Induced T cells specific for melanoma antigens not present in the vaccine have antitumor activity.

<p>(<b>A-C</b>) Mice were inoculated with B16-OVA or B16 tumor on the right or left flank separately followed by immunization with 1×10⁶ DC-shA20-FAP (AF), DC-shA20-OVA (AO), DC-shA20-FAP-OVA (AFO) or PBS 5 days later (n = 5 per group). B16-OVA (<b>A</b>) and B16 (<b>B</b>) tumor growth was measured. The AFO and the AF vaccine had anti-B16 activity with AFO being superior (p<0.05). (<b>C</b>) Kaplan-Meier survival curve (AO vs AF, p<0.05; AO vs AFO, p<0.05). (<b>D,E</b>) Mice were inoculated with B16-OVA followed by immunization with 1×10⁶ AF, AO, AFO, and PBS 5 days later. 3 weeks later, splenocytes were prepared and cultured in vitro in the presence of B16-OVA tumor lysate and IL2 for 5 days and their cytolytic activity was evaluated in standard subjected ⁵¹Cr release assay against B16-OVA (<b>D</b>) or B16 (<b>E</b>). Splenocytes isolated from AF and AFO vaccinated mice had significant cytolytic activity B16. *AFO vs AF and AO, p<0.05; **AF and AO vs PBS, p<0.05;***AFO and AF vs AO, p<0.05).</p

DC-shA20-FAP-TRP2 vaccine increases FAP- and TRP2-specific CD8-positive T cells in tumors.

<p>Mice were inoculated with B16 tumors followed by immunization with 1×10⁶ DC-shA20-FAP (AF), DC-shA20-TRP2 (AT), DC-shA20-FAP-TRP2 (AFT) or PBS on day 5. Tumor tissues were dissected 3 weeks post vaccination (n = 5). (<b>A</b>) Representative FACS analysis of infiltrating CD8+ T cells. (<b>B</b>) Summary data of all mice showing a significant increase (p<0.05) of CD8+ T cells in mice vaccinated with AFT (n = 8 per group). (<b>C</b>) TILs were restimulated with DCs transduced with FAP (DC-Lv-FAP) or TRP2 (DC-LV-TRP2) followed by intracellular staining of IFN-γ. One of 2 representative experiments in shown.</p

A Vaccine That Co-Targets Tumor Cells and Cancer Associated Fibroblasts Results in Enhanced Antitumor Activity by Inducing Antigen Spreading

<div><p>Dendritic cell (DC) vaccines targeting only cancer cells have produced limited antitumor activity in most clinical studies. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To co-target CAFs and tumor cells we developed a new compound DC vaccine that encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2). DC-shA20-FAP-TRP2 vaccination induced robust FAP- and TRP2-specific T-cell responses, resulting in greater antitumor activity in the B16 melanoma model in comparison to monovalent vaccines or a vaccine encoding antigens and a control shRNA. DC-shA20-FAP-TRP2 vaccination enhanced tumor infiltration of CD8-positive T cells, and induced antigen-spreading resulting in potent antitumor activity. Thus, co-targeting of tumor cells and CAFs results in the induction of broad-based tumor-specific T-cell responses and has the potential to improve current vaccine approaches for cancer.</p></div

DC-shA20-FAP-TRP2 vaccine has potent antitumor activity.

<p>(<b>A</b>) RT-PCR for FAP and GAPDH of B16 cell line, and day 5 B16 tumors. (<b>B,C</b>) Mice were inoculated with B16 followed by immunization with 1×10⁶ DC-shA20-FAP (AF), DC-shA20-TRP2 (AT), DC-shA20-FAP-TRP2 (AFT), DC-shCo-FAP-TRP2 (ACT) or PBS on day 5 (n =  5 per group). (<b>B</b>) Cotargeting FAP and TRP2 with AFT resulted in the greatest antitumor response (AF vs AFT, p<0.05; AT vs AFT, p<0.05). (<b>C</b>) Kaplan-Meier survival curve (AF vs AFT, p<0.05; AT vs AFT, p<0.05).</p

DC-shA20-FAP-TRP2 vaccine induces antigen spreading.

