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. 2004 Jun 28:1:14.
doi: 10.1186/1742-4690-1-14.

Trans-dominant cellular inhibition of DC-SIGN-mediated HIV-1 transmission

Li Wu  1 , Thomas D Martin, Yoon-Chi Han, Sabine K J Breun, Vineet N KewalRamani
Affiliations

Trans-dominant cellular inhibition of DC-SIGN-mediated HIV-1 transmission

Li Wu et al. Retrovirology. .

Abstract

Background: Dendritic cell (DC) transmission of human immunodeficiency virus (HIV) to CD4+ T cells occurs across a point of cell-cell contact referred to as the infectious synapse. The relationship between the infectious synapse and the classically defined immunological synapse is not currently understood. We have recently demonstrated that human B cells expressing exogenous DC-SIGN, DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin, efficiently transmit captured HIV type 1 (HIV-1) to CD4+ T cells. K562, another human cell line of hematopoietic origin that has been extensively used in functional analyses of DC-SIGN and related molecules, lacks the principal molecules involved in the formation of immunological synaptic junctions, namely major histocompatibility complex (MHC) class II molecules and leukocyte function-associated antigen-1 (LFA-1). We thus examined whether K562 erythroleukemic cells could recapitulate efficient DC-SIGN-mediated HIV-1 transmission (DMHT).

Results: Here we demonstrate that DMHT requires cell-cell contact. Despite similar expression of functional DC-SIGN, K562/DC-SIGN cells were inefficient in the transmission of HIV-1 to CD4+ T cells when compared with Raji/DC-SIGN cells. Expression of MHC class II molecules or LFA-1 on K562/DC-SIGN cells was insufficient to rescue HIV-1 transmission efficiency. Strikingly, we observed that co-culture of K562 cells with Raji/DC-SIGN cells impaired DMHT to CD4+ T cells. The K562 cell inhibition of transmission was not directly exerted on the CD4+ T cell targets and required contact between K562 and Raji/DC-SIGN cells.

Conclusions: DMHT is cell type dependent and requires cell-cell contact. We also find that the cellular milieu can negatively regulate DC-SIGN transmission of HIV-1 in trans.

