC-SIGN was determined by measuring the detectable percentage of cells that bound fluorescent beads, using flow cytometry on a FACSCalibur instrument. The fluorescent bead adhesion assay has been described in detail previously 59.

HIV-1 capture and transmission assays were performed as described previously 5. In brief, donor cells (2.5 × 10⁵) were incubated with 1 × 10⁵ infectious units (IU) of pseudotyped HIV-Luc/ADA in a total volume of 400 μl for 2 h at 37°C to allow adsorption of the virus. After 2 h, cells were washed with 1 ml of PBS and cocultured with Hut/CCR5 target cells (1 × 10⁵) in the presence of 10 μg of polybrene in 1 ml of culture medium. Cell lysates were obtained 2 days after infection and analyzed for luciferase activity with a commercially available kit (Promega). HIV-1 enhancement assays were performed using limiting amounts of HIV-1 (1 × 10⁴ IU) during incubation with donor cells, and target cells were added directly to the coculture without removing the virus present in the culture medium. To assay DC-SIGN-blocking agents, cells were preincubated with either mannan (20 μg/ml; Sigma) or MAb against DC-SIGN (10 μg/ml) or mouse IgG control (10 μg/ml) for 30 min at 37°C before virus addition.

To investigate whether cell-cell contact between donor cells and target cells was critical for HIV-1 transmission, transwell cell culture plates with inserts of polycarbonate membranes of 3-μm pore size (Costar) were used in HIV-1 capture and transmission assays to separate donor cells from target cells.

To test mixed donor cells for effects on DMHT, equal amounts (1.25 × 10⁵, 1:1 ratio) of two donor cell types with or without DC-SIGN were mixed prior to the 2-h incubation of HIV-Luc/ADA (1 × 10⁵ IU) at 37°C. After virus incubation, these cells were washed and cocultured with Hut/CCR5 target cells in the presence of polybrene as described above for the HIV-1 capture and transmission assays. As a control for the susceptibility of Hut/CCR5 cells to direct infection by HIV-1, parental Raji or K562 cells were mixed with Hut/CCR5 cells (1 × 10⁵, 1:1 ratio), then incubated with HIV-Luc/ADA (2.5 × 10⁴ IU) for 2 h at 37°C, washed with PBS, and cultured in the presence of polybrene (10 μg/ml). Cell lysates were obtained 2 days after infection and analyzed for luciferase activity. Hut/CCR5 cells alone (1 × 10⁵) were used as a control.