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Background:  Immature dendritic cells (iDCs) can capture human immunodeficiency virus type 1 (HIV-1) and mediate transfer of virus particles to T cells. The mechanism by which this transfer occurs, however, remains unclear.

Methods:  iDCs were derived from CD14⁺ monocytes by differentiation in GM-CSF and IL-4 containing media. Kinetic experiments were performed using CXCR4- (Lai) or CCR5- (NL4-3/Ba-L) tropic virus isolates. Luciferase expressing HIV-reporter viruses were used in single cycle of replication assays. Exosomes from virus-exposed iDC supernatants (sup) were isolated via HLA-DR1-conjugated magnetic beads or by differential centrifugation and floatation on linear sucrose gradients. Exosome preparations were analyzed for exosomal and viral proteins by Western blotting, examined for p24^gag content by ELISA, or used in infectivity transfer studies with T cells or MAGI/CCR5 cells.

Results:  HIV-1 captured by iDCs was endocytosed and protected from trypsin digestion while kinetic experiments demonstrated a constitutive release of captured virus particles into cell-free supernatants sup. Moreover, cell-free sup derived from iDCs 24 hours post-virus exposure were infectious to T cells. FACS analysis showed no signs of DC maturation as a consequence of virus exposure. Electron microscope (EM) analyses revealed the presence of virus particles within multivesicular endosomal bodies (MVB) following acute infection suggesting exocytosis as a potential mechanism for captured particle release. Differential centrifugation and sucrose-gradient analysis revealed association of HIV-1 with HLA-DR1⁺ vesicles in DC-sup. Furthermore, we could isolate all of the infectivity in DC sup with HLA-DR1⁺ magnetic beads. Interestingly, HLA-DR1⁺ exosome fractions were 80-fold more infectious per nanogram (ng) of p24^gag than cell-free virus stocks.

Conclusions:  Our data suggest that HIV-1 captured by iDCs is transmitted to T cells, without new infection, by exosome-associated exocytosis. Virus particles were found in MVB following endocytosis, and accumulated in iDC sup over time. These particles were infectious and could be enriched by HLA-DR1 (exosome marker) magnetic-bead separation. Since HIV-containing exosome fractions were more infectious than cell-free virus stocks, we hypothesize that the nature of the intracellular compartment that HIV-1 particles migrate to within virus-capturing cells can significantly impact the efficacy of cross-infection of T cells.

Keywords: transmission; dendritic cells; exocytosis