Figure 1
Figure 1. Human B-CLL cells express high levels of TLR9 and can be potently activated by CpG-B ODNs. Purified B-CLL cells from CLL patients 1 to 5 in Table 1 were used and tested. (A) RT-PCR results of TLR1-10 expression profile in B-CLL cells from 5 CLL patients. (B) RT-PCR results of TLR9 mRNA expression and (C) Western blot results of TLR9 protein expression in B-CLL cells from 5 patients were compared with that of normal B and T cells from 5 healthy blood donors, and normalized to the expression of β-actin. Data are presented as the mean ± SD in the adjacent bar diagram. *P < .01, B-CLL cells and B cells versus T cells. **P < .01, B-CLL cells versus B cells. (D) Morphologies of fresh and day 3 cultured B-CLL cells with or without CpG-A or CpG-B ODN were examined. Images of B-CLL cells in cultures were acquired on an Olympus CKX41 inverted microscope with an Olympus DP12 digital microscope camera at 200× magnification. Images of Giemsa staining of B-CLL cells were acquired on a Zeiss Axioplan 2 Upright Microscope (Carl Zeiss Inc) with a Spot RT color digital camera (Diagnostic Instruments), captured with a Zeiss Plan-Apochromat 63×/1.4 oil objective lens at 630× magnification, and processed with Spot advanced 4.6 software. (E) Expression of surface markers (shaded histogram) on fresh or day 3 cultured B-CLL cells with or without CpG-A or CpG-B ODN was analyzed by flow cytometry, indicated with MFI number, and overlaid with isotype control (unshaded histogram). Data are representative results from 1 of 5 CLL patients. (F) MFI fold increases of the indicated surface molecules on day 3 cultured versus uncultured B-CLL cells. Data are the mean ± SD from 5 independent experiments. *P < .01. (G) MFI fold increases of CD40 expression on day 3 cultured B-CLL cells with CpG-A or CpG-B ODNs versus B-CLL cells cultured in media only. Data are the mean ± SD from 5 independent experiments. *P < .01. (H) Graded doses of irradiated day 3 cultured B-CLL cells with or without CpG ODNs were used as stimulators to allogeneic T cells in MLR assays. Data are representative results with B-CLL cells from 4 CLL patients. The error bars represent the SD of triplicates. The independent experiments were performed with B-CLL cells derived from different CLL patients

Human B-CLL cells express high levels of TLR9 and can be potently activated by CpG-B ODNs. Purified B-CLL cells from CLL patients 1 to 5 in Table 1 were used and tested. (A) RT-PCR results of TLR1-10 expression profile in B-CLL cells from 5 CLL patients. (B) RT-PCR results of TLR9 mRNA expression and (C) Western blot results of TLR9 protein expression in B-CLL cells from 5 patients were compared with that of normal B and T cells from 5 healthy blood donors, and normalized to the expression of β-actin. Data are presented as the mean ± SD in the adjacent bar diagram. *P < .01, B-CLL cells and B cells versus T cells. **P < .01, B-CLL cells versus B cells. (D) Morphologies of fresh and day 3 cultured B-CLL cells with or without CpG-A or CpG-B ODN were examined. Images of B-CLL cells in cultures were acquired on an Olympus CKX41 inverted microscope with an Olympus DP12 digital microscope camera at 200× magnification. Images of Giemsa staining of B-CLL cells were acquired on a Zeiss Axioplan 2 Upright Microscope (Carl Zeiss Inc) with a Spot RT color digital camera (Diagnostic Instruments), captured with a Zeiss Plan-Apochromat 63×/1.4 oil objective lens at 630× magnification, and processed with Spot advanced 4.6 software. (E) Expression of surface markers (shaded histogram) on fresh or day 3 cultured B-CLL cells with or without CpG-A or CpG-B ODN was analyzed by flow cytometry, indicated with MFI number, and overlaid with isotype control (unshaded histogram). Data are representative results from 1 of 5 CLL patients. (F) MFI fold increases of the indicated surface molecules on day 3 cultured versus uncultured B-CLL cells. Data are the mean ± SD from 5 independent experiments. *P < .01. (G) MFI fold increases of CD40 expression on day 3 cultured B-CLL cells with CpG-A or CpG-B ODNs versus B-CLL cells cultured in media only. Data are the mean ± SD from 5 independent experiments. *P < .01. (H) Graded doses of irradiated day 3 cultured B-CLL cells with or without CpG ODNs were used as stimulators to allogeneic T cells in MLR assays. Data are representative results with B-CLL cells from 4 CLL patients. The error bars represent the SD of triplicates. The independent experiments were performed with B-CLL cells derived from different CLL patients

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