B16 melanomas evade antitumor immunity by the loss of epitope presentation and the acquisition of tumor resistance to granzyme B.

Melanomas exhibit the highest rate of heterogeneity among cancer cell types. In this study, we tested the two types of B16 melanoma cells (B16-S0-1 and B16-S1-1) showing resistance to antitumor immunity. These cells expressed Trp2 protein. Contrary to B16 and B16-S0-1 cells, B16-S1-1 cells failed to stimulate IFN-γ responses in Trp2-specific CD8+ T cells, suggesting that B16-S1-1 cells may have lost the ability to present antigen to Ag-specific CTLs in the context of MHC class I molecules. However, B16-S0-1 cells exhibited active Stat3 and decreased Bcl-2 expression, which were found to be not associated with immune escape. B16-S0-1 cells were more resistant to granzyme B-mediated caspase activation and apoptosis than B16 cells. Thus, these data show that B16 cells escape antitumor immune responses through the loss of epitope presentation to CTLs and the acquisition of tumor cell resistance to granzyme B-mediated caspase activation.

B16 melanoma control by anti-PD-L1 requires CD8+ T cells and NK cells: application of anti-PD-L1 Abs and Trp2 peptide vaccines.

Anti-programmed death ligand 1 (PD-L1) therapy has been beneficial in treating patients with certain cancers. Here, we tested whether anti-PD-L1 therapy is effective for controlling different types of tumors using animal models of TC-1, MC38 and B16. We found that, despite PD-L1 expression, anti-PD-L1 therapy showed little and some antitumor activity in the TC-1 and MC38 models. However, anti-PD-L1 therapy exhibited a more dramatic antitumor effect in the B16 model. This difference in antitumor responses was likely associated with the CD8 + T cell infiltration status of tumor tissues. In the B16 model, CD8 + T cells and to a lesser degree NK cells were found to be responsible for the antitumor response of anti-PD-L1 therapy, as determined by immune cell subset depletion. In particular, CD8 + T cells from B16-bearing mice produced an IFN-γ in response to B16 cells and citrate phosphate buffer-treated B16 cell peptide elutes but not to an immunodominant class I epitope, Trp2180-188, suggesting that CD8 + T cells that recognize neoantigens were induced in B16 tumor-bearing mice and then reactivated by anti-PD-L1 for tumor control. When B16 tumor-bearing mice were treated with anti-PD-L1 in combination with Trp2180-188 peptide vaccines, they displayed significantly more tumor control than either single therapy. Taken together, these studies show that B16 melanomas are more effectively controlled through reactivation of tumor-infiltrating lymphocytes by anti-PD-L1 therapy. Moreover, combined therapy using anti-PD-L1 and Trp2 peptide vaccines is more beneficial for controlling B16 melanomas through reactivation of neoantigen-specific CD8 + T cells and induction of Trp2-specific CD8 + T cells.

CD8+ T Cells Directed Against a Peptide Epitope Derived From Peptidoglycan-Associated Lipoprotein of Legionella pneumophila Confer Disease Protection.

Legionella pneumophila, an intracellular bacterium, may cause life-threatening pneumonia in immunocompromised individuals. Mononuclear cells and antibodies have been reported to be associated with the host defense response against L. pneumophila. This study is to determine whether Legionella peptidoglycan-associated lipoprotein (PAL)-specific CD8+ T cells are directly associated with protection against L. pneumophila, with a focus on potential epitopes. Synthetic peptides derived from PAL of L. pneumophila were obtained and tested through in vitro and in vivo cytotoxic T lymphocyte (CTL) assays for immunogenicity. PAL DNA vaccines or a peptide epitope with or without CpG-oligodeoxynucleotides (ODN) was evaluated for protection against L. pneumophila infection in animal models. When mice were immunized with DNA vaccines expressing the PAL of L. pneumophila, they were significantly protected against a lethal challenge with L. pneumophila through induction of antigen-specific CD8+ CTLs. Of the 13 PAL peptides tested, PAL92-100 (EYLKTHPGA) was the most immunogenic and induced the strongest CTL responses. When mice were immunized with the PAL92-100 peptide plus CpG-ODN, they were protected against the lethal challenge, while control mice died within 3-6 days after the challenge. Consistent with lung tissue histological data, bacterial counts in the lungs of immunized mice were significantly lower than those in control mice. Also, the amino acid sequence of PAL92-100 peptides is conserved among various Legionella species. To our knowledge, this study is the first to demonstrate that PAL92-100-specific CD8+ T cells play a central role in the host defense response against L. pneumophila.

