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1.
Figure 3

Figure 3. Uptake of FITC-OVA by BMDC is not enhanced by treatment with CTB. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

BMDC treated for 10 min at either 37°C with PBS (untreated), 1 μg/ml of LPS, 1 μg/ml of Pam3CSK, 5 μg/ml of LT-IIa-B5, or 5 μg/ml of CTB were incubated with FITC-OVA (0.2 mg/ml) for 10 min, washed with PBS, and stained with an APC-conjugated anti-CD11c mAb. FITC fluorescence of CD11c-positive, 7-AAD-negative cells was measured by flow cytometry. Data are presented as the MFI. Error bars denote one standard deviation from the mean obtained from triplicate cultures. Key: **, statistical difference from the control at P<0.01; ***, statistical difference from the control at P<0.001; ns, no statistical difference from the control.

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.
2.
Figure 1

Figure 1. Uptake of FITC-OVA by BMDC is enhanced by LT-IIa-B5. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

BMDC incubated for 10 min at either 37°C or on ice with 5 μg/ml of LT-IIa-B5 or PBS (Untreated) were treated with FITC-OVA (0.2 mg/ml) for 10 min, washed with PBS, and stained with an APC-conjugated anti-CD11c mAb. The Mean Fluorescent Intensity (MFI) of CD11c-positive, 7-AAD-negative cells was obtained using flow cytometry. Triplicate samples were employed for each experiment. A. Histographical analysis of uptake of FITC-OVA by BMDC. Data from one of three independent experiments are shown. B. Graphical comparison of the uptake of FITC-OVA by BMDC. Data from one of three independent experiments is shown. Error bars denote one standard deviation of the mean obtained from triplicate samples. Key: ***, statistical difference (P<0.001) between the treated population and the untreated control.

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.
3.
Figure 5

Figure 5. Cytokine production in BMDC induced by treatment with LT-IIa-B5. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

BMDC (5×106) derived from wt C57BL/6 mice (WT) or C57Bl/6 TLR2-/- mice (TLR2-/-) were incubated for 6 hrs at 37°C with PBS (Untreated), 100 ng/ml of LPS, or with 5 μg/ml of LT-IIa-B5. Total RNAs isolated from the BMDC were analyzed by qRT-PCR for mRNA encoding IL-12p40, TNF-α, IL-10, IL-4, and IFN-γ. Data are presented as fold change in expression of the cytokine from expression of that cytokine in the untreated BMDC. Error bars denote one standard deviation from the mean obtained from triplicate cultures. Key: +, TLR2-proficient BMDC; -, TLR2-deficient BMDC; white bars, BMDC treated with LPS; black bars, BMDC treated with LT-IIa-B5; *, statistical difference from the untreated control (P<0.05).

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.
4.
Figure 4

Figure 4. Expression of surface co-stimulatory receptors by wt and TLR2-deficient BMDC after treatment with LT-IIa-B5. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

BMDC (1×106) derived from wt C57BL/6 mice or C57Bl/6(TLR2-/-) mice were incubated for 24 hr at 37°C with, PBS (Untreated), 1.0 μg/ml of LPS, 1.0 μg/ml Pam3CSK, or 5.0 μg/ml of LT-IIa-B5. CD11c+ cells were analyzed by flow cytometry for expression of CD40, CD80, CD86, and MHC-II. A. Histographic analysis of expression of CD40, CD80, CD86, and MHC-II. Data from one of three independent experiments are shown. The MFI values for CD40, CD80, CD86, and MHC-II are denoted within each histogram. B. Graphical comparison of the expression of CD40, CD80, CD86, and MHC-II. Error bars denote one standard deviation from the mean. Key: wt, BMDC from TLR2-proficient C57Bl/6 mice; TLR2-/-, BMDC from TLR2-deficient mice; ***, statistical difference from the untreated control (P<0.001); **, statistical difference from the untreated control (P<0.01); ns, not significant.

