Paclitaxel-exposed ovarian cancer cells induce cancer­specific CD4+ T cells after doxorubicin exposure through regulation of MyD88 expression.

Ovarian cancer has the highest mortality rate among gynecological malignancies due to high chemoresistance to the combination of platinum with taxane. Immunotherapy against ovarian cancer is a promising strategy to develop from animal-based cancer research. We investigated changes in the immunogenicity of paclitaxel-exposed ovarian cancer cells following exposure to other chemotherapeutic drugs. Murine ovarian surface epithelial cells (MOSECs) showed some resistance to paclitaxel, a first-line therapy for ovarian cancer. However, MOSECs pre-exposed to paclitaxel died through apoptosis after incubation with doxorubicin or cisplatin for 2 h. Injected into mice, the paclitaxel-exposed MOSECs post-treated with doxorubicin induced more MOSEC-specific CD4(+) T cells and extended survival for a greater time than MOSECs treated with paclitaxel alone; and bone marrow-derived dendritic cells (BMDCs) expressed higher levels of co-stimulatory molecules and produced IL-12 after co-culture with paclitaxel-exposed MOSECs treated with doxorubicin. We also observed that in paclitaxel-exposed MOSECs treated with doxorubicin, but not cisplatin, the expression of MyD88 and related target proteins decreased compared to paclitaxel-exposed MOSECs only, while in BMDCs co-cultured with these MOSECs the expression of myeloid differentiation primary response gene 88 (MyD88) increased. These findings suggest that paclitaxel pre-exposed cancer cells treated with doxorubicin can induce significant apoptosis and a therapeutic antitumor immune response in advanced ovarian cancer.

Cancer cells containing nanoscale chemotherapeutic drugs generate antiovarian cancer-specific CD4+ T cells in peritoneal space.

Owing to the poor prognosis of patients with ovarian cancer, new treatment strategies immediately need to be developed. Although several immunotherapeutic approaches have been examined for the treatment of advanced stage ovarian cancer, their implementation in clinical practice remains low. We previously showed doxorubicin-treated murine ovarian cancer cells [murine ovarian surface epithelial cells (MOSECs)] are able to deliver drug to adjacent cells in vivo to eradicate tumor cells. In this study, we hypothesized that irradiated tumor cell treated with anticancer drugs may kill other cancer cell by cell to cell contact and also by generating antitumor immune responses. The MOSECs treated with anticancer drugs (doxorubicin and cisplatin) died through apoptosis, and this was increased in accordance with the dose of drug. The cleaved caspase-3 expression was significantly increased in the MOSECs coexposed with doxorubicin and cisplatin. Anticancer drug-treated MOSECs generated MOSEC-specific CD4 T-cell immune responses. Bone marrow-derived dendritic cells expressed upregulated IL-12p40 mRNA but IL-6 and IL-10 mRNA downregulated after coculture with MOSECs cotreated with doxorubicin and cisplatin. Furthermore, the mice vaccinated with MOSECs cotreated with doxorubicin and cisplatin had enhanced antitumor immunity and prolonged survival. We also observed that CD4 T cells and natural killer cells are essential for the antitumor immunity generated by vaccination with anticancer drug-loaded MOSECs. These findings suggest that irradiated MOSECs treated with anticancer drugs could be a new immune-therapeutic strategy against advanced ovarian cancers.

Dendritic cells stimulated with outer membrane protein A (OmpA) of Salmonella typhimurium generate effective anti-tumor immunity.

Gram-negative bacterial outer membrane proteins (Omps) have an important role in pathogenesis and signal reception. We previously reported that Acinetobacter OmpA (AbOmpA) induced maturation of bone marrow-derived dendritic cells (BMDCs) and that AbOmpA-primed DCs produced IL-12 which generated Th1 CD4(+) T-cells. We analyzed the effects of Salmonella typhimurium OmpA (OmpA-Sal) on dendritic cell (DC) maturation in the present study, and determined that tumor antigen-pulsed DCs stimulated with OmpA-Sal induced anti-tumor responses in a mouse model. OmpA-Sal activated BMDCs by augmenting expression of MHC class II and of the co-stimulatory molecules CD80 and CD86. RT-PCR revealed that IL-12(p40) gene expression is highly augmented in OmpA-Sal-stimulated BMDCs. DNA (CRT/E7) vaccination combined with OmpA-Sal stimulation generated more antigen-specific CD8(+) T-cells in the present study. Certain antigen-pulsed BMDCs stimulated with OmpA-Sal induced strong PADRE-specific CD4(+) and E7-specific CD8(+) T-cell responses. In addition, BMDCs stimulated with OmpA-Sal (OmpA-Sal-BMDCs) and pulsed with both E7 and PADRE peptide generated greater numbers of E7-specific CD8(+) effector and memory T-cells than those pulsed with E7 peptide alone. E7- and PADRE-expressing OmpA-Sal-BMDC vaccines resulted in significant long-term protective anti-tumor effects in vaccinated mice. Our data suggested that E7- and PADRE-expressing BMDCs that were matured in the presence of OmpA-Sal might enhance anti-tumor immunity and support the therapeutic use of OmpA-Sal in DC-based immunotherapy.

