Candidate microbicides block HIV-1 infection of human immature Langerhans cells within epithelial tissue explants

Abstract

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1(Ba-L) infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08-4.77%). HIV-1-infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1-1 microg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1(Ba-L) (an R5 HIV-1 strain) more efficiently infected LC-T cell cocultures when compared with HIV-1(IIIB) (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.

Figure 1

Experimental set-up for infecting LCs within epithelial tissue explants and for evaluating potential microbicides. (A) Suction blisters. (B) Blister roofs, i.e., epithelial tissue explants, floating in PBS after surgical removal. (C) 50-μl droplets containing HIV-1 before LC infection (droplets containing drugs in the microbicide experiments appeared identical). (D and E) Epithelial tissue explants being draped over HIV-1 droplets. (F) Epithelial tissue explants floating in complete medium after HIV-1 infection. The pictures shown are representative of a typical experiment.

Figure 2

HIV-1 infection of single crawl-out LC demonstrated by flow cytometry. Representative intracellular HIV-1 p24 and surface MHC class II mAb double-staining of LCs that have emigrated from HIV-1_Ba-L–exposed (A and C) or uninfected (B) epithelial tissue explants, demonstrating 0.85% productively infected cells in A and low nonspecific staining in B. Dead cells were excluded from all analyses. HIV-1–infected cells stained with an isotype control Ab were always negative (data not shown). (C) Plot of percentage of HIV-1 p24⁺ LCs (with background staining subtracted) obtained in 31 separate experiments showing the range of intracellular HIV-1 p24 positivity depending on the skin donor.

Figure 3

Downregulation of CD4 and CD83 on HIV-1–infected LCs. LCs that had emigrated from HIV-1_Ba-L–exposed skin were double stained for the surface antigens shown and intracellular HIV-1 p24. Dead cells were excluded from all analyses. Data shown are representative of at least three experiments.

Figure 4

HIV-1–infected LCs are responsible for transmitting high levels of infection to cocultured CD4⁺ T cells. (A, top left) Tissue explants were exposed to two HIV-1 primary isolates, HIV-1_CJI.3 (R5) or HIV-1_CI-8 (R5X4), on the basal epithelial (BE) side or the stratum corneum (SC) side, washed, and then floated in media containing allogeneic CD4⁺ T cells. (B) electron micrograph taken from an LC–T cell coculture 7 d after HIV-1_CI-8 exposure of explants showing numerous HIV-1 virions surrounding and budding from a CD4⁺ T cell (×64,000; inset ×440,000). (A, bottom left) Epidermal cell suspensions were made immediately after HIV-1 exposure of epithelial tissue explants to HIV-1_CJI.3, and half of the suspensions were then depleted of LCs by immunomagnetic bead separation. Nondepleted epidermal cells (EC) and LC-depleted epidermal cells (EC-LC) were then cocultured with allogeneic CD4⁺ T cells. (A, bottom right) Emigrated LCs were collected from media 2 d after HIV-1_CJI.3 exposure of epithelial tissue explants, and half of the cells were exposed to trypsin treatment and the other half left untreated. Trypsin-treated LCs and untreated LCs were then cocultured with allogeneic CD4⁺ T cells. HIV-1 infection levels were assessed by measuring HIV-1 p24 protein content by ELISA in coculture supernatants collected every other day (A, all panels). Data shown are representative of at least three separate experiments that showed similar results.

Figure 4

HIV-1–infected LCs are responsible for transmitting high levels of infection to cocultured CD4⁺ T cells. (A, top left) Tissue explants were exposed to two HIV-1 primary isolates, HIV-1_CJI.3 (R5) or HIV-1_CI-8 (R5X4), on the basal epithelial (BE) side or the stratum corneum (SC) side, washed, and then floated in media containing allogeneic CD4⁺ T cells. (B) electron micrograph taken from an LC–T cell coculture 7 d after HIV-1_CI-8 exposure of explants showing numerous HIV-1 virions surrounding and budding from a CD4⁺ T cell (×64,000; inset ×440,000). (A, bottom left) Epidermal cell suspensions were made immediately after HIV-1 exposure of epithelial tissue explants to HIV-1_CJI.3, and half of the suspensions were then depleted of LCs by immunomagnetic bead separation. Nondepleted epidermal cells (EC) and LC-depleted epidermal cells (EC-LC) were then cocultured with allogeneic CD4⁺ T cells. (A, bottom right) Emigrated LCs were collected from media 2 d after HIV-1_CJI.3 exposure of epithelial tissue explants, and half of the cells were exposed to trypsin treatment and the other half left untreated. Trypsin-treated LCs and untreated LCs were then cocultured with allogeneic CD4⁺ T cells. HIV-1 infection levels were assessed by measuring HIV-1 p24 protein content by ELISA in coculture supernatants collected every other day (A, all panels). Data shown are representative of at least three separate experiments that showed similar results.

Figure 5

Preferential infection of HIV-1_Ba-L in LC–T cell cocultures when compared directly with HIV-1_IIIB. Known amounts, as determined by HIV-1 p24 content, of HIV-1_Ba-L (an R5 virus) and HIV-1_IIIB (an X4 virus) were incubated with epithelial tissue explants, washed, and floated in media containing either allogeneic (A–C) or autologous (C) CD4⁺ T cells. For data shown in C, high titers (1:100 for HIV-1_Ba-L and 1:15 for HIV-1_IIIB) of each HIV-1 strain were used for infection. Infection levels were assessed by measuring HIV-1 p24 protein content by ELISA in coculture supernatants collected every other day. Data shown are representative of at least three separate experiments that showed similar results.

Figure 6

Preincubation of epithelial tissue explants with AOP-RANTES specifically blocks subsequent HIV-1_Ba-L and HIV_CJ1.3, but not HIV-1_IIIB, infection in LC and LC–T cell cocultures in a dose-dependent manner. Epithelial tissue explants were incubated with 1, 10, or 100 nM of AOP-RANTES, a CCR5 ligand, for 20 min before exposure to 1:100 HIV-1_Ba-L (A and B) or HIV_CJ1.3 (C), both R5 viruses, or 1:15 HIV-1_IIIB (D), an X4 virus. After infection, explants were washed and floated in media alone (A) or in media containing allogeneic CD4⁺ T cells (B–D). HIV-1 infection levels were assessed by intracellular HIV-1 p24 staining (A) or by measuring HIV-1 p24 protein content by ELISA in coculture supernatants collected every other day (B–D). Data shown are representative of at least three separate experiments that showed similar results.

Figure 7

Preincubation of epithelial tissue explants with CAP blocks subsequent HIV-1 infection in LC and LC–T cell cocultures in a dose-dependent manner. Epithelial tissue explants were incubated with 1 or 10 mg/ml of CAP, a candidate microbicide, for 20 min before exposure to HIV-1. After infection, explants were washed and floated in media alone (A) or in media containing allogeneic CD4⁺ T cells (B and C). HIV-1 infection levels were assessed by intracellular HIV-1 p24 staining (A) or by measuring HIV-1 p24 protein content by ELISA in coculture supernatants collected every other day (B and C). Data shown are representative of at least three separate experiments that showed similar results.