Fig. 3: Modulation of CD73 expression and adenosine levels during pDC-MM cell interactions.

A pDCs were co-cultured with autologous patient MM cells for 24 h, followed by multicolor flow analyses to assess the pDC-induced change in CD73 expression on MM cells. CD138⁺ MM cells were examined using flow cytometry, and Median Fluorescence Intensity (MFI) of CD73 expression was determined using anti-CD73 Abs conjugated to AlexaFluor-488, in the presence or absence of pDCs. Left Panel: Representative histograms show CD73 expression in MM cells cultured in the presence (red) and absence (green) of pDCs. [Black histogram: Isotype control Ab]. Right panel: Normalized CD73 expression in MM cells cultured in the presence and absence of pDCs. Data were quantified from the histogram analyses shown in the left panel (mean ± SD; p = 0.008; MFI: 1.2-fold change in pDC-MM vs MM; Data obtained from analysis of seven MM patient BM samples). B pDCs were co-cultured with autologous patient MM cells for 24 h, followed by multicolor flow analyses to assess the changes in CD73 expression on CD303 + MM BM pDCs. Median Fluorescence Intensity (MFI) of CD73 expression was determined using anti-CD73 Abs conjugated to AlexaFluor-647. Representative histograms show CD73 expression in MM BM pDCs cultured in the presence (blue) or absence (green) of MM cells. (Shaded histogram: Isotype control Abs). C MM- BM pDCs were co-cultured with MM cells in the presence and absence of anti-CD73 Abs (0.5 µg/ml) for 2 days; supernatants were collected and analyzed for adenosine levels using a fluorometric adenosine assay kit. The percentage of soluble adenosine in isotype-Ab- versus anti-CD73 Ab-treated MM cells, MM BM pDCs, both alone, or in pDC-MM coculture (mean ± SD, p < 0.05; n = 3) is shown. The quantification of data showing the amount of adenosine in supernatants (pmol/sample) for MM-pDC coculture versus MM cells alone (p = 0.0167). (Note: The isotype treatment is indicated as a very low level of anti-CD73 Ab in the semi-log plot).