Regulation of the translation activity of antigen-specific mRNA is responsible for antigen loss and tumor immune escape in a HER2-expressing tumor model.

Tumor cells tend to behave differently in response to immune selective conditions. Contrary to those in therapeutic antitumor conditions, tumor cells in prophylactic antitumor conditions lose antigen expression for antitumor immune escape. Here, using a CT26/HER2 tumor model, we investigate the underlying mechanism(s). We selected tumor cell variants (CT26/HER2-A1 and -A2) displaying resistance to antitumor protective immunity and loss of HER2 antigen expression. These immune-resistant cells failed to induce Ag-specific IgG and IFN-γ responses while forming tumors at the same rate as CT26/HER2 cells. RT-PCR, qRT-PCR, PCR, Western blot and DNA sequencing analyses demonstrated that HER2 expression was inhibited at the post-transcriptional level in these immune-resistant cells, suggesting that tumor cells may escape antitumor immunity through the post-transcriptional regulation of antigen gene expression. The proteasome and lysosomal protein degradation pathways were not responsible for antigen loss, as determined by an inhibitor assay. Finally, HER2 mRNA was found to be not present in the monosomes and polysomes of CT26/HER2-A2 cells, as opposed to CT26/HER2 cells, suggesting that the translation activity of HER2 mRNAs may be suppressed in these immune-resistant cells. Taken together, our results report a new mechanism by which tumor cells respond to antitumor protective immunity for antitumor immune evasion.

Optimized Gemcitabine Therapy in Combination with E7 Peptide Immunization Elicits Tumor Cure by Preventing Ag-Specific CTL Inhibition in Animals with Large Established Tumors.

The role of chemotherapeutic agents in tumor immunotherapy is still controversial. In this study, we test using a TC-1 tumor model whether gemcitabine plus E7 peptide vaccine regimens (E7 peptides+CpG-ODN+anti-4-1BB Abs) may result in tumor cure in mice with large established tumors, with a focus on their effects on Ag-specific cytotoxic T lymphocyte (CTL) and myeloid-derived suppressor cell levels. Gemcitabine inhibited tumor growth by its direct cytotoxicity to tumor cells in vivo. E7 peptide vaccine regimens enhanced Ag-specific CTL lytic and antitumor therapeutic activity. Initial combination therapy using gemcitabine and E7 peptide vaccine regimens resulted in tumor regression with tumor relapse in animals with large established tumors, which appeared to result from the suppression of Ag-specific CTL activity by gemcitabine treatment. However, optimization of gemcitabine therapy by reducing its dose and frequency led to complete tumor regression without any recurring tumors in all tested mice even after discontinuation of therapy, possibly due to Ag-specific CTL responses. Thus, this study shows that the optimal dose and therapy frequency of gemcitabine are critical for achieving tumor cure in tumor-bearing animals undergoing E7 peptide vaccine regimen therapy, mainly by preventing CTL suppression. These findings may have implications for designing peptide-based therapeutic vaccines in cancer patients undergoing chemotherapy.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein.

PURPOSE: The goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones. MATERIALS AND METHODS: For hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953-7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection. CONCLUSION: This study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production.

PURPOSE: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies. MATERIALS AND METHODS: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone. CONCLUSION: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Therapeutic Tumor Control of HER2 DNA Vaccines Is Achieved by an Alteration of Tumor Cells and Tumor Microenvironment by Gemcitabine and Anti-Gr-1 Ab Treatment in a HER2-Expressing Tumor Model.

