Fig. 1. Characterization of MDDC. Monocytes were differentiated into immature MDDC for 6 days in the presence of IL-4 and GM-CSF. The cell profile was analyzed by flow cytometry for the expression of CD14, HLA-DR, CD1a, CD80, and CD83 on immature versus mature MDDC [i.e., activated with LPS (1 μg/ml) for 2 days] and also for the expression of CD4, DC-SIGN, HLA-DR, and CD80 in HIV-infected DCs versus uninfected.

Fig. 2. Kinetics of DC infection by HIV-1 clades B, C, and A/E. DCs were obtained from peripheral blood of healthy donors after culture of monocytes for 6 days in medium supplemented with IL-4 and GM-CSF. Cells were exposed for 2 h at 37 °C to an MOI of 0.1 of HIV-1 clades B, C, and A/E and washed twice with PBS to remove unbound virus. Supernatants from HIV-infected or uninfected DC were harvested 2, 6, 24, and 72 h after the infection. The presence of p24 Gag antigen was detected by ELISA from serial dilutions of supernatants; the data are corrected for supernatant dilutions obtained with donors #152 and #160. ● = Subtype B; ■ = subtype C;  = subtype A/E.

Fig. 3. ANOVA of HIV-1 infection at 72 h postinfection. Immature MDDCs from four healthy donors were infected or not with subtype B, C, or A/E at an MOI of 0.1 for 2, 6, 24, and 72 h. RNA was extracted from cells harvested at each time point for the infected and uninfected samples and subjected to microarray analysis. ANOVA analysis was performed on 3700 genes that show differential expression with respect to subtypes and 65 genes were selected (P < 0.05). Ratios were expressed in terms of uninfected samples. Log₂ ratios are depicted in green (downregulated) or red (upregulated). The magnitude of the regulation is illustrated by the intensity of the color. Genes were clustered into 2 basic groups. Cluster 1 is composed of genes upregulated in HIV-1 subtypes C and A/E and dowregulated in HIV-1 subtype B. Cluster 2 consisted of genes upregulated by HIV-1 subtype B and downregulated by HIV-1 subtypes C and A/E. (B) 3D plot of the first 3 principle components of the genes depicted in panel A showing subtypes distribution. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Functional categories of genes modulated by HIV-1 infection. Immature MDDCs from four healthy donors were infected or not with subtype B, C, or A/E at an MOI of 0.1 for different times. RNA was extracted from cells harvested at each time point for the infected and uninfected samples and subjected to microarray analysis. A SAM analysis was performed on 3700 genes in which changes in gene expression were obtained by normalizing the values of the HIV-1-infected MDDCs against the noninfected MDDCs. A set of 656 genes was selected as being significantly altered by HIV-1 regardless of the time points and subtypes. A total of 8 functional categories were assigned and obtained through search tools NCBI and DAVID. The total number of genes within each functional category including all time points (i.e., 2, 6, 24, and 72 h postinfection) is represented by the pie chart.

Fig. 5. Gene expression profiling of the immediate phase (2 h) of HIV-1 infection in DCs. A SAM analysis on samples of immature MDDCs infected with HIV-1 was performed as described in Materials and methods. A set of 101 genes was selected as repressed or activated (between a 2-fold and a 4-fold change) by HIV-1 at 2 h postinfection. Genes depicted in green (A) or red (B), respectively, were further divided into 8 functional categories. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Gene expression pattern representing the early phase (6 and 24 h) of HIV-1 infection in immature MDDCs. A SAM analysis on samples of immature MDDCs infected with HIV-1 was performed as described in Materials and methods. A set of 121 genes was selected as repressed or activated (between a 2-fold and a 4-fold change) by HIV-1 at 6 h and 42 genes at 24 h postinfection. Downregulated and upregulated genes are depicted in green or red, respectively, and were further divided into 8 functional categories. Panels A and B show genes regulated at 6 h, whereas panel C shows genes altered at 24 h postinfection. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Gene expression pattern in the late phase (72 h) of HIV-1 infection in immature MDDCs. A SAM analysis on samples of immature MDDCs infected with HIV-1 or not was performed as described in Materials and methods. A set of 92 genes was selected as repressed or activated (between a 2-fold and a 4-fold change) by HIV-1 at 72 h postinfection. Downregulated and upregulated genes are depicted in green (A) or red (B), respectively, and were further divided into 8 functional categories. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Microarray results were validated against the RTPCR in log₂ units, as detailed in Materials and methods

The calculations were performed by normalizing to the GAPDH and the noninfected samples given by: ddCT = dCT(Sample) − dCT(noninfected), where dCT(Sample) = CT(Sample) − CT(GAPDH) and dCT(noninfected) = CT(noninfected) − CT(GAPDH). The P values were calculated using a two-tailed t test, and assuming that the variances were unknown.