Rv0652-induced cytokine production is partially mediated by MyD88- and TRIF-pathways.

<p>DCs derived from WT, MyD88^−/−, and TRIF-deficient mice were treated with Rv0652 (1 ug/ml) or LPS (200 ng ng/ml) for 24 h. The supernatants were harvested and ELISA was performed to test TNF h. The supernatants were harvested and ELISA was performed to test TNF-alpha, IL-6, and IL-1beta production. Data are means and SEM of 3 experiments. **<i>P</i><0.01 and ***<i>P</i><0.001, compared to WT cultures.</p

Rv0652-treated DCs pulsed with OVA_257–264 protects mice against E.G7 tumor challenge.

<p>C57BL/6 mice were challenged s.c with 3×10⁵ E.G7 or EL4 tumor cells into the right flank area. For administration of DCs (1×10⁶ cells/mice), mice were injected intravenously with PBS, iDC, DCs-OVA (DCs pulsed with OVA_257–264), Rv0652-DCs-OVA (Rv0652-treated DCs pulsed with OVA_257–264), or LPS-DCs-OVA (LPS-treated DCs pulsed with OVA_257–264) on days 1, 3 and 5 after the tumor challenge. Following the tumor challenge with E.G7 (A) and EL4 (B), tumor growth was monitored by measuring the diameter of the tumor every 2 days. **<i>P</i><0.01, each group, n = 10. (C) Survival of mice with an E.G7 tumor challenge after injection of OVA peptide-pulsed and Rv0652-treated DCs, n = 10 mice/group. <i>P</i> value calculated by Kaplan-Meyer log-rank test between two groups of mice injected with DCs-OVA and Rv0652-DCs-OVA, respectively.</p

Rv0652-treated DCs induce proliferation of CD4⁺ and CD8⁺ T cells and a Th1 response.

<p>Transgenic OVA-specific CD8⁺ T cells (A-i) and OVA-specific CD4⁺ T cells (B-i) were isolated and stained using CFSE. These cells were then co-cultured for 96 h with untreated DCs, DCs pulsed with OVA h with untreated DCs, DCs pulsed with OVA_257–264, Rv0652 (1 ug/ml)-treated DCs pulsed with OVA_257–264, or LPS (200 ng ng/ml)-treated DCs pulsed with OVA_257–264. T-cell proliferation was assessed using flow cytometry and the percentage of proliferating cells is shown in each panel. Bar graphs show the percentage of OVA-specific CD8⁺ T cells (A-ii) and OVA-specific CD4⁺ T cells (B-ii) from 3 independent experiments. ***<i>P</i><0.001 compared to T cells/OVA pulsed-DCs. (C) IFN-gamma production was measured in each culture supernatant from Fig. 5A and 5B after 24 h of culture by performing an ELISA. Data are the mean and SEM of 3 experiments. ***<i>P</i><0.001 compared to the value from T cell/OVA peptide-pulsed DCs.</p

A Potential Protein Adjuvant Derived from <i>Mycobacterium tuberculosis</i> Rv0652 Enhances Dendritic Cells-Based Tumor Immunotherapy

<div><p>A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from <i>Mycobacterium tuberculosis</i> Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production (TNF-alpha, IL-1beta, and IL-6) that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4⁺ and CD8⁺ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8⁺ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.</p></div

Rv0652 enhances the migration of DCs <i>in vitro</i> and <i>in vivo</i>.

<p>(A) DCs were treated with the indicated concentrations of Rv0652 or LPS (200 ng ng/ml) for 24 h. The percentage of CCR7 h. The percentage of CCR7⁺CD11c⁺ DCs was analyzed by flow cytometry. The number in each panel indicates the percentage of positive cells (i). Bar graphs show the percentage of CCR7-expressing CD11c⁺ cells, representing 4 independent experiments. *<i>P</i><0.05 and ***<i>P</i><0.001 compared to the media control (ii). (B) DCs were treated with Rv0652 or LPS for 24 h and then subjected to an <i>in </i><i>vitro</i> transwell chemotaxis assay using media alone or media containing CCL19 (300 ng ng/ml). Recorded data are the mean and SEM of 4 experiments. ***<i>P</i><0.001, compared to untreated DCs. (C) CFSE-labeled Rv0652 (1 ug/ml)- or LPS (200 ng ng/ml)-treated DCs were injected subcutaneously into the hind leg footpad of mice, the cells were recovered from popliteal LNs 72 h later and then analyzed. Data are the mean and SEM of 4 experiments. **<i>P</i><0.05 compared to untreated DCs.</p

Rv0652 binds to TLR4 but not TLR2, and induces phenotypic and functional maturation of DCs.