Therapeutic control of tumors is challenging as they tend to alter their biological functions and microenvironment. In a CT26/HER2 tumor model, HER2 DNA vaccines and even anti-PD-L1 Abs failed to display antitumor therapeutic activity while inducing Ag-specific cytotoxic T lymphocyte (CTL) activity. To clarify this contradictory finding, we selected tumor cells (CT26/HER2-1) from one tumor-bearing animal in the therapeutic model. CT26/HER2-1 cells behaved similar to wild-type CT26/HER2 cells in their HER2 expression, immune cell stimulation for IFN-γ production, and antitumor immune sensitivity. A similar finding was obtained with additional CT26/HER2-2, -3, -4, -5, and -6 cells from the therapeutic model, suggesting that a lack of antitumor therapeutic activity of HER2 DNA vaccines might be ascribed to a factor in the tumor microenvironment, but not to an alteration in tumor cell functions. When tumor-bearing mice were depleted of myeloid-derived suppressor cells (MDSCs) by anti-Gr-1 Ab treatment, they displayed HER2 vaccine-mediated antitumor activity, suggesting a role of MDSCs in blocking antitumor activity. Moreover, when tumor-bearing mice were treated with gemcitabine, they displayed HER2 vaccine-mediated antitumor activity, suggesting that cytotoxic drug treatment makes tumor cells susceptible to lysis by CTLs. Thus, these studies show that therapeutic control of HER2 DNA vaccines can be achieved by anti-Gr-1 Ab treatment through MDSC depletion and by gemcitabine treatment through sensitization of tumor cells to CTL-mediated killing in this model. These findings may have implications for achieving therapeutic control of CTL-resistant tumors in cancer therapy.

Tumor regression is mediated via the induction of HER263-71- specific CD8+ CTL activity in a 4T1.2/HER2 tumor model: no involvement of CD80 in tumor control.

In the CT26/HER2 and 4T1.2/HER2 tumor models, CT26/HER2 cells form tumors that continue to grow, whereas 4T1.2/HER2 cells form tumors that eventually regress. Here, we investigated the differences in the behaviors of these two cell lines. When immune cells from 4T1.2/HER2 tumor-bearing animals were stimulated with HER2 class I peptides, they displayed a 2-fold increase in IFN-γ levels, in response to the peptides, HER263-71 and HER2342-350. In contrast, extremely high levels of antigen-non-specific IFN-γ production were observed with immune cells and sera from CT26/HER2 tumor-bearing mice. However, IFN-γ had no effect on tumor progression in the CT26/HER2 model, as determined by an IFN-γ knockout assay. 4T1.2/HER2 tumor-bearing mice displayed CTL activity in response to HER263-71 but not to HER2342-350, whereas no such induction was observed in CT26/HER2 tumor-bearing mice. When 4T1.2/HER2 cell-challenged mice were depleted of CD8+ T cells, they lost their tumor-regressing activity, suggesting an antitumor role of HER263-71-specific CD8+ CTLs in the control of this tumor type. CT26/HER2 cells also expressed CD80. However, CD80-transfected 4T1.2/HER2 and CD80-non-expressing CT26/HER2 cells failed to alter their tumorigenicity, suggesting no role of CD80 in tumor control. Despite increased levels of myeloid-derived suppressor cells in the tumor, they were not associated with tumor progression in the CT26/HER2 model, as determined by a cell depletion assay. Overall, these data show that, contrary to CT26/HER2 tumors, 4T1.2/HER2 tumors regress via the induction of HER263-71-specific CD8+ CTLs and that CD80 is not associated with the regression of these tumors.

Intratumorally Establishing Type 2 Innate Lymphoid Cells Blocks Tumor Growth.

