Immature dendritic cell-derived exosomes can mediate HIV-1 trans infection

Abstract

Immature dendritic cells (DCs) capture HIV type 1 (HIV-1) and can transmit captured virus particles to T cells. In this report, we show that HIV-1 particles captured by DCs can be transmitted to T cells by exocytosis without de novo infection. Captured HIV-1 particles were rapidly endocytosed to tetraspan protein (CD9, CD63)-positive endocytic compartments that were reminiscent of multivesicular endosomal bodies. Furthermore, some of the endocytosed virus particles were constitutively released into the extracellular milieu in association with HLA-DR1(+), CD1b(+), CD9(+), and CD63(+) vesicles (exosomes) and could initiate productive infections of CD4(+) target cells. Surprisingly, the exocytosed vesicle-associated HIV-1 particles from DCs were 10-fold more infectious on a perparticle basis than cell-free virus particles. These studies describe a previously undescribed mechanism of DC-mediated HIV-1 transmission and suggest that virus particle trafficking to multivesicular endosomal bodies and subsequent exocytosis can provide HIV-1 particles captured by DCs an avenue for immune escape.

Fig. 1.

HIV-1 particles captured by DCs can escape degradation and are released into the extracellular milieu. DCs were exposed to HIV/Lai (A) or NL4 –3/Ba-L (B) virus particles at 37°C or 4°C for 2 h, washed, and incubated with trypsin. Cells were lysed immediately and the p24^gag content of the lysates determined by an ELISA. The data are reported as relative virus binding to DCs after trypsin treatment to that observed in the absence of trypsin and are the mean ± standard deviations of three independent experiments. DCs exposed to HIV/Lai (C) or NL4 –3/Ba-L (D) virus particles at 37°C were untreated or incubated with trypsin before culture for a period of 24 h. Cells and cell-free supernatants were harvested periodically and the p24^gag content determined by ELISA. The data reported are percent p24^gag left associated with cells or present in supernatants over time and are the mean of three independent cultures.

Fig. 2.

Exocytosed HIV-1 capsids present in DC-derived supernatants are infectious. DCs were exposed to Lai-luc or SFV-luc virus particles (moi = 0.001), washed, and placed in culture by themselves or cocultured with T cells. Supernatants, harvested 24 h after virus exposure from DC-alone cultures, were incubated with Jurkat T cells. Cells were harvested 3 days postinitiation of culture, lysed, and lysates used for assaying luciferase activity. The data reported are from infections of three independent cultures ± standard deviations and are representative of four independent experiments.

Fig. 3.

Microscopy analysis for HIV-1 particles in DCs suggests localization of captured HIV-1 particles in MVB-like compartments. DCs exposed to Lai for 3 h were fixed either for thin-section electron microscopy (A and B) or for immunofluorescence microscopy (C–K). (A) An overview of an infected immature DC. Note the presence of virus particles at the surface (white arrowhead), in macropinosomes (black arrow), and in endosomal compartments (black arrowhead). B shows the presence of a mature HIV-1 particle found tethered to the limiting membrane of a MVB (arrow). Cells were coimmunostained for CD9 (C), CD63 (F), or 414 (I) and p24^gag (D, G, and J) and counterstained with DAPI. Merged images (red, CD9, CD63 or 414; green, p24^gag) are shown in E, H, and K. (Scale bar, 10 μM in C–K.)

Fig. 4.

Infectious HIV-1 particles in DC supernatants are associated with exosomes. (A) Vesicle-enriched fractions obtained by differential centrifugation and flotation on linear sucrose gradients were analyzed by Western blotting for the presence of MHC Class II. Densities of the different gradient fractions are indicated at the bottom. (B) The p24^gag content of the sucrose density gradient fractions was determined by an ELISA and plotted against the density of the fractions. (C) HLA-DR1⁺ vesicle fractions isolated from 24-h supernatants of HIV-luc-exposed DCs were incubated with T cells. Luciferase activity of lysed extracts was determined 3 days after infection. The data reported are the mean of three independent cultures ± standard deviations and are representative of three independent experiments. (D) DCs exposed to Lai-luc virus were left untreated or treated with trypsin for 10 min at 37°C before return to culture. Twenty-four-hour supernatants harvested from these cultures were incubated with magnetic beads conjugated to either normal mouse sera, HLA-DR1, CD1b, CD9, CD1d, or CD14, and the positively eluted fractions incubated with MAGI-CCR5 cells. The number of blue foci (indicative of the number of infectious virus particles associated with eluted fractions) was determined 2 days after infection. The data reported are the mean of three independent cultures ± standard errors of mean and are representative of two independent experiments. The absence of a histogram indicates that no infectivity was detected.

Fig. 5.

Exosome-associated HIV is more infectious on a per-particle basis. (A) Unfractionated (whole supernatant), HLA-DR1⁺, or CD14⁺ fraction from 24-h supernatants harvested from Lai-luc exposed DCs was incubated with MAGI-CCR5 cells, and the number of blue foci counted 2 days after infection. The data are reported as the relative number of infectious particles present in HLA-DR1⁺ or CD14⁺ fractions to that present in unfractionated (whole) supernatants and are the means of five independent experiments ± standard deviation. (B) CD14⁺ or HLA-DR1⁺ immunoisolated fractions from DC supernatants were left untreated or incubated with either anti-gp120 mAb (2G12, 10 μg/ml) or normal mouse serum before initiation of infection of MAGI-CCR5 cells. The data reported are the average of two independent experiments, each performed in triplicate, ± standard error of mean. (C) Cell-free virus derived from transient transfections of HEK293T cells or HLA-DR1⁺ fractions derived from 24-h supernatants of virus-exposed DCs was incubated with MAGI-CCR5 cells. The number of blue foci was determined 2 days after infection and the data reported as the number of infectious particles per ng of p24^gag (mean ± standard deviation of five independent experiments performed with three independently generated virus stocks). (D) A proposed model for DC-derived exosome-mediated HIV-1 trans infection.