These data confirm our in vivo results and show that a soluble factor, released in eye tumors but not in normal eyes, was able to counteract the antiproliferative effect of CpG motifs. Figure 3 PIOL supernatant counteracts in vitro antiproliferative effect of CpG-ODNs on A20.IIA malignant B cells. 104 cells were stimulated for 72 hours with various concentrations of CpG or control ODNs in concentrations find more ranging from 0.003 to 60 μg/mL or with medium alone and with the presence of supernatant from (A) PBS 1X injected eyes (PIE),
(B) SCL, (C) PCL, or (D) PIOL. The incorporation of the [3H] thymidine was measured by a scintillation counter. *P < 0.05; **P < 0.01. The data shown are representative results from 1 of 3 experiments. Figure 4 Soluble molecule present in PIOL but not in normal ocular microenvironment is able to abrogate in vitro effect of CpG-ODNs in a dose-dependent manner. 104 cells were stimulated for 72 hours with CpG or control ODNs at 30 μg/mL and in the presence of several diluted doses of control supernatant (PIE) or PIOL supernatant (1X, 1/20, 1/35, 1/50, 1/75, 1/100, 1/200, 1/500). The incorporation of the [3H] thymidine was measured by a scintillation counter. *P < 0.05; **P < 0.01. The PIOL microenvironment did not modify either TLR9 expression or the internalization of CpG-ODNs by tumor cells To investigate the possibility that
the loss of the CpG-ODNs antitumor action was associated with modulation of TLR9 expression, we used flow cytometry to compare TLR9 expression on A20.IIA cells after incubation XL184 solubility dmso with supernatant from medium alone, PIOL or PIE. No differences were found between these conditions (Figure 5A). Figure 5 The PIOL microenvironment did not modify TLR9 expression or internalization of CpG-ODNs by tumor cells. (A) 104 A20.IIA cells were incubated with PIOL or PIE supernatant. 3 days later, cytometric analysis was performed of TLR9 expression by cells incubated with
PIOL supernatant, overlaid Sulfite dehydrogenase with isotype control and compared to TLR9 expression by cells incubated with PIE supernatant or medium alone. (B) 104 A20.IIA cells were incubated for 24 hours with medium alone or with PIOL or PIE supernatant and in the presence or absence of FITC-labeled CpG-ODNs at 3 μg/mL. FITC expression by A20.IIA tumor cells was analyzed by flow cytometry. Next we RG7420 purchase examined whether the PIOL molecular microenvironment inhibited internalization of CpG ODNs by tumor cells. FITC-labelled CpG 1826 ODNs were added for 24 hours at a concentration of 3 μg/mL to A20.IIA lymphoma cells in the presence of PIOL or PIE supernatant. Flow cytometric analysis indicated that FITC expression by tumor cells with PIOL supernatant was similar to that incubated with PIE supernatant (Figure 5B).These findings show that the addition of PIOL supernatant does not modify CpG internalization by lymphoma B-cells, even in vivo in our three model (data not shown).
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