Figure 5
ps20high CD4+ T cells form a higher frequency of conjugates, multiple conjugates and VS with HIV-1 infected donor cells. 1 × 105 DDAO vital dye labelled ps20inter (Figure 5A) and ps20high (Figure 5B) target cells were co-cultured with 60% 1 × 105 2044-infected donor cells for 1 hour on Poly-L-lysine coated glass cover slips. Cells were then fixed and stained with a PE anti-Gag (red) and a FITC anti-CD4 (green) Abs. The frequency of target cells that were involved in a single conjugate was defined as the percentage of dye-labelled target cells apposed to an HIV-1-infected donor cell. Representative high power fields captured at 63× magnification are shown, which depict single conjugates for between (A) J-ps20inter or (B) J-ps20high cells and HIV-infected donor cells. Left panels depict conjugates with no HIV-Gag CD4 polarization to conjugate interface. Right panels depict conjugates with HIV-Gag and CD4 polarization (yellow) to conjugate interfaces. (C) Picture shows a representative high power field of dye labelled targets (white arrows) involved in multiple conjugates, defined as a target cell apposed to two or more HIV-1-infected donor cells. (D) Picture shows a representative high power field of a polysynapse, defined as a cell with two or more virological synapses (yellow) at conjugate interfaces. (E) Picture shows a representative high power field of remote contacts (RC) (filopodial bridges or nanotubes) that connect uninfected target cells to HIV-infected targets. (F) Mean frequency of J-ps20inter vs. J-ps20high cells involved in single conjugate, multiple conjugates, or which contain virological synapses, polysynapses or are in contact through remote contacts are shown. A total of 600 random target cells were assessed across quadruplicate experiments. (G) Qualitative analysis of the junction diameter of a conjugates was measured using LEICA TCS SP2 software, where the diameter was measured across the medial section of a conjugate. Graph depicts the mean conjugate interface diameter (μM) between J-ps20inter vs. J-ps20high cells and HIV-1-infected donor cells. A total of at least 30 conjugates per population were measured across quadruplicate experiments. Asterisk denotes statistically significant data as calculated using an unpaired t-test (*P ≤0.05).