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. 2007 Jul;81(13):6858-68.
doi: 10.1128/JVI.02472-06. Epub 2007 Apr 18.

Activated CD34-derived Langerhans cells mediate transinfection with human immunodeficiency virus

Kelly M Fahrbach  1 , Sheila M Barry, Seyoum Ayehunie, Sarah Lamore, Mitchell Klausner, Thomas J Hope
Affiliations

Activated CD34-derived Langerhans cells mediate transinfection with human immunodeficiency virus

Kelly M Fahrbach et al. J Virol. 2007 Jul.

Abstract

Langerhans cells (LCs) are a subset of dendritic cells (DCs) that reside within epidermal and mucosal tissue. Because of their location, LCs are potentially the first cells to encounter human immunodeficiency virus (HIV) during sexual transmission. We report that LCs purified from CD34(+)-derived DCs can facilitate the transinfection of target cells but only after activation. Virions were observed in an intracellular compartment that contains several tetraspanins, in addition to the unique LC markers langerin and CD1a. This reveals that the trafficking of HIV within LCs is reminiscent of that which occurs in mature monocyte-derived DCs and that it varies with the activation state of the cell. The observation that activated LCs can mediate transinfection suggests a potential role for these cells in the known increase in HIV transmission associated with sexually transmitted infections that would cause inflammation of the genital lining.

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Figures

FIG. 1.
FIG. 1.
Characterization of LC-specific traits for CD34+-derived DC-100 cells. (A) Flow cytometry of DC-100 cells to determine surface expression of langerin and CD1a. Unstained cells were used as a control for comparison to DC-100 cells stained with antibodies to langerin and CD1a. (B) Electron micrograph of LC within DC-100 population. An example of Birbeck granules is boxed and enlarged.
FIG. 2.
FIG. 2.
Characterization of DC-100 cells following activation. (A) Surface expression of maturation markers HLA-DR, CD80, CD83, and CD86 was measured in untreated cells (first column) and after activation with LPS (second column), TNF-α (third column), or both (fourth column). Marker expression was compared to that of unstained controls (first row). (B) Expression of CD1a and langerin was measured in unactivated (untreated) and activated (LPS plus TNF-α) DC-100 cells. (C) Expression of DC-SIGN in untreated and LPS-plus-TNF-α-treated DC-100 cells.
FIG. 3.
FIG. 3.
Infectivity profiles of unsorted DC-100 cells. (A) Unactivated and activated LCs were exposed to X4-tropic or R5-tropic HIV, and infection of the cells was determined by level of luciferase activity (relative light units). The level of infection was compared to that occurring in target p4-2 cells directly exposed to virus. (B) Unactivated (open circles) and activated (open squares) DC-100 cells were exposed to increasing concentrations of X4-tropic HIV prior to coculture with target cells. Infection was determined by measuring luciferase activity of samples (relative light units). p4-2 targets exposed to virus (asterisks) defined background infectivity. Cocultures with mature MDDCs acted as a positive control (open triangles). Any infection of effector cells was determined by analysis of cells cultured with virus and not exposed to targets. These controls include unactivated DC-100 cells alone (closed circles), activated DC-100 cells (closed squares), and mature MDDCs (closed triangles). Each data point represents the average of three samples with error bars representing the standard errors of the means. Representative results of four independent experiments are shown.
FIG. 4.
FIG. 4.
Sorting and infectivity profiles of CD1ahi and CD1alo/neg DC-100 cells. (A) DC-100 cells were stained with FITC-conjugated CD1a antibody and sorted to isolate CD1alo/neg and CD1ahi DC-100 cells. From left to right, dot plots show unsorted, CD1ahi, and CD1alo/neg DC-100 cells. (B and C) Luciferase activity measured in cocultures of unactivated (closed squares) and activated (open squares) CD1ahi LCs with target cells. Background levels were defined by virus-exposed p4-2 targets (asterisks). (B) Cocultures of unactivated (closed diamonds) and activated (open diamonds) CD1alo/neg cells; (C) cocultures of immature (closed triangles) and mature (open triangles) MDDCs. Each data point represents the average of three samples with error bars representing the standard errors of the means. Each graph is representative of three independent experiments.
FIG. 5.
FIG. 5.
Enhanced transinfection by activated LCs involves internalized virions. (A) Luciferase activity measured in cultures of p4-2 target cells with unactivated and activated CD1ahi LCs that were previously exposed to virus and washed to remove unbound virus. Immature and mature MDDCs were also pulsed with virus and served as negative and positive controls, respectively. (B) Unactivated and activated LCs were pulsed with virus for 2 h, washed to remove unbound virus, and then exposed to 0.05% trypsin plus 0.53 mM EDTA for 5 min at 37°C to remove surface-bound virions. Luciferase activity was measured as described above. Each data point represents the average of three samples with error bars representing the standard errors of the means. Representative results from four independent experiments are shown.
FIG. 6.
FIG. 6.
Characterization of MVB components in activated LCs. Unsorted unactivated and activated DC-100 cells were incubated with X4-tropic HIV with RFP-Vpr prior to fixing and staining. (A) Percentage of unactivated (light bars) and activated (dark bars) LCs with the representative tetraspanin associated with HIV. Error bars show standard errors of the means. (B) LCs were identified by positive staining for CD1a (blue). Virus was identified by expression of fluorescently tagged Vpr (pseudocolored green), and cells were then stained for CD81 (red). The boxed area is enlarged and color separated to show individual stains.
FIG. 7.
FIG. 7.
Association of CD1a and langerin in an MVB in activated LCs. (A) Deconvolution microscopy of DC-100 cells. Unsorted unactivated (left) and activated (right) DC-100 cells were incubated with X4-tropic HIV with RFP-Vpr (pseudocolored green) prior to coculture with CD4+ T cells. LCs were identified by positive staining for CD1a (blue) and CD4 (red), while T cells were only CD4 positive. The boxed area is enlarged and color separated to show individual stains. (B) DC-100 cells were treated as for panel A; however, LCs were identified by staining for langerin (blue). (C) Percentages of unactivated and activated LCs with CD1a associated with HIV. Error bars show standard errors of the means from four individual experiments; a P value of <0.05 is marked with an asterisk. (D) Percentages of cells with langerin associated with HIV in unactivated and activated LCs. Error bars show standard errors of the means from four individual experiments; a P value of <0.05 is marked with an asterisk.
FIG. 8.
FIG. 8.
Transfer of CD1a from activated LCs to T cells. (A) Unsorted unactivated (left) and activated (right) DC-100 cells were incubated with X4-tropic HIV with RFP-Vpr (pseudocolored green) prior to coculture with CD4+ T cells. LCs were identified by positive staining for CD1a (blue) and CD4 (red), while T cells were only CD4 positive. The boxed area includes a single, transferred virion within a T cell. Small panels show magnifications of virions and individual stains. (B) Percentages of unactivated and activated LCs that have transferred virions to T cells. (C) Percentages of virions transferred to T cells that show an association with CD1a after coculture with unactivated or activated LCs. Error bars show standard errors of the means from two individual experiments; a P value of <0.05 is marked with an asterisk.
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