Human Retroviruses

Nasr, Najla, Harman, Andrew, Turville, Stuart, Cunningham, Anthony L, Cunningham, Anthony L., Najla Nasr, Andrew Harman, Stuart Turville, Anthony L. Cunningham

Dendritic cells (DC) present in the genital tract are one of the first cells to encounter HIV during sexual mucosal transmission. In addition they are able to efficiently transfer the virus to its main target cells, CD4(+) T-lymphocytes. As such an understanding of how HIV interacts with and manipulates DCs is of key importance for the design of mucosal vaccines and microbicides. However working with these cells is difficult for several reasons. Firstly, immature DCs are difficult to infect due to their high endocytic capacity and mature DCs are usually resistant to infection. Secondly, tissue DCs are inherently difficult to isolate, which results in small yields and the cells are prone to maturation as a result of extraction. Here we describe how to isolate CD1a expressing Langerhans cells from the epidermis and CD1a(+), CD14(+) and perhaps BDCA3(+) DCs from the dermis. We also describe how to produce the model monocyte-derived DC (MDDC) by cytokine stimulation of CD14(+) monocytes, which results in the production of large numbers of immature cells. We also describe methods by which high titer HIV stocks can be generated to infect a significant proportion of DCs and also methods for determining the titer of such stocks.