Article Figures & Data
Figures
- Figure 1.
Effect of OADv infection on MSCs immunophenotypic profile. A, MSCs expressing TLR-9 was analyzed by flow cytometry on OAdv-infected (black) or -uninfected (white) MSCs. Bars represent the mean ± SD of triplicates from three independent experiments. *, P < 0.05 by Mann–Whitney test. B, Luciferase activity was determined from OAdv-infected or -uninfected MSCs/pHAGE-NFκB-GFP/Luc cell lysates at different time-points after infection. Bars represent the mean ± SD of triplicates from three independent experiments. *, P < 0.05 by Mann–Whitney test. The supernatants from uninfected or 48 hours OAdv-infected MSCs were analyzed for the presence of secreted cytokines. C, Pixel intensity quantification is shown after Proteome Profiler Human Cytokine Array analysis. IL8, interleukin 8; MIF, Macrophage migration inhibitory factor. Bars represent the mean ± SD of spot duplicates from two independent experiments. D, Luminex analysis of cytokine concentration is shown. Bars represent the mean ± SD of duplicates from two independent experiments.
- Figure 2.
Proinflammatory cytokines production in autologous or allogeneic PBMCs cocultures with OAdv-infected MSCs. Uninfected or OAdv-infected (MOI = 50) MSCs from three different donors were cocultured with autologous or allogeneic human PBMCs at a PBMCs-to-MSCs ratio of 10 in triplicates. After 48 hours of coculture, culture medium was collected and cytokines level assessed by ELISA. The mean ± SD of triplicates from two independent experiments is shown (*, P < 0.05; ***, P < 0.001 by Kruskal–Wallis with Dunn post hoc test).
- Figure 3.
Effect of monocyte and/or NK-cell depletion on proinflammatory cytokines production in cocultures of OAdv-MSCs and allogeneic PBMCs. Allogeneic human PBMCs from the same donor were depleted for monocytes and/or NK cells, and cocultured with uninfected or OAdv-infected MSCs (PBMCs:MSC = 10). Nondepleted PBMCs alone or in coculture with OAdv-infected MSCs were used as control. After 48 hours of coculture, culture medium was collected and cytokines level assessed by ELISA. The mean ± SD of triplicates from two independent experiments is shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001 by Kruskal–Wallis with Dunn post hoc test).
- Figure 4.
CD4+ CD8+ T-cell and NK cell activation in cocultures of autologous or allogeneic human PBMCs with OAdv-infected MSCs. Uninfected or OAdv-infected MSCs were cocultured with autologous or allogeneic human PBMCs at a PBMCs-to-MSCs ratio of 10 in triplicates. After 48 hours of coculture, PBMCs were harvested and T cells and NK cells analyzed. A, Top, analysis of CD69 expression. Left, example of CD4+- and CD8+-positive cells expressing CD69 histograms in different coculture conditions. Middle, percentage of CD4+- and CD8+-positive cells expressing CD69. Right, CD69 mean fluorescent intensity of CD4+- and CD8+-positive cells. Bottom, analysis of CD25 expression. Left, example of CD4+- and CD8+-positive cells expressing CD25 histograms in different coculture conditions. Middle, percentage of CD4+ (left) and CD8+ (right)-positive cells expressing CD25. Right, CD25 mean fluorescent intensity of CD4+- and CD8+-positive cells. B, Top, analysis of CD69 expression. Left, example of CD56+CD16+-positive cells expressing CD69 histograms in different coculture conditions. Middle, percentage of CD56+CD16+-positive cells expressing CD69. Right, CD69 mean fluorescent intensity of CD56+CD16+-positive cells. Bottom, analysis of CD107a expression. Left, example of CD56+CD16+-positive cells expressing CD107a histograms in different coculture conditions. Middle, percentage of CD56+CD16+-positive cells expressing CD107a. Right, CD107a mean fluorescent intensity of CD56+CD16+-positive cells. C, Same experiment was performed allowing direct cell contact or using transwells system to separate OAdv-MSCs from allogeneic PBMCs. The percentage of CD4+, CD8+, or CD56+CD16+-positive cells expressing CD69 is represented. The mean ± SD of triplicates from two independent experiments is shown in all analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001 by Kruskal–Wallis with Dunn post hoc test).