A long-standing question in the field of tumor immunotherapy is how Th2 cytokines block tumor growth. Their antitumor effects are particularly prominent when they are secreted continuously in tumors, suggesting that Th2 cytokines may create a tumor microenvironment unfavorable for tumor growth independently of adaptive immunity. In this study, we show that local production of IL-33 establishes a high number of type 2 innate lymphoid cells (ILC2s) with potent antitumor activity. IL-33 promotes secretion of a massive amount of CXCR2 ligands from ILC2s but creates a tumor microenvironment where tumor cells express CXCR2 through a dysfunctional angiogenesis/hypoxia/reactive oxygen species axis. These two signaling events converge to reinforce tumor cell-specific apoptosis through CXCR2. Our results identify a previously unrecognized antitumor therapeutic pathway wherein ILC2s play a central role.

DNA vaccines, electroporation and their applications in cancer treatment.

Numerous animal studies and recent clinical studies have shown that electroporation-delivered DNA vaccines can elicit robust Ag-specific CTL responses and reduce disease severity. However, cancer antigens are generally poorly immunogenic, requiring special conditions for immune response induction. To date, many different approaches have been used to elicit Ag-specific CTL and anti-neoplastic responses to DNA vaccines against cancer. In vivo electroporation is one example, whereas others include DNA manipulation, xenogeneic antigen use, immune stimulatory molecule and immune response regulator application, DNA prime-boost immunization strategy use and different DNA delivery methods. These strategies likely increase the immunogenicity of cancer DNA vaccines, thereby contributing to cancer eradication. However, cancer cells are heterogeneous and might become CTL-resistant. Thus, understanding the CTL resistance mechanism(s) employed by cancer cells is critical to develop counter-measures for this immune escape. In this review, the use of electroporation as a DNA delivery method, the strategies used to enhance the immune responses, the cancer antigens that have been tested, and the escape mechanism(s) used by tumor cells are discussed, with a focus on the progress of clinical trials using cancer DNA vaccines.

MC32 tumor cells acquire Ag-specific CTL resistance through the loss of CEA in a colon cancer model.

We previously reported that MC32 cells resist carcinoembryonic antigen (CEA) DNA vaccination by losing their antigen presentation to Ag-specific CTLs in the context of MHC class I antigens in a colon cancer therapeutic model. In this study, we selected 2 tumor cells, MC32-S2-2 and MC32-S4-2, which have the ability to form tumors in CEA DNA vaccine-immunized mice. Wild type MC32 cells grew significantly less in CEA-immunized mice (with Ag-specific CTL lytic activity) than in control mice (with no Ag-specific CTL lytic activity). However, MC32-S2-2 and MC32-S4-2 cells grew at a similar rate in both control and CEA-immunized mice, confirming their resistant status against CEA DNA vaccination. MC32-S2-2 and MC32-S4-2 cells were not susceptible to lysis by CEA-specific CD8+ T cells. Moreover, when MC32-S2-2 and MC32-S4-2 cells were used as stimulating agents of CEA-specific immune cells for IFN-γ production, these cells failed to stimulate the induction of Ag-specific IFN-γ, suggesting a loss of tumor cell recognition by Ag-specific immune cells. However, MC32-S2-2 and MC32-S4-2 cells expressed MHC class I antigens in a manner similar to that of wild type MC32 cells. Finally, Western blot assay confirmed that in MC32-S2-2 and MC32-S4-2 cells, CEA expression remained absent but mouse CEA was expressed. Taken together, these data show that MC32 cells may also be able to achieve resistance to CEA-specific CTLs by antigen loss in this model.

A loss of antitumor therapeutic activity of CEA DNA vaccines is associated with the lack of tumor cells' antigen presentation to Ag-specific CTLs in a colon cancer model.

