Figure 3. CD36 expression on MDM cells cultivated in HEMA condition treated with recombinant Nef.

PBMCs were cultivated in HEMA condition for three days and for additional three days in presence of 50/mL rNef/myr (A) Representative histograms of the three populations (Lym, Ery, MDMs) analyzed for CD36 and CD4 expression by flow cytometry at six days expansion. The respective populations were identified as described in figure 2A and the fluorescence intensities in Nef-treated (solid grey histogram) compared to untreated (solid line) cells are shown. Matched isotype (dotted line) was used as control of non-specific fluorescence signals. In the column bar graphs on the right of the histograms are presented the MFI of cells stained with matched isotype control (iso), untreated cells (Ctr) and Nef-treated cells (Nef) stained with FITC- or APC-conjugated anti-CD36 and CD4 antibodies. The results are representative of ten (CD36) and four (CD4) independent experiments. MDMs were analyzed by flow cytometry for the expression of several specific markers, i.e. CD14, CD11c, CD86, CD68 and CD206. Representative histograms are shown in (B) and the fluorescence intensities of respective antibodies in Nef-treated (solid grey histogram) were compared to untreated (solid line) cells. The results are representative of five independent experiments. (C) Representative histograms of PBMC-derived MDMs analyzed by flow cytometry for the expression of TLR2 and TLR4. The MFI of Nef-treated (+ Nef) compared to untreated (untreated) cells was reported in the respective histograms. The results are representative of five independent experiments (*p<0.05). Matched isotype (dotted line or Ctr iso) was used as control of non-specific fluorescence signals. SYTOX Blue was used to exclude dead cells in all the experiments presented in this figure.