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.
5.
Figure 2

Figure 2. LT-IIa-B5-induced enhanced uptake of FITC-OVA by BMDC is time- and dose-dependent. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

A. Dose-dependence. BMDC incubated for 10 min at 37°C with 0.1, 0.5, 1, 3, 5, or 10 μg/ml of LT-IIa-B5 were treated with FITC-OVA (0.2 mg/ml) for 10 min, washed with PBS, and stained with an APC-conjugated anti-CD11c mAb. Data are presented as fold change in the MFI of the FITC fluorescence of CD11c-positive, 7-AAD-negative LT-IIa-B5 treated BMDC to the MFI of the FITC fluorescence of CD11c-positive, 7-AAD-negative untreated BMDC. Error bars denote one standard deviation from the mean obtained from triplicate cultures. B. Time dependence. BMDC incubated at 37°C for 10, 20, or 30 min and 1, 2, 12, or 24 hrs with 5 μg/ml of LT-IIa-B5 were treated with FITC-OVA (0.2 mg/ml) for 10 min, washed with PBS, and stained with an APC-conjugated anti-CD11c mAb. Data are presented as fold change of MFI of the FITC fluorescence of CD11c-positive, 7-AAD-negative LT-IIa-B5 treated BMDC to MFI of the FITC fluorescence of CD11c-positive, 7-AAD-negative untreated BMDC. Error bars denote one standard deviation from the mean obtained from triplicate cultures.

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.
6.
Figure 7

Figure 7. Enhanced uptake of FITC-OVA by BMDC induced by treatment with LT-IIa-B5 is dependent on TLR2. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

A. BMDC treated with PBS (untreated), 5 μg/ml of LT-IIa-B5, or 1 μg/ml of LPS were co-incubated with 10 μg/ml or 20 μg/ml of an anti-TLR2 blocking mAb (α-TLR2). After 10 min, BMDC were incubated with FITC-OVA (0.2 mg/ml), washed with PBS, and stained for CD11c. CD11c-positive cells were measured for FITC fluorescence. Data are presented as the MFI. Error bars denote one standard deviation from the mean obtained from triplicate cultures. Key: ***, statistically different from the untreated group or, for the anti-TLR2-treated cells, statistically different from the LT-IIa-B5-treated group (as denoted by the lines) (P<0.001); ns, not significant from the untreated group. B. BMDC derived from the bone marrow of wt C57Bl/6 mice or C57Bl/6(TLR2-/-) mice treated with PBS (Untreated), 5 μg/ml of LT-IIa-B5, or 1 μg/ml of LPS were incubated with FITC-OVA (0.2 mg/ml) for 10 min, washed with PBS, and stained for CD11c. Data are presented as the MFI with error bars denoting one standard deviation from the mean obtained from triplicate cultures. Key: +, TLR2-proficient BMDC; -, TLR2-deficient BMDC; +++, statistical difference from the matched (Untreated vs TLR2-deficient) control; ***, statistical difference between the wt and TLR2-deficient BMDC treated with LT-IIa-B5 (as denoted by the lines); ns, not significant to the matched untreated control or, in the case of the LPS-treated cells, no significant difference between the TLR2-proficient and the TLR2-deficient BMDC.

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.
7.
Figure 6

Figure 6. OVA-specific CD4+ T cells proliferation is enhanced by LT-IIa-B5. From: Enhanced antigen uptake by dendritic cells induced by the B pentamer of the type II heat-labile enterotoxin LT-IIa requires engagement of TLR2.

BMDC (5×104) were incubated for 30 min with PBS (Control), 1.0 mg/ml OVA (OVA), 1.0 mg/ml BSA, 5 μg/ml LPS and 1.0 mg/ml OVA (LPS + OVA), 5 μg/ml Pam3CSK and 1.0 mg/ml OVA (Pam3CSK + OVA), or with 20 μg/ml LT-IIa-B5 and 1.0 mg/ml OVA (LT-IIa-B5 + OVA). Similar experiments were performed in which BMDC were initially incubated with 1.0 g/ml OVA, washed, and subsequent treated with 5 μg/ml LPS (OVA, then LPS), 5 μg/ml Pam3CSK (OVA, then Pam3CSK), or 20 μg/ml LT-IIa-B5 (OVA, then LT-IIa-B5). After washing, BMDC were co-cultured with CFSE-stained naïve DO11.10 CD4+ T cells (1×106). Cells were harvested after 4 days, washed in PBS, stained for CD4, and the CFSE fluorescence of the DO11.10 CD4+ T cells was determined as a measure of proliferation. A. Histographical analysis of Ag-specific CD4+ T cells proliferation. The percentage of dividing cells in each population is denoted below the bracket in each histogram. B. Graphical comparison of Ag-specific CD4+ T cell proliferation. Error bars denote one standard deviation from the mean obtained from triplicate cultures. Key: +++ and ++ statistical differences from the control at P<0.001 and P<0.01 respectively. *** and **statistical differences from the matched column at P<0.001 and P<0.01 respectively.

Chang Hoon Lee, et al. Vaccine. ;28(21):3696-3705.

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