<p>(A) Fluorescence microscopic observation of Rv0652 binding on DCs. (i) DCs derived from WT, TLR2^−/−, and TLR4^−/− mice were treated with Rv0652 (0.5 ug/ml) for 1 h and stained with Alexa568 h and stained with Alexa568-conjugated anti-Rv0652 mAb and FITC-conjugated CD11c⁺ Ab. The percentage of labeled cells was analyzed by flow cytometry. The percentage of positive cells is shown in each panel. Bar graphs show the mean and standard error of the mean (SEM) for the percentage of Rv0652-Alexa568 labeled CD11c⁺ cells observed in 3 independent experiments. ***<i>P</i><0.001 compared to Rv0652-treated WT DCs. (ii) DCs derived from WT, TLR2^−/−, and TLR4^−/− mice were treated with Rv0652 (0.5 ug/ml) for 1 h, fixed, and stained with DAPI and Alexa568 h, fixed, and stained with DAPI and Alexa568-conjugated anti-Rv0652 mAb. One representative experiment out of 3 with similar results is shown. (B) Flow cytometry was used to analyze the expression of surface molecules on CD11c⁺ cells. The mean fluorescence intensity (MFI) and the percentage of positive cells are shown in each panel (i). Bar graphs show the percentage of surface molecule-expressing CD11c⁺ cells, representing 5 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 compared to medium control (ii). (C, D) ELISA was performed to test IL-12p70, IL-10, TNF-alpha, IL-6, and IL-1beta production in Rv0652- and LPS-treated DCs. The presented data are the mean and SEM of 3 experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 compared to untreated DCs. (E–F) DCs derived from WT, TLR2^−/−, and TLR4^−/− mice were treated with Rv0652 (1 ug/ml) or LPS (200 ng ng/ml) for 24 h. (E) Flow cytometry was used to analyze the expression of surface molecules on CD11c⁺ cells. The percentage of positive cells is shown in each panel (i). Bar graphs show the percentage of surface molecule-expressing CD11c⁺ cells, representing 4 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 compared with WT cultures (ii). (F) The supernatants were harvested and an ELISA was performed to test IL-1beta production. Data are means and SEM of 3 experiments. ***<i>P</i><0.001, compared to WT cultures.</p

Purity and endotoxin contamination of the rRv0652 protein and its effect on cell viability.

<p>(A) Recombinant Rv0652 protein was purified with NTA resin, subjected to SDS-PAGE, and analyzed by using Coomassie blue (CB) staining (left panel) and immunoblot assay using anti-histidine and anti-Rv0652 antibodies (right panel). (B) DCs were stimulated with vehicle, Rv0652 (1 ug/mL), or LPS (200 ng ng/mL), and treated with/without polymyxin B (10 ug/mL), proteinase K (5 ug/ml), or heat (100°C for 30 min min). Culture supernatants were harvested after 24 h, and IL h, and IL-1beta levels were assessed by an ELISA. Data are the mean and SEM of 3 experiments. ***<i>P</i><0.001. (C) DCs were treated with the indicated concentrations of Rv0652 or LPS (200 ng ng/mL) for 24 h, stained with Annexin V and PI, and analyzed by flow cytometry. Results are representative of 4 separate experiments with similar results. h, stained with Annexin V and PI, and analyzed by flow cytometry. Results are representative of 4 separate experiments with similar results.</p

Rv0652-treated DCs pulsed with OVA_257–264 enhances CTL activity and CD8⁺ T-cell tumor infiltration.

<p>C57BL/6 mice were immunized with PBS, iDC, DCs-OVA (DCs pulsed with OVA_257–264), Rv0652-DCs-OVA (Rv0652-treated DCs pulsed with OVA_257–264), or LPS-DCs-OVA (LPS-treated DCs pulsed with OVA_257–264) on days 1 and 7. (A) Splenocytes from the mice immunized with PBS or various DCs for 10 days were treated with OVA_257–264 (10 ug/ml) for 7 days and then co-cultured with EL4 (CFSE^low) or E.G7 (CFSE^high) cells. After 2 days, mixed lymphocyte tumor cultures were analyzed by flow cytometry. Histograms show antigen-specific lysis of tumor cells. The percentage in each panel indicates specific lysis of E.G7 cells (i). Bar graphs show the percentage of specific lysis of E.G7 cells representing 4 independent experiments (ii). ***<i>P</i><0.001. (B) Splenocytes (i) and tumor infiltrating CD8⁺ T cells (ii) from the mice immunized with PBS or various DCs for 10 days were treated with OVA_257–264 (10 ug/ml) for 24 h IL h IL-2 and IFN-gamma production was measured in supernatants by ELISA. Data are the mean and SEM of 4 experiments. *<i>P</i><0.05 and ***<i>P</i><0.001. (C) C57BL/6 mice were challenged s.c. with E.G7 tumor cells to the right flank area. One, 3, and, 5 days after the tumor challenge, mice were injected intravenously with PBS, iDC, DCs-OVA (DCs pulsed with OVA_257–264), or Rv0652-DCs-OVA (Rv0652-treated DCs pulsed with OVA_257–264) (n = 3). At day 20, the cells in the tumor area were analyzed for the presence of CD8⁺CD69⁺ T cells. The percentage in each panel indicates the infiltration of CD8⁺CD69⁺ T cells into the tumor area. Data are the mean and SEM of 3 experiments. **<i>P</i><0.05 and ***<i>P</i><0.001.</p

Effect of DOK-1 in OVA-induced tissue inflammation and mucus responses.

<p>(A) Lung sections were stained with hematoxylin and eosin, D-PAS, alcian blue for the evaluation of inflammatory cells and airway mucus responses. ×40 of original magnification. Con, non OVA challenged; OVA, OVA-challenged with empty lentiviral vector; DOK_ShRNA, OVA-challenged with DOK_ShRNA knockdown; DOK, OVA-challenged with DOK-1 overexpression. At least 4 mice were included in each group. (B) Inflammatory index that scored parenchymal inflammation. At least 4 mice were included in each group. *P<0.05 (C) Mucus index evaluated by morphometric analysis representing alcian blue stained mucus cells (percentage of positive cells) in airway epithelial cells. At least 4 mice were included in each group. *P<0.05.</p

Inflammatory cell counts in the lungs from mice challenged with OVA and DOK-1 shRNA.

<p>Inflammatory cell counts in the lungs from mice challenged with OVA and DOK-1 shRNA.</p