Human colon cancers express carcinoembryonic antigen (CEA). Thus, CEA has been considered as a potential vaccine target for immune therapy against colon cancer. In this study, CEA DNA vaccines plus anti-4-1BB Abs treatment was found to increase Ag-specific CTL activity and antitumor protective responses to MC32 cells. However, CEA DNA vaccines alone displayed few antitumor therapeutic effects while significantly inducing Ag-specific CTL responses. Anti-4-1BB Abs alone displayed antitumor therapeutic effects. Intratumoral electroporation with IL-12 cDNA also showed antitumor therapeutic activity against MC32 cells in a CD8+ T cell-dependent and CEA-non-specific manner, suggesting that established MC32 cells are still susceptible to CTL-mediated killing. Finally, our in vitro assays (Western blot assay, IFN-γ, CTL and apoptosis assays, FACS analysis) and animal studies demonstrated that a lack of antitumor therapeutic activity of CEA DNA vaccines might result from acquisition of tumor cell resistance to Ag-specific CTL-mediated killing through the loss of tumor cells' antigen presentation to Ag-specific CTLs. Taken together, these data show that MC32 cells may resist CEA DNA vaccination by their loss of antigen presentation to CEA-specific CTLs in the therapeutic model.

Alterations of colonic contractility in an interleukin-10 knockout mouse model of inflammatory bowel disease.

BACKGROUND/AIMS: Inflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10(-/-)) mice. METHODS: Prepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining. RESULTS: The spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10(-/-) mice compared to IL-10(+/+) mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10(-/-) mice compared to IL-10(+/+) mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10(-/-) mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10(-/-) mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased. CONCLUSIONS: These results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.

Flagellin enhances tumor-specific CD8⁺ T cell immune responses through TLR5 stimulation in a therapeutic cancer vaccine model.

Tumor antigen (TA)-specific immunotherapy is an emerging approach for cancer treatment. Potent adjuvants are prerequisites to the immunotherapy for overcoming the low immunogenicity of TAs. We previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, has potent adjuvant activity in various vaccination models. In this study, we investigated whether the FlaB protein could be a potent adjuvant for a human papillomavirus 16 E6 and E7 (E6/E7) peptide-based anticancer immunotherapy. We used an E6/E7-expressing TC-1 carcinoma implantation animal model and tested TA-specific immunomodulation by FlaB. We co-administered the E6/E7 peptide either with or without FlaB into TC-1 tumor-bearing mice and then analyzed the antitumor activity of the peptide. FlaB significantly potentiated specific antitumor immune responses elicited by the peptide immunization, as evidenced by retarded in vivo tumor growth and significantly prolonged survival. We noticed that TC-1 cells do not express Toll-like receptor 5 (TLR5) on their surface and the TLR5 signaling pathway in TC-1 cells was not responsible for the antitumor effect of FlaB. FlaB potentiated the CTL activity and Ag-specific IFN-γ production of CD8(+) T cells from the draining lymph node and spleen. In addition, this antitumor activity was abrogated following the in vivo depletion of CD8(+) T cells and in TLR5 knockout (KO) or MyD88 KO mice. These results suggest that flagellin could enhance TA-specific CD8(+) CTL immune responses through TLR5 stimulation in cancer immunotherapy.

DNA vaccines targeting human papillomavirus-associated diseases: progresses in animal and clinical studies.

Human papillomavirus (HPV) infection is a major cause of cervical cancer and its precancerous diseases. Cervical cancer is the second deadliest cancer killer among women worldwide. Moreover, HPV is also known to be a causative agent of oral, pharyngeal, anal and genital cancer. Recent application of HPV structural protein (L1)-targeted prophylactic vaccines (Gardasil® and Cervarix®) is expected to reduce the incidence of HPV infection and cervical cancer, and possibly other HPV-associated cancers. However, the benefit of the prophylactic vaccines for treating HPV-infected patients is unlikely, underscoring the importance of developing therapeutic vaccines against HPV infection. In this regard, numerous types of therapeutic vaccine approaches targeting the HPV regulatory proteins, E6 and E7, have been tested for their efficacy in animals and clinically. In this communication, we review HPV vaccine types, in particular DNA vaccines, their designs and delivery by electroporation and their immunologic and antitumor efficacy in animals and humans, along with the basics of HPV and its pathogenesis.