- Figure 5.
Antitumor efficacy of OAdv-loaded MSCs in vitro. A431-GL and FaDu-GL cancer cell lines were cocultured with OAdv-infected or -uninfected MSCs in the presence or absence of allogeneic PBMCs (PBMCs:cancer cell ratio = 10) in triplicates. After 5 days in coculture, live cancer cells were counted by flow cytometer. Cytotoxicity was expressed as the percentage of live cancer cells on cocultures, normalized to the number of cancer cells cultured alone (*, P < 0.05 by Kruskal–Wallis with Dunn post hoc test). Results from two independent experiments are shown.
- Figure 6.
Antitumor efficacy of OAdv-loaded MSCs in vivo. A, Athymic nu/nu mice bearing subcutaneous A549 tumors were intraperitoneally injected with PBS, MSCs, OAdv, or MSCs previously infected with OAdv (n = 7 mice per group). Left, the mean of tumor growth ± SEM is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 OAdv versus PBS group; #, P < 0.05 OAdv versus MSCs; &, P < 0.05 OAdv versus MSCs/OAdv group by Kruskal–Wallis with Dunn post hoc test. Right, the presence of OAdv genomes in tumors at the end of the experiment was assessed by real-time PCR. B, NSG mice bearing subcutaneous A549 tumors received an intravenous injection of human allogeneic PBMCs except one group (n = 7 mice per group). Next day, mice were intraperitoneally injected with PBS, MSCs, OAdv, or MSCs previously infected with OAdv. Left, the mean of tumor growth ± SEM is shown. *, P < 0.05; **, P < 0.01 allogeneic PBMCs + MSC/OAdv versus PBS group by Kruskal–Wallis with Dunn post hoc test. Right, the presence of OAdv genomes in tumors at the end of the experiment was assessed by real-time PCR. Bottom, IHC staining of E1A of a representative tumor from each group is shown. The arrow points to positive virus E1A staining. C, NSG mice bearing subcutaneous A431 tumors were treated as described previously. The mean of tumor growth ± SEM is shown. *, P < 0.05; **, P < 0.01 allogeneic PBMCs + MSC/OAdv versus PBS group; #, P < 0.05 allogeneic PBMCs + MSC/OAdv versus MSCs; &, P < 0.05, allogeneic PBMCs + MSC/OAdv versus OAdv group, by Kruskal–Wallis with Dunn post hoc test. D, NSG mice bearing subcutaneous A549 tumors received an intravenous injection of human allogeneic PBMCs or PBMCs previously depleted for monocytes or NK cells (n = 7 mice per group). Next day, mice were intraperitoneally injected with PBS, or MSCs previously infected with OAdv. Right, the mean of tumor growth ± SEM is shown (#, P < 0.05, allogeneic PBMCs + MSC/OAdv vs. allogeneic PBMCs-NK− + MSC/OAdv group; &, P < 0.05, allogeneic PBMCs + MSC/OAdv vs. allogeneic PBMCs-Mo− + MSC/OAdv group by Kruskal–Wallis with Dunn post hoc test). Left, analysis of the percentage of CD56+ cells and CD14+ cells in PBMC and NK- or monocytes-depleted PBMCs samples, respectively.
Additional Files
Supplementary Data
- Supplementary Figures S1-S2 - Supplementary Figure 1. Cytokine array; Supplementary Figure 2. Effect of TLR-9 knock-down on OAd-infected MenSCs properties.
- Supplementary Material and Methods - Supplementary Material and Methods
Volume 18, Issue 1