<p>Mice were inoculated with B16 tumors followed by immunization with 1×10⁶ DC-shA20-FAP (AF), DC-shA20-TRP2 (AT), DC-shA20-FAP-TRP2 (AFT) or PBS on day 5. TILs and Splenocytes were prepared 3 weeks post vaccination (n = 5). (<b>A</b>) TILs were restimulated with DC-Lv-TRP2, DC-Lv-TRP1, DC-Lv-TYR, DC-Lv-gp100, DC-Lv-MART1 or mock transduced DCs (PBS) followed by intracellular staining of IFN-γ. FACS analysis and a cumulative bar graph are shown. TILs isolated from AFT vaccinated mice had the highest frequency of B16-speciifc T cells (p<0.05). (<b>B</b>) Splenocytes were subjected to IFN-γ Elispot assays. DC-Lv-TRP2, DC-Lv-TRP1, DC-Lv-TYR, DC-Lv-gp100, DC-Lv-MART1 or mock transduced DCs (PBS) were used as APCs. There was a significant increase (p<0.05) of T cells specific for TRP1, TYR, gp100, and MART1 only in AFT vaccinated mice.</p

Tumor- and FAP- co-targeted DC induce T-cell activation.

<p>(<b>A</b>) Scheme of lentiviral constructs. (<b>B and C</b>) Mouse BM-DCs were transduced with lentivirus and FAP and TRP2 expression (<b>B</b>) and A20 expression (<b>C</b>) were detected by RT-PCR or Q-PCR individually. (<b>D and E</b>) Mice were immunized with 1×10⁶ lentivirus-transduced BM-DCs in 25 µl sterile PBS or PBS control through footpad 14 days post vaccination splenocytes were prepared and CD8+ (<b>D</b>) and CD4+ (<b>E</b>) T cells selected. The frequency of FAP- and TRP2-specific T cells was determined using IFN-γ ELISPOT assays (n = 2; assay performed in triplicates). AF, lentiviral vector coexpressing an A20-specific short-hairpin RNA (shRNA) and FAP; AT, lentiviral vector coexpressing A20-shRNA and TRP2; AFT, lentiviral vector coexpressing A20-shRNA, FAP, and TRP2; CFT, lentiviral vector coexpressing GFP-shRNA, FAP, and TRP2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBS, phosphate-buffered saline.</p

Synthesis, crystal structures and DNA-binding properties of Zn(II), Cd(II), Mn(II), Cu(II) complexes based on a thiazole derivative and carboxylic acids

By single-crystal X-ray diffraction (XRD) analysis, five complexes, catena-[(µ-2,2-bis(4-carboxyphenyl)hexafluoropropane)-(2-(pyidin-2-yl)-1,3-benzothiazole)-aqua-zinc(II)] (1), catena-[(µ-2,2-bis(4-carboxyphenyl)hexafluoropropane)-(2-(pyidin-2-yl)1,3-benzothiazole)-aqua-cadmium(II)] (2), catena-[hepta(µ-2,2-bis(4carboxyphenyl)hexafluoropropane)-diaqua-bis(2-(pyidin-2-yl)-1,3-benzothiazole)-tri-manganes(II)] (3), catena-[(µ-benzene-1,2,4,5-tetracarboxylato)-(2-(pyidin-2-yl)-1,3benzothiazole)copper(II)] (4) and catena-[(µ-3-nitrobenzene-1,2-dicarboxylato)-(2-(pyidin-2-yl)1,3-benzothiazole)-aqua-zinc(II)] (5), were characterized in this research. In these five complexes, H2hfipbb = 2,2-bis(4-carboxyphenyl)hexafluoropropane, H4btec = 1,2,4,5-benzenetetracarboxylic acid, H2NPTA = 3-nitrophthalic acid and bpt = 2-(pyridin-2-yl)benzo[d]thiazole). Complexes 1 and 2 occur in the monoclinic system with space group C2/c, 4 and 5 occur in the monoclinic system with space group P21/c, and 3 occurs in the triclinic system with space group P-1. The interaction between CT-DNA (calf thymus DNA) and those complexes was examined using UV spectroscopic analysis, fluorescent spectroscopic analysis, circular dichroism spectroscopy (CD) and viscosity analysis. This research reveals that those complexes have strong ability to interact with DNA, and they all bind to DNA in an intercalation mode, thus affecting the structure of DNA. This binding mode can arise from the interaction between the benzothiazole metal complexes and DNA. Hence, we hope to provide some scientific research bases for studying benzothiazole complexes and designing new thiazole anticancer drugs.</p

Reduction in cell-associated luciferase activity closely reflects the killing activity of T killer cells against tumor targets.

