Figure 5

Effect of MSC-secreted factors on HIV-1 reactivation from T-cells. We investigated whether MSCs can reactivate HIV-1 using two well-established T-cell line models, i.e. ACH2 and J-Lat (9.2). (A) Effect of exposure to ASC-CM or BMSC-CM on virus reactivation from ACH2 cells. As compared to unstimulated ACH2 cells (black bars) exposure to 50% CM from both ASCs (gray bars) and BMSCs (white bars) increased HIV-1 p24 production (pg/mL) at 3, 5 and 7-days. (B) Western immunoblot analysis of lysates from the ACH2 cells cocultured with ASCs was further confirmed HIV-1 activation, where increased expression of HIV-1 Nef protein was clearly evident. Relative band intensity (presented the numbers to the corresponding lanes) was compared by the Image-J software (version 1.50). (C) A representative image of ASCs cocultured with ACH2 cells. Following 7-days of coculture, unlike the ACH2 cultures alone (left) the cocultured ACH2 cells preferentially adhered to the underlying ASCs (right). (D) As compared to ACH2 cells alone, significantly higher HIV-1 p24 production was observed when ACH2 cells were cocultured with ASCs. (E) ASC-mediated latency reactivation was investigated using the J-Lat (9.2) cell line, which expresses GFP from an integrated HIV-1 provirus. In coculture experiments, the J-Lat (9.2) cells were found to directly interact with ASCs (right) as compared to themselves (left). (F) A representative image of GFP expression in J-Lat (9.2) cells, alone (left) or following ASC coculture (right). (G) Fold change in GFP-positive J-Lat (9.2) cells, in the absence or presence of ASCs. Error bars show ±SEM and significant changes are represented as P-values (*p < 0.01, **p < 0.001). The latency-reactivation potential of ASCs was corroborated using two established T-cell line models of HIV-1 latency.