CD4+ TH1 cells generated by Ii-PADRE DNA at prime phase are important to induce effectors and memory CD8+ T cells.

The requirement for CD4 T cells in priming and maintaining cytotoxic T-lymphocyte responses presents a long-standing paradox in cellular immunology. In this study, we used sequential coadministration of a DNA vaccine encoding an invariant (Ii) chain in which the class II-associated Ii-peptide region is replaced with CD4 T-helper epitope, PADRE [Pan human leukocyte antigen-DR reactive epitope (Ii-PADRE)] or Bcl-xL with a DNA vaccine encoding Sig/E7/LAMP-1 to verify the role of CD4 T cells for the generation of effectors and memory E7-specific CD8 T-cell immune responses. Sequential vaccination, with Ii-PADRE+Sig/E7/LAMP-1 priming followed by Bcl-xL+Sig/E7/LAMP-1 boosting led to generation of E7-specific CD8 T cells, and was nearly equivalent in effect to coadministration with Ii-PADRE+Sig/E7/LAMP-1 or Bcl-xL+Sig/E7/LAMP-1 at both prime and boost. The mice vaccinated with the Ii-PADRE+Sig/E7/LAMP-1 prime-Bcl-xL+Sig/E7/LAMP-1 boost regimen exhibited better long-term E7-specific immune responses and tumor prevention effects in vivo than the mice vaccinated with the reverse sequential coadministration. After CD4 T-cell depletion, mice primed with Ii-PADRE+Sig/E7/LAMP-1 generated low numbers of E7-specific CD8 T cells and suppressed long-term memory CD8 T-cell response regardless of the sequence or combination of DNA vaccines administered. Mice primed with Bcl-xL+Sig/E7/LAMP-1 only suppressed long-term memory CD8 T-cell response after depletion of CD4 T cells before priming. Our findings suggest that activated CD4 T cells at prime phase are important to generate the antigen-specific CD8 T-cell immune responses and CD4 T cells, which are naive or activated, play a role to maintain the long-term memory responses.

Doxorubicin enhances CD4(+) T-cell immune responses by inducing expression of CD40 ligand and 4-1BB.

Chemotherapy agents have adverse immunotherapeutic effects secondary to inhibition of hematopoietic stem cell proliferation, particularly in committed lymphoblast. Certain anti-cancer drugs have immuno-modulatory properties, although mechanisms are still not fully understood. In the current study, we explored the effects of doxorubicin on peripheral blood CD4(+) and CD8(+) T-cell responses pre- and post-stimulation. Doxorubicin treatment alone had no effects on peripheral blood T lymphocytes and regulatory T-cells in vivo and in vitro. However, CD4(+) T-cells were resistant to doxorubicin and demonstrated more robust proliferation than CD8(+) T-cells after doxorubicin pre-treatment. CD40 ligand and 4-1BB expression on the surface of CD4(+) T-cells were increased after TCR-ligation activation; however, expression on CD8(+) T-cells was not increased. Dendritic cells cultured in the presence of activated CD4(+) T-cells pre-treated with doxorubicin had greater survival rates than those cultured with activated CD8(+) T-cells. Doxorubicin pre-treatment followed by anti-CD3epsilon+anti-4-1BB activation led to proliferation of CD4(+) T-cells and no proliferative effects on CD8(+) T-cells. Our results collectively suggest that doxorubicin pre-treatment in cancer patients may be a more effective way to enhance anti-cancer immune responses by increased antigen-specific CD4(+) Th1 immune responses.

Methotrexate-incorporated polymeric nanoparticles of methoxy poly(ethylene glycol)-grafted chitosan.

We prepared methotrexate (MTX)-encapsulated polymeric nanoparticles using methoxy poly(ethylene glycol) (MPEG)-grafted chitosan (ChitoPEG) copolymer. MTX-encapsulated polymeric nanoparticles of ChitoPEG copolymer has around 50-300nm in particle size and showed spherical shape when observed by transmission electron microscope (TEM). In (1)H nuclear magnetic resonance (NMR) study, the specific peaks of MTX and chitosan as a drug carrying inner-core disappeared at D(2)O and only the specific peak of MPEG was observed, while specific peaks of MPEG, MTX, and chitosan appeared in DCl/D(2)O mixtures. These results indicated that MTX was complexed with chitosan and then core-shell type nanoparticles had formed in aqueous environment, i.e., MTX/chitosan complexes composed of inner-core and MPEG composed of outer-shell of the nanoparticles. Loading efficiency of MTX in the polymeric nanoparticles was 94% (w/w) of ChitoPEG-1, 91.1% (w/w) of ChitoPEG-2, 90.1% (w/w) of ChitoPEG-3 and 65.2% (w/w) of ChitoPEG-4, expectively. The higher the drug feeding ratio, the higher the drug content and the lower the loading efficiency. The higher the MPEG graft ratio in the copolymer, the lower the drug content and loading efficiency. Drug contents evaluated by (1)H NMR were the same as found by UV spectrophotometer.