<p>Mock-transduced (<b>A</b>) or cTCR-transduced (<b>B</b>) splenocytes were added to 4T1-Her2 cells at the indicated ratio. Cells were harvested 72 later for quantification of luciferase activity. <b>C</b>. Using the formula: % specific luc reduction = (% luc reduction from engrafted T-cell)/(% luc reduction from control T-cell)×100, the data from <b>A</b> and <b>B</b> were converted into percentage of specific luciferase release and plotted.</p

The Role of IL-12 Signaling in Enhanced Anti-HIV Immunity

<div><p>(A and B) In vivo injection with IL-12 preferentially enhanced gp120-specific CTL and Th responses induced by SOCS1-silenced DCs. C57BL/6 mice were immunized with 1 × 10⁶ of HIV gp120-pulsed (50 μg/ml), transduced DCs derived from BM of WT mice or IL-12 receptor KO mice with ex vivo TNFα maturation (50 ng/ml). On days 1, 3, and 5 after DC immunization, murine IL-12 (1 μg/mouse, Peprotech) was administered intraperitoneally. CD8⁺ T cells (A) or CD4⁺ T cells (B) isolated 2 wk later from the pooled splenocytes of immunized mice (2–3 each group) were subjected to IFN-γ ELISPOT assays. An irrelevant protein, OVA, was used as a negative control. Representative data from two independent experiments are presented. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DC versus LV-SOCS1-siRNA-DC + IL-12, or IL12R KO LV-SOCS1-siRNA-DC + IL-12 versus LV-SOCS1-siRNA-DC + IL-12.</p> <p>(C and D) gp120-specific CTL and Th responses induced by SOCS1-silenced DCs or Ad-IL-12-DCs. BM-derived DCs from WT mice were transfected with LV-SOCS1-siRNA (MOI of 5) or Ad-IL-12 with various MOIs of 10–1,000 or cotransfected with LV-SOCS1-siRNA (MOI of 5) and Ad-IL-12 (MOI of 10) for 4 h. DCs derived from BM of IL-12 receptor KO mice were cotransfected with LV-SOCS1-siRNA (MOI of 5) and Ad-IL-12 (MOI of 10) for 4 h. Groups of C57BL/6 mice were immunized with 1 × 10⁶ of gp120-pulsed (50 μg/ml), transfected DCs with ex vivo TNFα maturation. CD8⁺ T-cells (C) or CD4⁺ T cells (D) isolated 2 wk later from the pooled splenocytes of immunized mice (2–3 each group) were subjected to IFN-γ ELISPOT assays. An irrelevant protein, OVA, was used as a negative control. Representative data from two independent experiments are presented. <i>P</i> < 0.01, Ad-IL-12/SOCS1-siRNA-DC versus IL-12-DCs, or Ad-IL-12/SOCS1-siRNA-DC versus IL12R KO Ad-IL-12/SOCS1-siRNA-DC.</p></div

The expression of the human version shhS1/FliC potently activates human monocyte-derived DCs.

<p>Human monocyte-derived DCs were transduced with Ad vectors at an MOI of 250 or stimulated with different TLR agonists. <b>A & B.</b> 24 h later, culture media were collected from Ad-transduced DCs (<b>A</b>) or TLR-transduced DCs (<b>B</b>) for evaluation of the representative cytokines by ELISA. <b>C & D</b>. 24 h later, cultures were washed and replaced with fresh medium. The concentrations of representative cytokines in culture medium 3 days after the wash were examined by ELISA. Data are representative of three repeated experiments. *<i>p</i><0.01, **<i>p</i><0.05.</p