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Figures

Figure 1
Figure 1
DC-SIGN transmission of HIV-1 requires donor and target cell contact Pseudotyped HIV-Luc/ADA (1 × 105 IU) was preincubated with Raji or Raji/DC-SIGN donor cells (2.5 × 105) for 2 h at 37°C; the cells were washed with 1 ml of PBS and then cocultured with Hut/CCR5 target cells (1 × 105) in the presence of 10 μg of polybrene in 1 ml of culture medium. Transwell cell culture plates with polycarbonate membrane inserts (3 μm pore size) were used in the capture and transmission assays to separate HIV-1-pulsed donor cells from target cells as illustrated. Cell lysates were obtained 2 days after infection and analyzed for luciferase activity. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second.
Figure 2
Figure 2
Variable efficiency of HIV-1 transmission mediated by different DC-SIGN-expressing cell lines (A) DC-SIGN expression in Raji and K562 cell lines. Parental cells and DC-SIGN transfectants were stained with MAb against DC-SIGN as described [5]. On all histograms, the gray curve represents staining with an isotype control antibody, whereas the filled black curve represents DC-SIGN MAb staining. The mean fluorescence intensity of DC-SIGN staining is shown in the inset of the histograms. One representative experiment out of three is shown. Cells maintained consistent DC-SIGN expression levels throughout the analyses. (B) Adhesion of HIV-1 to DC-SIGN-expressing cells. Cells were incubated with pseudotyped HIV-Luc/ADA containing 20 ng of CA-p24 for 2 h at 37°C, washed extensively, lysed with 0.5% Triton X-100, and quantified with p24 ELISA kits. HIV-1 absorbed by Raji/DC-SIGN cells was normalized as 100% (170 pg of recovered CA-p24 in this experiment). The relative percentage of adsorbed p24 was the average of three separate samples. One representative experiment of six is shown. (C) Adhesion of ICAM-3 to DC-SIGN-expressing cells. The percentage of the cells bound to ICAM-3 was measured by flow cytometry using a fluorescent bead adhesion assay as described [5]. Adhesion of ICAM-3 to DC-SIGN-negative parental cells was less than 2%. Mouse IgG represents an isotype control antibody. One representative experiment of three is shown. (D) Capture and transmission of HIV-Luc/ADA by different donor cells. Donor cells pulsed with HIV-1 (1 × 105 IU) were washed before coculturing with Hut/CCR5 target cells as described for Figure 1. DC-SIGN-negative parental cells were used as controls. Donor cells were preincubated with either mannan (20 μg/ml) or MAb against DC-SIGN (10 μg/ml), respectively, before virus addition. Mouse IgG (10 μg/ml) was used a control antibody. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second. (E) DC-SIGN enhancement of trans-infection by HIV-Luc/ADA. Donor cells pulsed with HIV-1 (1 × 104 IU) were cocultured with Hut/CCR5 target cells without removing unbound virus present in the culture medium. DC-SIGN-negative parental cells were used as controls. Donor cells were preincubated with either mannan (20 μg/ml) or MAb against DC-SIGN (10 μg/ml), respectively, before virus addition. Mouse IgG (10 μg/ml) was used as a control antibody. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second.
Figure 3
Figure 3
Manipulation of MHC class II and LFA-1 expression does not affect DMHT HLA-DR surface expression of (A) K562 and K562/DC-SIGN cells that were transfected with the construct pWT-CIITA compared with (B) Raji and Raji/DC-SIGN cells that were transfected with the construct pDN-CIITA (300–1130). On all histograms, the gray curve represents staining with an isotype control antibody, whereas the filled black curve represents HLA-DR MAb staining. The mean fluorescence intensity is shown in the inset of the histograms. (C) Expression of DC-SIGN, HLA-DR, and LFA-1 in donor cells. Raji/DC-SIGN and K562/DC-SIGN cells that co-express either HLA-DR, LFA-1, or both were singly stained with PE-conjugated isotype control Ab or MAb against DC-SIGN, HLA-DR, or LFA-1. Antibody staining (FL2) is depicted by the histogram plots along the x axis. (D) Transmission of HIV-Luc/ADA by DC-SIGN-expressing donor cells that were manipulated for HLA-DR and LFA-1 expression. The HIV-1 capture and transmission assay was performed as described for Figure 1. Hut/CCR5 cells were used as targets and DC-SIGN-negative parental cells were used as controls. +, positive expression; -, negative. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second.
Figure 4
Figure 4
Trans-dominant cellular inhibition of DC-SIGN transmission of HIV-1 (A) Transmission of HIV-1 by Raji/DC-SIGN cells was inhibited in the presence of K562 cells. Donor cells alone (1.25 × 105) or two mixed types of donor cells (1.25 × 105 for each, 1:1 ratio) as indicated were incubated with HIV-Luc/ADA. The HIV-1 capture and transmission assay using transwell plates was performed as described for Figure 1. Hut/CCR5 cells were used as targets and DC-SIGN-negative parental cells were used as controls. +, K562 cells present; -, absent. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second. (B) Direct infection of Hut/CCR5 targets cells with HIV-Luc/ADA in the presence of the Raji or K562 cells. Hut/CCR5 cells alone (1 × 105) or mixed with Raji or K562 cells (1 × 105 for each, 1:1 ratio) were incubated with HIV-Luc/ADA (2.5 × 104 IU) for 2 h at 37°C, washed with PBS, then cultured 2 days before lysis. One representative experiment out of two is shown. (C) Trans inhibition of HIV-1 transmission mediated by Raji/DC-SIGN cells requires contact with K562 cells. The HIV-1 capture and transmission assay using the transwell plates was performed as described for Figure 1. The different types of donor cells were either mixed or separated by the permeable membrane as illustrated. DC-SIGN-negative parental cells were used as controls. Hut/CCR5 cells were used as target cells, which were co-cultured with donor cells below the membrane inserts. K562 cells were placed on top of the membrane inserts in the separated donor cell group. +, K562 cells present; -, absent. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown.
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