STAT3 Activation in Combination with NF-KappaB Inhibition Induces Tolerogenic Dendritic Cells with High Therapeutic Potential to Attenuate Collagen-Induced Arthritis

Figure 2

Constitutive repression of NF-κB favours the acquisition of tolerogenic properties by DCs, which attenuate CIA development. Bone marrow-derived precursors from DBA/1J mice were cultured in conditions to differentiate to DCs and transduced on days 4 and 5 with lentiviral vectors (MOI 10) encoding for IκBαSR or with empty PGK-Cre vectors (control) in the presence of polybrene. At day 6, DCs were either left without treatment (iDC) or treated with 500 ng/ml LPS for 24 h (mDC). (a, b) Levels of mRNA encoding for cytokines were evaluated in transduced DCs by quantitative real-time RT-PCR and normalized with the levels of gapdh mRNA. (a) Levels of mRNA encoding for different cytokines are represented as relative units. Values are from three or more independent experiments carried out in duplicate. , , and by one-way ANOVA followed by Tukey’s post hoc test. (b) Data presented as the fold increase of mRNA levels in mDCs relative to mDCs transduced with control vectors. Values are from three or more independent experiments carried out in duplicate. , which indicates no differences between cytokine mRNA levels in mDCs transduced with IκBαSR and in mDCs transduced with control vectors, is represented by a dotted line. by unpaired two-tailed Student’s -test. (c) After lentiviral transduction, DCs were cocultured with naive CD4⁺ T lymphocytes (at DC : T-cell ratio of 1 : 5) in the presence of anti-CD3ε antibody and incubated for 5 d. Afterward, the percentage of Treg lymphocytes was evaluated by intracellular immunostaining of Foxp3 in the CD4⁺ gate. To determine the percentage of Th1 and Th17 lymphocytes, cells were restimulated with PMA and ionomycin in the presence of brefeldin A for the last 4 h of culture and the extent of IFN-γ and IL-17 was assessed by intracellular cytokine immunostaining and quantified by flow cytometry in the CD4⁺-gated population. Dot plots of Foxp3 versus CD4 (top panels) and IFN-γ versus IL-17 in the CD4⁺ population (bottom panels) are shown. Representative data from three independent experiments is shown. The percentage of cells present in each quadrant is indicated. (d) After transduction, mDCs were loaded with 40 μg/ml of CII for 18 h and then intravenously transferred ( transduced DCs/mouse) into DBA/1J mice at day 35 after CIA induction. The disease score was evaluated along with CIA development as described in Materials and Methods. Data represents from eight animals per group. by Mann–Whitney test. (e) After transduction, DCs were not pulsed with any exogenous antigen and intravenously administered ( transduced DCs per animal) to DBA/1J mice at day 35 after CIA induction. Experimental groups receiving DCs transduced with IκBαSR or with the empty PGK-Cre control vector (empty vector) were also compared with a group of mice that did not receive DCs (without DCs). The disease score was evaluated along with CIA development as described in Materials and Methods. Data represents from four animals per group. No significant differences were detected between different experimental groups.

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