DC Transfer of R5 and X4 HIV-1: Blood versus thymus. (A) Method used to detect transfer of HIV-1. DC were infected with R5 (NL(AD8)-nef/EGFP) or X4 (NL4-3-nef/EGFP) HIV-1. Mock PBMC and thymocytes were cultured in parallel. DC were either cultured alone for the duration of the culture period (5 days), or at 24 h post-infection mock PBMC were added to the blood DC and mock CD3hi/CD3lo thymocytes were added to the thymic DC at a 1:1 ratio. (B) Results from a single experiment demonstrate the gating strategy to determine the number of EGFP+ cells following R5 infection of thymic pDC (left panels) and mDC (right panels) cultured alone or in the presence of CD3hi thymocytes. In some experiments, thymocytes were treated with 0.1 μM AZT prior to co-culture with DC. Representative plots from 4 donors are shown. (C) Blood (left hand panels) and thymic (right hand panels) DC infected with either R5 (upper row) or X4 (lower row) HIV-1. Each symbol represents the mean of duplicate experiments from a single donor. The edges of the boxes are the 25 and 75 percentiles, the horizontal line in the box is the median and the whiskers extend to the minimum and maximum data points. * indicates a p value of < 0.05 as determined by the Wilcoxon signed rank test. The results for 5-6 donors are shown. (D) Thymic pDC were infected with R5 HIV-1. At 24 h post-infection, uninfected CD3hi thymocytes were added to the pDC at a 1:1 ratio. Culture was continued for 4 days, at which time the pDC-thymocyte co-cultures were labelled with CD3, and the number of EGFP+CD3+ thymocytes was determined by flow cytometry. Columns represent the mean of 2 experiments ± SEM.