Beraprost enhances production of antigen-specific IgG isotypes without modulating germinal center B cell generation and the affinity maturation.

Immune regulating functions of lipid mediators are being recognized. Prostaglandins (PGs) are derived from phospholipid by cyclooxygenase-2 (COX-2) in inflammatory tissues. Based upon our previous data that imply an immune modulating activity of prostacyclin, we hypothesized that PGs promote the humoral immune responses in vivo. To test this hypothesis, we examined the effect of a prostacyclin analog beraprost and PGE2 on the antibody responses that were induced by immunizing mice with keyhole limpet hemocyanin (KLH). Beraprost was used due to the extremely unstable property of prostacyclin. Our results showed that beraprost indeed promoted the production of anti-KLH antibodies, which was dose-dependent and specific to beraprost because PGE2 did not modulate the antibody response compared with controls. The enhancing effect of beraprost was reproduced in the secondary responses, suggesting that memory B cell generation during the primary response was significantly affected by beraprost. Analysis of the isotypes of anti-KLH antibodies revealed that beraprost stimulated the production of IgA and IgG subisotypes but not IgM. However, germinal center B cell generation and the affinity of anti-KLH antibodies were not affected by beraprost administration. To confirm these results we immunized COX-2 knockout mice with KLH and analyzed whether the results with wild-type mice were reflected in the absence of PGs. The primary and secondary antibody responses were significantly impaired in the KO animals. The levels of anti-KLH IgG subisotypes and IgA were severely reduced in KO mice whereas those of IgM were comparable to controls. These results reveal an unrecognized function of PG in the humoral immune responses.

Combined stimulation of TLR9 and 4.1BB augments Trp2 peptide vaccine-mediated melanoma rejection by increasing Ag-specific CTL activity and infiltration into tumor sites.

Peptide vaccines are a clinically applicable therapy shown to be effective in tumor control. In this study, Trp2 peptides plus CpG-oligodeoxynucleotide treatment was found to induce Ag-specific IFN-γ and CD8+CTL responses, and antitumor activity against large established melanoma (tumor size, 7mm). A combination of anti-4.1BB antibodies with Trp2 peptides+CpG-oligodeoxynucleotide increased the antitumor cure rate from 0% to 75%. This effect was concomitant with greater induction of Ag-specific CD8+CTLs and their infiltration into the tumor sites, highlighting the importance of combined stimulation of TLR9 and 4.1BB for achieving tumor eradication. These findings may have implications for designing peptide-based therapeutic vaccines for cancer-patients.

Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.

Electroporation driven delivery of both an IL-12 expressing plasmid and cisplatin synergizes to inhibit B16 melanoma tumor growth through an NK cell mediated tumor killing mechanism.

Combined therapy using chemotherapeutic drugs and immunotherapeutics offers some promise for treating patients with cancer. In this study, we evaluated whether cisplatin delivered by intratumoral (IT)-electroporation (EP) might enhance antitumor activity against established B16 melanoma and whether further addition of intramuscular (IM)-EP of IL-12 cDNA to IT-EP of cisplatin might augment antitumor therapeutic activity, with a focus on the underlining antitumor mechanism(s). When tumor (7 mm)-bearing animals were treated locally with cisplatin by IT-EP, they showed tumor growth inhibition significantly more than those without IT-EP. Moreover, IL-12 cDNA delivered by IM-EP was also able to inhibit tumor growth significantly more than control vector delivery. This tumor growth inhibition was mediated by NK cells, but not CD4+ T or CD8+ T cells, as determined by immune cell subset depletion and IFN-γ induction. Moreover, concurrent therapy using IT-EP of cisplatin plus IM-EP of IL-12 cDNA displayed antitumor therapeutic synergy. This therapeutic synergy appeared to be mediated by increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. Taken together, these data support that cisplatin delivery by IT-EP plus IL-12 gene delivery by IM-EP are more effective at inducing antitumor therapeutic responses through increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. This combined approach might have some implication for treating melanoma in patients.